A sensitive and specific DNA probe for detection and identification of bovine herpesvirus 4 (BHV-4) was developed. Cloned fragments from a library of HindIII fragments of the BHV-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sentitivity and specificity in detecting viral DNA by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral DNA, and detected viral DNA in 0.001 microgram of total DNA extracted from BHV-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral DNA in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of BHV-4. The DNA probes did not hybridize to total DNA prepared from uninfected bovine cells or from cells infected with BHV-1, BHV-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive DNA purification procedures. This simplified procedure also resulted in specific detection of field isolates of BHV-4 and prototype strains of BHV-4.

Each of 5 US-origin serotypes of bluetongue virus (BTV) was inoculated into a separate pair of sheep. The duration of each animal's ensuing viremia was monitored, using a BTV serogroup-specific nested polymerase chain reaction (PCR) method and an embryonating chicken egg (ECE) inoculation procedure. Mean duration of viremia was 100 and 38 days for the PCR and ECE methods, respectively. This difference was significant (P < 0.001) and documents a more prolonged viremia in virus-exposed sheep than has been reported. A dual internal oligonucleotide solution hybridization procedure was developed for the rapid (2 hours) colorimetric detection and identification of BTV-specific PCR products. This enzyme-linked oligonucleotide sorbent assay (ELOSA) relied on annealing of separate biotinylated and fluoresceinated probes to the amplified BTV nucleic acid; these complexes were captured on streptavidin-coated microtitration wells and were detected, using a horseradish peroxidase-labeled antifluorescein antibody conjugate. End-point dilution analyses of PCR products indicated that the ELOSA was more sensitive than gel electrophoretic or comparable colorimetric slot-blot hybridization techniques. The BTV PCR-ELOSA system represents a more sensitive and expeditious means of diagnosing BTV-induced viremia than does the ECE procedure currently used. The combination of ELOSA with PCR should facilitate practical application of nucleic acid technology to diagnostic veterinary medicine.

Dogs have been used extensively as an experimental model for studying musculoskeletal disorders. Many of these studies incorporated sequential radiography to quantitate a particular treatment's effect, using the contralateral bone as the control condition. The contralateral bone can be used as a control only if there is bilateral symmetry between right and left limbs. We performed radiography (craniocaudal and lateromedial views) on 10 pairs of humeri, radii, femora, and tibiae from dogs, using an alignment jig, to radiographically determine homotypic geometric variations of long bones. The bones were divided into 5 regions: proximal epiphysis, proximal metaphysis, diaphysis, distal metaphysis, and distal epiphysis. The total bone diameter, medullary diameter, and cortical width (total of medial + lateral cortex or total of cranial + caudal cortex) were measured at specified slices throughout each of these regions. Fourteen of 540 homotypic comparisons revealed significant differences between right and left bones at either a slice or region. Although there were only 14 significant differences between right and left bones at any region or slice, measurements were more precise with lower coefficients of variation in the diaphyseal and epiphyseal regions. Homotypic differences in diaphyseal and epiphyseal regions were < 5.3 and 7.5%, respectively power = 0.8). In metaphyseal regions, however, homotypic differences were larger; these differences could have been as large as 11.5% for total bone diameter, 15.6% for medullary diameter, and 80.0% for cortical width without achieving significant differences between populations (power = 0.8). This study validated the concept of using the contralateral limb as the control condition in orthopedic studies using dogs, particularly when evaluating the geometric properties of long bones radiographically.

