Serial sections of bone and soft tissue from the metacarpophalangeal joints of 2 mature and 2 immature horses were evaluated for substance P immunoreactive sensory nerve fibers. Formalin-fixed specimens were sectioned, either nondemineralized or demineralized with formic acid or EDTA. Rabbit antiserum to substance P (SP) was used in the strep. tavidin-biotin-peraxidase complex method for immunolocalization of SP antigen, and staining with 3,3'- diaminobenzidine was used for permanent identification of SP fibers. Abundant sensory nerve fibers were identified in the joint capsule, synovial membrane subintimal layers, collateral ligaments, suspensory ligament and distal sesamoidean ligament attachments to the sesamoid bones, and the periarticular periosteal layers. Sparse SP-immunoreactive nerve fibers were found in subchondral bone plates of the metacarpus, proximal first phalanx, and dorsal articular surface of the sesamoid bones. Most SP fibers were associated with blood vessels in the small cancellous spaces and haversian canals of the subchondral bone. The deeper marrow spaces contained increased numbers of SP sensory fibers; a few appeared in small groups and as several SP-immunoreactive fibers in a larger nerve. Cortical bone contained only a few SP fibers in the haversian canals. Substance P fibers were not identified in the osteocytic lacunae, canaliculi, or the bony lamellae of the haversian systems of the subchondral bone plate, and its extension to the metaphyseal and diaphyseal cortical bone. Equine metacarpophalangeal joint soft tissues have an abundant sensory nerve supply, similar to that of other species. However, the subchondral bone plate also has sparse sensory nerve fibers, which is a unique finding, and may help explain signs of bone pain associated with disease states of the fetlock.

Cerebrospinal fluid and serum were obtained from 17 adult, healthy llamas (9 males, 1 castrated male, and 7 females). Osmolality; activities of lactate dehydrogenase and creatine kinase; and concentrations of glucose, sodium, chloride, potassium, total protein, and albumin were determined in serum and CSF. Total and differential cell counts were determined in CSF, and electrophoresis of CSF proteins was performed. Total nucleated cell count was low, 0 to 3/microliter, which is lower than that reported for other domestic species and is similar to values in healthy people. Differential leukocyte percentages were disparate depending on the degree of blood contamination. Blood contamination influenced the percentage of neutrophils and eosinophils in CSF. Samples with few erythrocytes had differential leukocyte distribution similar to that of other species: mostly lymphocytes, fewer monocytoid cells, and scant neutrophils. Older llamas had a few eosinophils in the CSF. Total protein, albumin, and gamma-globulin concentrations in llamas were similar to values in cattle and were higher than values in most domestic species. Glucose concentration in CSF was approximately 40% of the value in serum (nonruminant animals and people typically have CSF glucose concentration that is approximately 60 to 80% of the serum glucose concentration). Sodium and Cl concentrations in CSF were higher than those in serum, whereas K concentration was lower in CSF, compared with serum. Activities of creatine kinase and lactate dehydrogenase in CSF were markedly lower than those in serum, and the ranges of values in this group of healthy llamas were narrow.

The alpha 1-proteinase inhibitors of trypsin, Spi1, Spi3A, and Spi3B, in bronchoalveolar lavage fluid (BALF) and serum of horses were separated by electrophoresis, and their proportions were quantified in 12 control horses and 12 with chronic obstructive pulmonary disease (COPD). A significantly lower proportion of Spi3B (P < 0.05) and higher proportion of Spi1 (P < 0.02 to P < 0.01) were detected in BALF, compared with serum, in control and COPD-affected horses and appeared to be attributable to reduced Spi3 activity in BALF. There was no significant difference between the control and COPD groups in this respect, indicating that the decrease in Spi3 may be a physiologic phenomenon. The differences observed may be associated with proteolytic damage to or preferential complex formation by Spi3.

Mean phosphorus (P) content in bovine rib bone was 102.9, 108.3, and 182.7 mg/g of bone on fresh, dry, and ash weight bases, respectively. Values for calcium (Ca) were 194.3, 203.7, and 344.6 mg/g, respectively, and for magnesium (Mg) were 5.3, 5.5, and 9.4 mg/g, respectively. Mean percentage of ash in rib bone was 59.12%. Expected concentrations of Ca, P, and Mg were determined on fresh, dry, and ash weight bases and for 3 age groups, 3 breeds, and bulls, females, and steers. On an ash weight basis, cattle 6 to 18 months old had 185.74 mg of P/g, 372.52 mg of Ca/g, and 12.37 mg of Mg/g. Those 19 to 36 months old had 182.02 mg of P/g, 322.35 mg of P/g, and 8.09 mg of Mg/g. Those > 36 months old had 174.80 mg of P/g, 340.36 mg of Ca/g, and 6.62 mg of Mg/g. Steers had 183.93 mg of P/g, 352.73 mg of Ca/g, and 10.15 mg of Mg/g. Females had 178.47 mg of P/g, 320.28 mg of Ca/g, and 6.5 mg of Mg/g. Males had 176.15 mg of P/g, aH on an ash weight basis. Dairy breeds were found to have 186.08 mg of P/g, 351.25 mg of Ca/g, and 10.47 mg of Mg/g. Cattle of mixed breeding had 177.42 mg of P/g, 341.28 mg of Ca/g, and 6.54 mg of Mg/g. The Africander breed of beef cattle had 167.07 mg of P/g, all on an ash weight basis.

