Serological diagnosis of brucellosis is still a great challenge due to the infeasibility of discriminating infected animals from vaccinated ones, so it is necessary to search for diagnostic biomarkers for differential diagnosis of brucellosis. Cell division cycle 42 (Cdc42) from sheep (Ovis aries) (OaCdc42) was cloned by rapid amplification of cDNA ends (RACE), and then tissue distribution and differential expression levels of OaCdc42 mRNA between infected and vaccinated sheep were analysed by RT-qPCR. The full-length cDNA of OaCdc42 was 1,609 bp containing an open reading frame (ORF) of 576 bp. OaCdc42 mRNAs were detected in the heart, liver, spleen, lung, kidneys, rumen, small intestine, skeletal muscles, and buffy coat, and the highest expression was detected in the small intestine. Compared to the control, the levels of OaCdc42 mRNA from sheep infected with Brucella melitensis or sheep vaccinated with Brucella suis S2 was significantly different (P < 0.01) after 40 and 30 days post-inoculation, respectively. However, the expression of OaCdc42 mRNA was significantly different between vaccinated and infected sheep (P < 0.05 or P < 0.01) on days: 14, 30, and 60 post-inoculation, whereas no significant difference (P > 0.05) was noted 40 days post-inoculation. Moreover, the expression of OaCdc42 from both infected and vaccinated sheep showed irregularity. OaCdc42 is not a good potential diagnostic biomarker for differential diagnosis of brucellosis in sheep.

The early interaction of Lawsonia intracellularis with host cells was examined with the use of porcine ileum models. Two conventional swine were anesthetized, and ligated ileum loops were prepared during abdominal surgery. The loops were inoculated with 10⁸ L. intracellularis or saline. After 60 min, samples of each loop were processed for routine histologic and electron microscopic study. Histologic and ultrathin sections of all the loops appeared normal, with no apposition of bacteria and host cells or bacterial entry events in any loop. Portions of ileum from a single gnotobiotic piglet were introduced as xenografts into the subcutis of each flank of 5 weaned mice with severe combined immunodeficiency disease. After 4 wk, 10⁸ L. intracellularis were inoculated into each of 4 viable xenografts with a sterile needle; the other 3 viable xenografts received saline. Histologic and ultrathin sections of all the xenografts 3 wk after inoculation showed relatively normal porcine intestinal architecture, with normal crypts, crypt cell differentiation, and low villous structures; the xenografts treated with the bacteria also showed intracytoplasmic L. intracellularis within crypt and villous epithelial cells. Thus, entry of L. intracellularis into target epithelial cells and multiplication may not be sufficient alone to directly cause cell proliferation. A proliferative response may require active division of crypt cells and differentiation in conjunction with L. intracellularis growth.

Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals, This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 X 10(6) viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 degrees C and 5% CO2. Histologic and histochemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22. Glycosaminoglycan (GAG) assay by dimethylmethylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P < 0.0001) increase in total GAG content was observed by day 3 (0.329 micrograms/mg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 +/- 0.062 micrograms/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations. The CS content significantly (P = 0.0002) increased for the first 14 days of incubation, then a plateau was observed for the remainder of the study. Peak CS content was 0.354 +/- 0.037 micrograms/mg. Concentration of KS reached a plateau earlier than did CS content, with peak amount of 0.193 +/- 0.027 micrograms/mg on day 10. Fluctuations in KS content were not significant until an increase on day 22. Chondrocytes actively populated the collagen implants, increasing in number and synthesizing matrix GAG epitopes over the 22 days of incubation. These results indicate that chondrocyte-laden porous collagen matrices may be suitable cartilage analogue materials and the optimal metabolic time for transfer to cartilage defects is 10 to 14 days.

Hexosamine concentration, DNA concentration, and [35S]sulfate incorporation for articular cartilage obtained from various sites in the metacarpophalangeal and carpal joints of horses were measured. The same measurements were made on the repair tissue filling full-thickness articular defects in the intermediate carpal bone and on cartilage surrounding partial-thickness defects 6 weeks after the defects were created arthroscopically. Cellularity (measured as DNA concentration), proteoglycan content (measured as hexosamine concentration), and proteoglycan synthesis (measured as [35S]sulfate incorporation) varied according to the site sampled. Cartilage from the transverse ridge of the head of the third metacarpal bone and the radial facet of the third carpal bone had the lowest hexosamine concentration, whereas rate of proteoglycan synthesis was lowest in cartilage from the transverse ridge of the head of the third metacarpal bone and the distal articular surface of the radial carpal bone. Repair tissue filling a full-thickness cartilage defect at 6 weeks was highly cellular. It was low in proteoglycan content, but was actively synthesizing these macromolecules. In contrast, the cartilage surrounding a partial-thickness defect was unchanged 6 weeks after the original defect was made.

Cell proliferation kinetic values were established for the hair root matrix of primary anagen follicles of 14 Beagles and 4 Cocker Spaniels with healthy skin and 9 Cocker Spaniels with primary idiopathic seborrhea. Indices were established by intradermal pulse labeling with tritiated thymidine, followed by cutaneous biopsy and autoradiography. The hair root matrix cell labeling index was 23.4 +/- 3.5% for Beagles, 24.4 +/- 4.0% for healthy Cocker Spaniels, and 24.9 +/- 4.3% for seborrheic Cocker Spaniels. These values indicate a rapidly proliferating cell population. Differences among these cell kinetic data for the 3 groups of dogs were not statistically significant. Although significant cell kinetic differences have been reported for other epidermal structures (interfollicular epithelium, upper hair follicle external root sheath, sebaceous glands) in seborrheic Cocker Spaniels, proliferation of hair root matrix cells apparently remains unaffected.