Keratan sulfate (KS) is a glycosaminoglycan, distribution of which is confined mostly to hyaline cartilage. As such, it is a putative marker of hyaline cartilage catabolism. In experiment 1, a focal osteochondral defect was made arthroscopically in 1 radial carpal bone of 2 ponies, and in 2 other ponies, chymopapain was injected into the radiocarpal joint to induce cartilage catabolism. Sequential and concurrent plasma and synovial fluid concentrations of KS were measured, up to 13 months after induction of cartilage injury, to determine whether changes in KS concentrations reflected cartilage catabolism. In experiment 2, a large, bilateral osteochondral defect was made in the radial carpal bones of 18 ponies, which were subsequently given postoperative exercise and/or injected intra-articularly with 250 mg of polysulfated glycosaminoglycan (PSGAG). Medication was given at surgery, then weekly for 4 weeks. Blood samples were collected and synovial fluid was aspirated before surgery, when medication was given, and at postmortem examination (postoperative week 17). The KS concentration was measured in these fluids to determine whether changes in KS concentration indicated an effect of joint treatment. In experiment 1, the concentration of KS in synovial fluid was highest 1 day after joint injury, and the concentration in plasma peaked 2 days after joint injury. For ponies receiving chymopapain intra-articularly (generalized cartilage catabolism), a fivefold increase over baseline was observed in the concentration of KS in plasma (peak mean, 1.2 microgram/ml), and a tenfold increase over baseline in synovial fluid (peak mean, 2.0 mg/ml) was observed. On average, these maxima were threefold higher than values in fluids of ponies with osteochondral defects (focal cartilage disease). In experiment 2, nonexercised ponies had lower KS concentration (as a percentage of the preoperative concentration) in synovial fluid than did exercised ponies at all postoperative times, and at postoperative week 17, this effect was significant (P < 0.05). This may be related to decreased turnover of KS in articular cartilage attributable to stall confinement and late increase in turnover related to exercise. Seventeen weeks after surgery, synovial fluid from exercised, medicated ponies had significantly (P < 0.05) higher KS content than did fluid from exercised, nonmedicated ponies. This indicated that exercise, when combined with medication, may increase KS release from articular cartilage. Synovial fluid from medicated joints of nonexercised ponies had significantly (P < 0.05) lower KS concentration than did synovial fluid from nonmedicated joints of nonexercised ponies. This indicated that, in nonexercised joints, medication with PSGAG may have decreased either release of KS from the articular cartilage into the synovial fluid or inhibited synthesis of KS. Concentration of KS in synovial fluid was not related clearly to the development of osteoarthritis in these ponies. Exercise or medication did not affect plasma KS concentration, and synovial fluid and plasma KS concentrations were not correlated. Data indicated that KS concentration in plasma and synovial fluid may be increased in acute, marked, generalized articular cartilage catabolism and that KS turnover in cartilage of joints with large osteochondral defects was affected by intra-articular PSGAG and postoperative exercise.

To study increase of the Salmonella population in the cecum of chickens infected with Eimeria tenella, quantitative changes in mannose residues on the cecal mucosa were investigated. Inhibition of S typhimurium adherence to the cecum by a 2% carbohydrate (D-mannose, D-galactose, L-fucose, alpha-methyl-D-glucoside) in phosphate-buffered saline solution was examined. Only D-mannose had inhibitory effects. Whereas, D-galactose had somewhat enhancing effects on adherence of S typhimurium to the cecal mucosa of uninfected germ-free chickens. In infected and uninfected chickens, D-mannose inhibited adherence of S typhimurium. D-mannose significantly (P < 0.05) increased adherence of Bacteroides sp. In infected and uninfected chickens, D-mannose did not have any effect on adherence of Clostridium perfringens and Bifidobacterium thermophilum. Under microscopic observation, only concanavalin A and Lens culinaris agglutinin, of 8 lectins examined, were recognized as lectin-positive staining lines or spots in the cecal mucosa, indicating presence of mannose residues on the cecal mucosa. In E tenella-infected chickens, lectin-positive staining was seen strongly on the coarse surface of damaged cells and at the bottom of the crypts. These results indicate that coccidial infection may induce increase of mannose residues on the intestinal surface and allow adhesion of more salmonellae to the intestine.