The effect of right paralumbar fossa exploratory celiotomy and omentopexy on peritoneal fluid constituents was studied in 22 adult dairy cows. Six cows were eliminated on the basis of physical examination findings (n = 2), surgical findings (n = 2), or inability to obtain a sufficient volume of peritoneal fluid (n = 2). Sixteen cattle had normal results of Csc and serum biochemical analysis, and a minimum of 1 ml of peritoneal fluid was obtained by abdominocentesis. Abdominocentesis was repeated on days 1, 2, and 6 after surgery. Statistical analysis for repeated measures was performed, using a significance level of P < 0.05. Stage of gestation was evaluated for interaction with time. Mean total nucleated cell count was 3,200 cells/1 before surgery, was significantly increased 2 days after surgery (16,336 cells/microliter), and continued to increase through day 6 (20,542 cells/microliter). Mean polymorphonuclear cell count was 1,312 cells/microliter before surgery and was significantly higher at 2 (11,043 cells/microliter) and 6 (10,619 cells/microliter) days after surgery. Mean lymphocyte count was 254 cells/microliter before surgery and was significantly increased 2 days (1,911 cells/microliter) after surgery. By day 6, lymphocyte numbers were similar to preoperative values. Mean mononuclear cell count was 770 cells/microliter before surgery and was significantly increased on days 1 (3,084 cells/microliter), 2 (3,285 cells/microliter and 6 (2,349 cells/microliter) after surgery. Mean eosinophil numbers were 1,388 cells/microliter before surgery and were significantly increased on day 6 (6,347 cells/microliter) only. Interaction between time and stage of gestation was found only for specific gravity and total protein concentration. In general, specific gravity and total protein concentration increased after surgery (mean before surgery, 1.016 and 3.6 g/dl; mean after surgery, 1.021 and 5.6 g/dl). Left paralumbar fossa celiotomy performed 7 days after surgery did not reveal complications of repeated abdominocentesis, and pregnancy status was unchanged. Peritoneal fluid constituents are highly variable after exploratory celiotomy and omentopexy in cattle. However, results of this study may provide a reference for interpretation of postoperative peritoneal fluid sample findings in cattle.

High molecular weight (MW) hyaluronate (HA) is an integral part of synovial fluid (SF), regulating many important physiologic and pathophysiologic mechanisms. Many of its effects depend on, or are reflected in, the concentration and MW of HA. High-performance liquid chromatography was used to assess simultaneously the concentration and MW of HA in SF obtained from horses with various arthritides: acute traumatic arthritis; chronic traumatic arthritis, including degenerative joint disease (DJD); and infectious arthritis. The size-exclusion column was calibrated, using appropriate HA concentration and MW standards, before the high-performance liquid chromatographic assays of the SF samples. Calibration of the column disclosed that the maximal limit for MW estimation of HA was around 3 million. In control joints, MW of HA ranged from 2 to 3 X 10(6) (mean 2.5 X 10(6)) and did not differ significantly from MW of HA in SF from horses with acute or chronic traumatic arthritis (mean 2 x 10(6); range 1.5 to 3 x 10(6)). Interestingly, a small amount of HA of moderately high MW (approx 1 to 1.5 x 10(6)) was detected in chromatograms of SF from infected joints. This degree of polymerization of SF HA was significantly (P < 0.01) lower, compared with that for control joints. There was no difference in mean (+/- SD) concentration of HA between control joints and joints with acute or chronic traumatic arthritis (0.33 +/- 0.12 g/L vs 0.18 +/- 0.03 g/L or 0.23 +/- 0.12 g/L), indicating that SF HA concentration probably should not be used as a diagnostic marker for the condition. However, the SF HA concentration was significantly (P < 0.01) lower in joints with infectious arthritis (0.07 +/- 0.03 g/L) and in the joints with radiographic evidence of DJD (0.12 +/- 0.01 g/L), compared with control joints.

