Objective—To determine whether canine tumor cell lines express functional tissue factor and shed tissue factor-containing microparticles. Sample—Cell lines derived from tumors of the canine mammary gland (CMT12 and CMT25), pancreas (P404), lung (BACA), prostate gland (Ace-1), bone (HMPOS, D-17, and OS2.4), and soft tissue (A72); from normal canine renal epithelium (MDCK); and from a malignant human mammary tumor (MDA-MB-231). Procedures—Tissue factor mRNA and antigen expression were evaluated in cells by use of canine-specific primers in a reverse transcriptase PCR assay and a rabbit polyclonal anti-human tissue factor antibody in flow cytometric and immunofluorescent microscopic assays, respectively. Tissue factor procoagulant activity on cell surfaces, in whole cell lysates, and in microparticle pellets was measured by use of an activated factor X-dependent chromogenic assay. Results—Canine tissue factor mRNA was identified in all canine tumor cells. All canine tumor cells expressed intracellular tissue factor; however, the HMPOS and D-17 osteosarcoma cells lacked surface tissue factor expression and activity. The highest tissue factor expression and activity were observed in canine mammary tumor cells and pulmonary carcinoma cells (BACA). These 3 tumors also shed tissue factor-bearing microparticles into tissue culture supernatants. Conclusions and Clinical Relevance—Tissue factor was constitutively highly expressed in canine tumor cell lines, particularly those derived from epithelial tumors. Because tumor-associated tissue factor can promote tumor growth and metastasis in human patients, high tissue factor expression could affect the in vivo biological behavior of these tumors in dogs.

Objective—To determine whether trilostane or ketotrilostane is more potent in dogs and determine the trilostane and ketotrilostane concentrations that inhibit adrenal gland cortisol, corticosterone, and aldosterone secretion by 50%. Sample—24 adrenal glands from 18 mixed-breed dogs. Procedures—Adrenal gland tissues were sliced, placed in tissue culture, and stimulated with 100 pg of ACTH/mL alone or with 5 concentrations of trilostane or ketotrilostane. Trials were performed independently 4 times. In each trial, 6 samples (1 for each time point) were collected for each of the 5 concentrations of trilostane and ketotrilostane tested as well as a single negative control samples. At the end of 0, 1, 2, 3, 5, and 7 hours, tubes were harvested and media and tissue slices were assayed for cortisol, corticosterone, aldosterone, and potassium concentrations. Data were analyzed via pharmacodynamic modeling. One adrenal slice exposed to each concentration of trilostane or ketotrilostane was submitted for histologic examination to assess tissue viability. Results—Ketotrilostane was 4.9 and 2.4 times as potent in inhibiting cortisol and corticosterone secretion, respectively, as its parent compound trilostane. For trilostane and ketotrilostane, the concentrations that inhibited secretion of cortisol or corticosterone secretion by 50% were 480 and 98.4 ng/mL, respectively, and 95.0 and 39.6 ng/mL, respectively. Conclusions and Clinical Relevance—Ketotrilostane was more potent than trilostane with respect to inhibition of cortisol and corticosterone secretion. The data should be useful in developing future studies to evaluate in vivo serum concentrations of trilostane and ketotrilostane for efficacy in the treatment of hyperadrenocorticism.

Thirteen 3-week-old pigs that had been allowed to nurse for the first 16 to 18 hours after birth were orally inoculated with 1 X 10(6.5) TCID(50) of porcine rotavirus. All developed diarrhea, anorexia, and vomiting by postinoculation (PI) hour 30. These signs had abated by PI day 6. Villus blunting in the small intestine was most severe in the jejunum and ileum of pigs euthanatized between PI days 3 and 5. Villi had returned to nearly normal length by PI day 6, although fused villi were seen in a few locations in the distal portion of the jejunum and in the ileum. Virus was detected in the feces of inoculated pigs by isolation in cell cultures and by electron microscopy during the 7-day course of the experiment. There was 1 extraintestinal virus isolation from the lung of 1 pig at PI day 2. Infection and disease developed in the presence of serum-neutralizing antibody obtained by nursing seropositive sows. There was no significant change in neutralizing antibody titers in the 3-week-old pigs over the course of the experiment. In this experimental work, a model to study rotavirus infection in 3-week-old pigs has been developed.

The continuous use of antibiotics for tissue culture adapted vaccines production has led to the increase in the bacterial resistance to these antibiotics. This study aims to evaluate the antimicrobial potential of thyme (Thymus vulgaris) and clove (Syzygium aromaticum) on bacterial and fungal contamination, in the production of tissue culture vaccines. The active agents in each plant were extracted by the conventional extraction technique using ethanol and water as solvents followed by concentration (steam distillation and boiling). The antimicrobial activities of different solvent extracts were determined in well agar diffusion technique using Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Candida albicans as model for gram positive, gram negative and fungal contamination, respectively. The cytotoxic effects of the different solvent extracts were tested on VERO and MDBK cell culture. The obtained results indicated that water and ethanolic extracts from thyme and clove plants showed significant antimicrobial activities (P < 0.05) as they could inhibit the growth of E. coli, S. aureus and Candida albicans. Ethanolic extract of thyme had the maximum zone of inhibition against E. coli (2.40±0.20) and Candida albicans (3.07±0.3), and the lowest inhibition zone against S. aureus (1.53±0.23), whereas the thyme water extract didn’t show any antimicrobial activity. The ethanolic extract of clove showed the greatest zone of inhibition against Candida albicans (2.63±0.2), E. coli (2.63±0.2), while the lowest was against S. aureus (1.87±0.3). Water extract of clove showed the greatest zone of inhibition against E. coli and S. aureus (1.93±0.4, and 2.47±0.1), respectively and 0.97±0.1 against Candida albicans. The ethanolic extracts of thyme and clove showed changes in the cell wall until concentration 1 mg/ml for clove and 10 µg/ml for thyme on VERO cells; while the cytotoxic effect on MDBK cells was observed till the concentration of 100 µg/ml for clove and thyme water extracts. In conclusion, the antimicrobial potential of clove  water extract on bacterial and fungal contaminant could replace antibiotics in the production of tissue culture vaccines at a concentration of 10 µg/ml.