Concentrations of 3 alpha-hydroxylated bile acids were measured in serum and urine of clinically normal (healthy) cats (n = 6), cats with severe hepatic lipidosis (n = 9), and cats with complete bile duct occlusion (n = 4). Bile acid concentrations were measured by use of a gradient flow high-performance liquid chromatography procedure with an acetonitrile and ammonium phosphate mobile phase and an in-line postanalytic column containing 3 alpha-hydroxysteroid dehydrogenase and a fluorescence detector. Specific identification of all bile acid peaks was not completed; unidentified moieties were represented in terms of their elution time (in minutes). Significant differences in serum and urine bile acid concentrations, quantitative and proportional, were determined among groups of cats. Cats with hepatic lipidosis and bile duct occlusion had significantly (P greater than or equal to 0.05) greater total serum and urine bile acids concentrations than did healthy cats. The proportion of hydrophobic bile acids in serum, those eluting at greater than or equal to 400 minutes, was 1.9% for healthy cats, 3.3% for cats with lipidosis, and 5.4% for bile duct-obstructed cats. Both groups of ill cats had a broader spectrum of unidentified late-eluting serum bile acids than did healthy cats; the largest spectrum developed in bile duct-occluded cats. The trihydroxy-to-dihydroxy serum bile acids ratio was 8.8:1 for healthy cats; 24.1:1 for cats with lipidosis: and 20:1 for cats with bile duct obstruction. There was a paucity of glycine-conjugated bile acids in all cats and small quantities of secondary bile acids in ill cats. A significantly (P < 0.05) smaller proportion of unconjugated primary bile acids was detected in sera from ill cats. Serum taurolithocholic acid was detected only in small quantities in cats of each group. There was significantly increased quantity, but lower proportion, of trihydroxy-cholestanoic acid in serum from ill cats, compared with healthy cats. A significantly (P < 0.05) greater proportional amount of unidentified moieties eluting at 130 and 277 minutes was detected in urine of cats with hepatic lipidosis; we believe that the unidentified moiety eluting at 277 minutes is tau- tauroallocholic acid. Large proportional amounts of taurocholic and cholic acids were detected in urine of all cats, but ill cats had significantly (P < 0.05) greater quantities (quantitatively and proportional). Ill cats had significantly (P < 0.05) more taurocholic than cholic acid in urine. Because taurine is an essential amino acid for cats and is a necessary daily dietary constituent, large urinary losses of taurine in conjugated bile acids may further compromise the health of anorectic cats with severe hepatic lipidosis.

Although feline immunodeficiency virus (FIV) and the unrelated retrovirus feline leukemia virus (FeLV) are associated with acquired immune deficiency in cats, experimental and field evidence indicates that coinfection with both viruses may lead to more serious disease syndrome. A third feline retrovirus, feline syncytium-forming virus (FeSFV), which is far more prevalent than either FIV or FeLV and is considered nonpathogenic in nature, is consistently coisolated from sick, FIV-infected cats. To determine the potential role of FeSFV in enhancement of FIV-mediated disease, persistent FeSFV infection was established in 14 of 24 nine-month-old cats. Four months later, half the FeSFV-infected and half the noninfected cats were inoculated with blood obtained from a cat persistently infected with the Petaluma strain of FIV. At postinoculation week 17, 1 male cat infected with only FIV died of bacterial bronchopneumonia that could have been attributed to FIV-induced acquired immune deficiency-like syndrome. However, none of the remaining cats had clinical illness, whether infected with either virus alone or coinfected with both viruses. As early as postinoculation week 6, decreases were observed in the CD4+ to CD8+ T-lymphocyte ratio of both groups of cats inoculated with FIV. Infection with FeSFV had no effect on the CD4, to CD8+ T-cell ratio. Mitogen stimulation assays and total WBC count were unaffected by FeSFV infection, although an increase in numbers of neutrophils from FeSFV-infected cats was consistent, especially when compared with the decrease observed after FIV infection. Overall, results of the study indicate that coinfection with FeSFV may not enhance progression of the FIV-induced early stages of disease and that the positive correlation between FeSFV and FIV-induced acquired immune deficiency-like syndrome is attributable to a common mode of transmission, rather than to a synergistic effect of coinfection.