Serologic testing to detect persistent IgG titer directed at Salmonella lipopolysaccharide (LPS) has proven useful in detecting Salmonella carrier cattle without clinical signs of disease and in seroepidemiologic studies. Although little cross-reactivity exists between most Salmonella serogroups, groups B (O1, 4 [5], 12) and D (O1, 9, 12) share somatic (LPS cell wall) antigens O1 and O12, which results in some cross-reactions. This may be unimportant in most instances, because group-B and group-D carriers need to be identified and culled. It may be desirable in some situations, such as when trying to control S dublin, to determine which serogroup is present in a given herd. For this reason, a procedure to produce a pure O9 group-D antigen was developed. Salmonella dublin (group D) was grown by use of standard procedures, and LPS was extracted by use of the phenol-water method. The LPS was then oxidized with sodium periodate, dialyzed, reduced with sodium borohydride, cleaved with hydrochloric acid, and again dialyzed. This procedure successfully cleaved the saccharides comprising O antigens 1 and 12, leaving a pure O9 ELISA antigen. Sera from cattle vaccinated or naturally infected with S typhimurium, S agona, and S schwarzengrund (all group B), S montevideo (group C1), and S dublin (group D) were tested by ELISA, using modified and unmodified antigens. When the ELISA antigen used was the chemically modified (pure O9) group-D antigen, elimination of cross-reactions confirmed the structural loss of cross-reacting O1 and O12 antigens.

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.

Collagen type I was purified from equine skin and flexor tendon, and type II collagen was purified from equine articular cartilage. The proteoglycans in these tissues were extracted, using guanidine HCl; the collagens were solubilized, using pepsin digestion, then were selectively precipitated with Nacl. Gel electrophoresis indicated that the precipitates contained only type I or type II collagen. Amino acid analysis indicated that collagen constituted > 97% of the total protein in the precipitates. Hydroxylation of proline was 42.0 t 0.6% (mean SEM) in alpha 1(I) and alpha 2(I), and was 48.1 +/- 1.3% in alpha 1(II) chains. The hydroxylation of lysine was 23.2 +/- 0.7% in alpha 1(I) and 34.1 0.9% in alpha 2(I) chains from tendon, and 49.6 +/- 4.3% in alpha 1(II) chains from cartilage. The cyanogen bromide (CB)-peptide patterns of chromatographically purified equine alpha 2(I) and alpha 1(II) chains were similar to those published previously for rat, bovine, and human alpha 2 and alpha 1 chains. However, the CB-peptide pattern of the equine alpha 1(I) chain resembled the guinea pig alpha 1(I) chain, which has no methionine between CB7 and CB6. Purified equine alpha 1(I)CB7,6 contained no methionine, methionine sulfoxide, or homoserine lactone. Mass of 42.26 kd was determined by use of mass spectrometry, and N-terminal sequence analysis established that the first 12 amino acids of this CB7,6 were identical to the sequence of human alpha 1(I)CB7. Because of this species specific difference in structure of the alpha 1(I) chain, equine Cb-peptides should be used as standards in studies of variations in the proportions of type I and type II collagens in equine tissues expressing the phenotype of fibrous tissue and cartilage.

Thirty-one clinically normal Cocker Spaniels, Miniature Schnauzers, and Doberman Pinschers (28 female, 3 male) 7 to 8 years old were uninephrectomized (month -2) to increase the risk of renal damage associated with reduction of renal mass. Two diets, differing principally in protein concentration, were used to test the hypothesis that high dietary protein intake causes renal damage in aging dogs. For 2 months after uninephrectomy, all dogs were fed diet A (18% protein). After glomerular filtration rate (GFR) was measured (month 0), 16 dogs were assigned to group A and were fed diet A for an additional 48 months. The other 15 dogs were assigned to group B, and were fed diet B (34% protein) for the subsequent 48 months. At 6-month intervals, GFR and urine protein-to-creatinine ratio (UP/C) were determined. At 48 months, terminal studies were done, survivors were euthanatized, and tissues were examined. Of 16 dogs in group A, 10 survived, compared with 13 of 15 in group B. Among survivors, a significant difference in GFR was not found between groups A and B, and decrease in GFR was not evident with time in either group. At 48 months, oral administration of casein caused minor acute effects on GFR and renal plasma flow in dogs of groups A and B. The UP/C values increased significantly (P = 0.001) from baseline values, but the increase was not progressive. The UP/C values were not affected by diet. Some dogs in both groups developed UP/C > 1.0. Morphologic studies performed on kidneys removed at -2 months (nephrectomy) and at 48 months (necropsy) revealed increased kidney weight in both groups at month 48, compared with month -2 (P = 0.003); at month 48, kidney weight change was significantly (P = 0.004) greater in group-B than in group-A dogs. Increased glomerular area at month 48, compared with month -2, was significantly (P= 0.000) related to time, but not to diet. Significant (P = 0.000) increase in glomerular mesangial matrix, interstitial fibrosis (P = 0.001), cell infiltration (P = 0.000), and lesions of the renal pelvis (P = 0.04) was observed between month -2 and month 48. Time, representing combined effects of uninephrectomy and aging, was the major factor responsible for the morphologic changes. Diet effects were significance (P = 0.008) for cell infiltration, but did not reach significance for mesangial matrix accumulation, fibrosis, or pelvic lesions. Kidney mineral analysis revealed no renal mineralization in either group between -2 and 48 months. Results indicated that GFR did not decrease with time during the geriatric period studied, but severity of renal lesions was increased. Effects of time and uninephrectomy, although not separable, were more important than those of dietary protein intake on progression of renal lesions.