Various types of human and nonhuman adenoviral (AdV) vectors are being used as gene delivery vectors in preclinical and clinical investigations. The objective of this study was to determine the ratio between the 2 best assays that would effectively address the variability in the titration of various AdV vectors in different cell lines and help obtain consistent results in preclinical and clinical studies using different AdV vectors. Here, we compared plaque-forming units, tissue culture infectious dose 50, focus-forming units (FFU), virus particle (VP) count, and genome copy number (GCN) of purified preparations of human AdV type C5, bovine AdV type 3, and porcine AdV type 3 to determine a correlation between infectious and noninfectious virus particles. Our results suggest that a VP:FFU or a VP:GCN ratio could accurately reflect the quality of an AdV preparation and could serve as an indicator to control batch-to-batch variability.

OBJECTIVE To determine the minimum infectious dose of porcine epidemic diarrhea virus (PEDV) in virus-inoculated feed. ANIMALS 30 crossbred 10-day-old pigs. PROCEDURES Tissue culture PEDV was diluted to form 8 serial 10-fold dilutions. An aliquot of stock virus (5.6 × 105 TCID50/mL) and each serial PEDV dilution were mixed into 4.5-kg batches of feed to create 9 PEDV-inoculated feed doses; 1 virus-negative dose of culture medium in feed was also created. Pigs were challenge exposed via oral administration of PEDV-inoculated feed, and fecal swab specimens were collected. All pigs were euthanized 7 days after challenge exposure; fresh tissues were collected and used for PCR assay, histologic examination, and immunohistochemical analysis. RESULTS The PCR cycle threshold (Ct) decreased by approximately 10 when PEDV was added to feed, compared with results for equivalent PEDV diluted in tissue culture medium. Pigs became infected with PEDV when challenge exposed with the 4 highest concentrations (lowest concentration to cause infection, 5.6 × 10(1) TCID50/g; Ct = 27 in tissue culture medium and 37 in feed). CONCLUSIONS AND CLINICAL RELEVANCE In this study, PEDV in feed with detectable Ct values of 27 to 37 was infective. The Ct was 37 for the lowest infective PEDV dose in feed, which may be above the limit of detection established for PEDV PCR assays used by some diagnostic laboratories. Overall, results indicated 5.6 × 10(1) TCID50/g was the minimum PEDV dose in feed that can lead to infection in 10-day-old pigs under the conditions of this study.

Thirteen pygmy goats (Capra hircus) from a herd naturally infected with Mycobacterium avium ss. paratuberculosis (MPTB) were monitored with 4 diagnostic assays for 2 to 15 mo. Cellular and humoral immune responses to the infection were assessed with assays of gamma interferon (IFNγ), serum antibody [enzyme-linked immunosorbent assay (ELISA) and agar gel diffusion (AGID)], and radiometric fecal culture. Microscopic examination and radiometric culture of tissue from 12 sites were performed at necropsy. Goats were considered infected if MPTB was isolated from any tissue sample collected at necropsy. Mycobacterial isolates were confirmed as MPTB with an IS900 polymerase chain reaction assay. Ten goats whose antemortem tests indicated infection carried heavy organism burdens at necropsy, both within and beyond the gastrointestinal system. False-negative ELISA, AGID, and/or culture results were obtained in 5 of the 10 confirmed cases during the study period. In 3 goats with sporadic fecal shedding of MPTB or detectable IFNγ response, or both, no abnormalities were detected at necropsy and no MPTB was isolated from the tissue samples; the antemortem fecal-culture and IFNγ results were thus considered false-positive. Diagnosticians should be alert to the possibility of both false-positive and false-negative test results for Johne's disease in goats. False-positive fecal-culture results may occur when a high prevalence of infection exists in the herd and the premises are likely to be heavily contaminated. The diverse antemortem testing patterns seen in these goats underscore the importance of using varied diagnostic assays serially or in parallel to increase the likelihood of identifying all infected goats.

Articular cartilage explants from 3 horses were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan on proteoglycan biosynthesis. Cultures were exposed to concentrations of 0, 50, or 200 microgram of the drug/ml for either 2 days or 6 days, and labeled with 35S, before measuring the content of sulfated proteoglycan in the culture media and in extracts of cartilage. In a second experiment, the explants were incubated with the isotope and subsequently exposed to the same concentrations of the polysulfated glycosaminoglycan for 4 days. Subsequently, the amount of remaining labeled proteoglycan was determined. Gel filtration chromatography was used to compare the hydrodynamic size of proteoglycans from the cartilage explants in each experiment. Polysulfated glycosaminoglycan caused a dose-dependent depression of sulfated proteoglycan synthesis, which was statistically significant after 6 days of exposure. Radioactive proteoglycan content in explants was similar in the experiment involving isotopic labeling prior to exposure to the drug. Proteoglycan monomer size was similar in all treatment groups. It was concluded that polysulfated glycosaminoglycan caused a modest depression in proteoglycan synthesis, had little effect on endogenous proteoglycan degradation, and did not influence the size of sulfated proteoglycans synthesized by normal equine chondrocytes in explant culture.

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (postfusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.