The genetic basis of drug-resistant strains of Actinobacillus pleuropneumoniae in Japan was studied. The A pleuropneumoniae strains AV277 and AV281 that belong to serotype 2 were resistant to streptomycin (SM) and sulfonamide (SA). Both strains had an 8.1-kilobase (kb) SM-SA plasmid that was previously classified in the H1 group. The AV177 (serotype 1) strain was resistant to SM, SA, ampicillin, and kanamycin (Km), but did not have any plasmids. The AV319 and AV324 (serotype 1) strains were resistant to Sm, SA, tetracycline (TC), and chloramphenicol (CP). The AV318 (serotype 12) strain was resistant to SM, SA, TC, minocycline, and CP. These 3 strains (AV319, AV324, and AV318) had a 4.3-kb SM-SA plasmid and a 5.2-kb CP plasmid. The 4.3-kb plasmid was classified in the H2 group. The AV263 (serotype 1) strain was resistant to SM, SA, KM, TC, and CP. It had a 5.2-kb CP plasmid and a 6.6-kb SM-SA-KM plasmid. Both plasmids did not replicate stably in Escherichia coli strains. The former 5.2-kb plasmid was mobilized in E coli strains by plasmid RP4, which belonged to incompatibility P with broad host range, but the latter 6.6-kb plasmid was not so mobilized. Three 5.2-kb CP plasmids isolated from strains AV319, AV324, and AV318, had the same restriction endonuclease pattern after digestion with Ava I and EcoRI. They coexisted with H1 group plasmids in the incompatibility test, and coexisted also with H2 group plasmids of the original A pleuropneumoniae strains. Results indicated that the 5.2-kb CP plasmids could be classified in a new incompatibility group, H3. In this study, 4 types of plasmids were isolated, but no plasmids encoded TC and minocycline resistance.

We used size-exclusion high-performance liquid chromatography (HPLC) to investigate the properties of the 2 isoforms of vitamin A-containing (holo) retinol-binding protein (RBP) in animals: the form that is bound to transthyretin (holo-TTR-RBP), and the form that does not bind to TTR (holo-free RBP). We also used radial immunodiffusion to measure immunologically active RBP (apo+ holo RBP). We compared the isoforms of RBP in animals with those of human beings to determine which animal is the best model of human RBP. Size-exclusion HPLC detected holo-free and holo-TTR-RBP in every animal species studied. Apparent concentration of holo-TTR-RBP varied among species: that of rabbits and dogs much greater than that of apes, sheep, goats, monkeys, rhinoceroses, felids, rats, human beings, and deer greater than that of pigs, zebra, and bison greater than that of penguins. Dogs have unusual RBP chromatograms; they have high concentration of RBP, but also appear to transport much of their vitamin A on proteins other than RBP, Human RBP antibody preparations could detect apo + holo RBP immunologic activity only in apes, monkeys, and felids. Apes and monkeys appeared to have complete cross-reactivity to human RBP antibodies. Felids may have substantial, but partial, cross-reactivity. Apes and monkeys appear to be the most relevant animal models for study of human RBP transport. However, there is a need for less-expensive models. Further research is needed, but in the interim, rats or sheep may be satisfactory for some purposes.

Nine adult female sheep were each surgically fitted with an Ivan and Johnston reentrant cannula in the cranial part of the duodenum just distal to the pylorus. By diversion (loss) of abomasal outflow, this model has been shown to consistently induce hypochloremic, hypokalemic metabolic alkalosis, accompanied by hyponatremia and dehydration. Each sheep was subjected to 3 treatment trials, each preceded by a 24-hour prediversion period, and a diversion period during which a syndrome of hypochloremia (68 +/- 2 mEq/L), hypokalemia, hyponatremia, and metabolic alkalosis was induced. Development of this syndrome was attributable to losses of large amounts of acid and electrolytes in the abomasal effluent. Mean total electrolyte contents of the effluent were: Cl-, 650 +/- 27 mEq; Na+, 388 +/- 23 mEq; and K+, 123 +/- 12 mEq, with total volume loss ranging from 3.6 to 10.0 L of gastric contents and pH ranging from 3 to 5. Decreases in plasma electrolyte concentrations also can be attributed to decreased intake, because anorexia developed shortly after the onset of diversion. Electrolyte losses in urine during diversion were minimal for Cl-(mean +/- SEM, 12.0 +/- 5.1 mEq), but were greater for Na+ (124.2 +/- 14.5 mEq) and K+ (185.1 +/- 31.2 mEq). Treatments consisted of 0.9% NaCl (300 mosm/ L), 3.6% NaCl (1,200 mosm/L, and 7.2% NaCl (2,400 mosm/L) administered over a 2-hour period, with the administered volume determined by the estimated total extracellular fluid Cl- deficit. Significant difference was not found among treatments, with all solutions resulting in return of clinicopathologic and physical variables to prediversion values within 12 hours of treatment. We concluded that rapid iv replacement of Cl-, with small volumes of hypertonic saline solution, is safe and effective for correction of experimentally induced hypochloremic, hypokalemic, metabolic alkalosis in sheep.