Cell proliferation kinetic values were established for the epidermis, hair follicle epithelium, and sebaceous glands of 10 Beagles and 4 Cocker Spaniels with healthy skin and coats. Values were established by intradermal pulse-labeling injections of [3H]thymidine, examination of cutaneous biopsied tissues, and autoradiography. The epidermal basal cell-labeling index was 1.41 +/- 0.46% for Beagles and 1.71 +/- 0.56% for Cocker Spaniels. The hair follicle basal cell-labeling index was 1.46 +/- 0.78 and 1.07 +/- 0.42%, respectively. Calculated epidermal cell-renewal time for viable layers of the epidermis was 23.38 +/- 5.93 days for Beagels and 20.97 +/- 4.92 days for Cocker Spaniels. Differences between cell kinetics data for the 2 breeds were not significant (P greater than 0.05). The basal cell-labeling index for the sebaceous gland was significantly (P = 0.009) lower for Cocker Spaniels (0.40 +/- 0.18%) than for Beagles (1.81 +/- 1.08%). Seemingly, epidermal and follicular cell proliferation kinetics in healthy dogs was similar between the 2 breeds, whereas sebaceous gland basal cells were less proliferative in healthy Cocker Spaniels than in healthy Beagles.

Cell proliferation kinetic values were established for the epidermis, hair follicle epithelium, and sebaceous glands of 8 Cocker Spaniels with primary idiopathic seborrhea. Values were established by intradermal pulse labeling injections of tritiated thymidine followed by cutaneous biopsy and autoradiography.The epidermal basal cell-labeling index was 4.96 +/- 0.97%, and the epidermal nucleated cell-labeling index was 3.33 +/- 0.71%. Calculated epidermal cell renewal time for the viable layers of the epidermis was 7.85 +/- 1.80 days. The hair follicle infundibulum basal cell-labeling index was 5.48 +/- 2.01%, and the sebaceous gland basal cell-labeling index was 5.94 +/- 4.15%. When compared with previously reported cell kinetic values for Cocker Spaniels and Beagles with healthy skin, these data indicate accelerated cellular proliferation in all 3 cutaneous structures in seborrheic Cocker Spaniels.

The use of cultured tissue has not yet become wide-spread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobble-stone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors. Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.

Piroxicam and other nonsteroidal anti-inflammatory drugs (NSAID) have antitumor activity against naturally acquired cancer in dogs and human beings, and against experimentally induced tumors in rodents. We are investigating potential mechanisms of NSAID anti-tumor activity. The direct cytotoxicity of piroxicam, indomethacin, and aspirin against 4, canine tumor cell lines (transitional cell carcinoma, squamous cell carcinoma, melanoma, and soft tissue sarcoma) was determined in short-term growth rate assays and in clonogenic assays. Piroxicam was evaluated alone and in combination with the lipoxygenase inhibitor zileuton, and in combination with the chemotherapeutic agents cisplatin and carboplatin. The 50% inhibitory concentrations (IC50) against melanoma cells in short-term growth rate assays were: 530 micromolar piroxicam, 180 micromolar indomethacin, and greater than 1 mM aspirin. These IC50 values were over 10 times greater than serum concentrations of these drugs that could safely be achieved in vivo. The IC50 of zileuton combined with piroxicam (280 micromolar) was not different from the IC50 of zileuton alone (230 micromolar; ANOVA P = 0.47) in melanoma cells. Similarly, addition of piroxicam did not alter the IC50 of either cisplatin (1.6 micromolar) or carboplatin (6.1 micromolar). These results suggest that NSAID, at serum concentrations achievable in vivo, do not have direct cytotoxicity against canine tumor cells tested. It is unlikely that the in vivo antitumor activity of NSAID is attributable to a direct cytotoxic effect.

Biological and biochemical characteristics of the leukotoxin of Fusobacterium necrophorum were determined. Culture supernatant of F necrophorum was toxic to polymorphonuclear neutrophilic leukocytes from cattle and sheep, but not to those from pigs and rabbits. Culture supernatant and sonicated bacterial cell fractions had low hemolytic activity and did not cause dermonecrosis in a guinea pig. Supernatant-derived leukotoxin was inactivated at 56 C for 5 minutes and became unstable at pH > 7.8 or < 6.6. Chemical treatment with 0.1% sodium dodecyl sulfate, 0.25% sodium deoxycholate, 5.2% sodium sulfide, or 0.25 mM titanium (III) citrate markedly decreased leukotoxicity. Enzymatic treatment with protease, trypsin, and chymotrypsin inactivated the toxin completely, whereas amylase had no effect. Use of protease inhibitors failed to prevent loss of leukotoxin activity. Using membrane partition chromatography and gel filtration, the estimated molecular weight of the toxin was > 300,000. On reduction and denaturation, the toxin dissociated into several components by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.