Genotypic characterisation of Giardia from domestic dogs in the USA.

The first large-scale urban survey of Giardia infections in dogs was undertaken in the USA. It involved several locations in the Western United States with Giardia isolates from microscopy-positive samples characterised by multi-locus PCR and sequencing. A high prevalence of Giardia was confirmed in asymptomatic domestic dogs, and for the first time, provides evidence that zoonotic assemblages/subgroups of Giardia occur frequently in domestic dogs living in urban environments, and more frequently than the dog specific assemblages.

Proteomic identification and profiling of canine lymphoma patients.

This study employed proteomic and bioinformatic approaches to identify serum biomarkers in canine lymphoma patients. Chilled serum samples derived from non-lymphoma (n = 92) and lymphoma (n = 87) patients were shipped from first opinion veterinary practices, subjected to ion exchange chromatography and analysed by surface-enhanced laser desorption ionization mass spectrometry. Nineteen serum protein peaks were identified between the two groups as being significantly different (P < 0.05) based upon their normalized ion intensities. Two biomarkers were identified that were capable of differentiating lymphoma and non-lymphoma patients. Analysis of the test data provided a positive predictive value (PPV) of 82%. A clinical follow-up study was carried out on 96 canine patients suspected of having lymphoma. Evaluation of this data gave a specificity value of 91%, sensitivity of 75%, PPV of 80% and negative predictive value of 88%. In conclusion, the expression pattern of two serum biomarkers has enabled serum samples to be classified into either lymphoma or non-lymphoma categories.

Identification of the rhesus macaque rhadinovirus lytic origin of DNA replication.

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.

Separation vs. replication of inactive and active centromeres in neoplastic cells.

The inactive centromeres in neoplastic and transformed cells exhibit premature separation at prophase or pro-metaphase. The factor(s) that control this behavior are not known. Using a human breast cancer cell line, MDA 435, and a transformed mouse cell line (L929), we studied the relationship between the sequence of centromere separation and the replication of centromeric region associated with the active and inactive centromeres. Whereas the inactive centromeres in L929 cells replicate their pericentric heterochromatin earlier than that associated with the active centromeres, those in MDA 435 cells exhibited no strong correlation between early separation and replication. A comparison between the intragenomic patterns of separation with replication of only active centromeres showed that the former is not dependent upon the latter in either L929 cells or MDA 435 cells. These studies indicate that, whereas inactive centromeres in neoplastic cells separate prematurely in different species, there is no uniformity in the control for replication nor does the timing of separation depend upon the timing of replication of the centric region.

Visualization of prekinetochore locus on the centromeric region of highly extended chromatin fibers: does kinetochore autoantigen CENP-C constitute a kinetochore organizing center?

Kinetochore is morphologically defined as a trilaminated, highly differentiated structure at the primary constriction of mitotic chromosomes. This subcellular organella is assumed to be composed of DNA and proteins. Immunoelectron microscopy has shown that centromere autoantigens CENP-C and CENP-B localize to the kinetochore inner plate and the underlying centromeric region respectively. We previously indicated that both are DNA-binding proteins that constitute centromeric heterochromatin throughout the cell cycle. Here, we tried to elucidate how these molecules are involved in the kinetochore/centromere organization in vivo by analyzing their morphological behavior in nuclei. Using immunofluorescence microscopy, we found that CENP-C remained as round discrete dots, whereas CENP-B displayed larger surrounding materials. To examine the CENP-C-binding locus on the genome, we prepared highly extended chromatin fibers and performed simultaneous immunofluorescence and fluorescence in situ hybridization. We obsreved that centromeric alphoid DNA, targeted by CENP-B, was highly dispersed, whereas the CENP-C antigen persisted as small dots well situated on the fibers. These features reminded us of the 'ball and cup' structure that had been presented for 'prekinetochore'. We propose here that CENP-C constitutes a 'kinetochore organizing center' tightly associating with DNA, whereas CENP-B heterochromatin offers the solid support during kinetochore maturation.

Concentration of enrofloxacin and its active metabolite in alveolar macrophages and pulmonary epithelial lining fluid of dogs.

The purpose of this study was to determine the concentration of enrofloxacin and its active metabolite, ciprofloxacin, in alveolar macrophages (AM) and epithelial lining fluid (ELF) of the lungs in comparison to plasma concentrations in healthy dogs. Eleven dogs were given a single oral dose (5 mg/kg) of enrofloxacin. Four hours later, plasma and bronchoalveolar lavage (BAL) fluid were collected. Cells were separated from the BAL fluid and lysed for determination of drug concentrations within AM. Supernatant was used to determine concentrations of drugs in ELF. Drug assays were performed by high-performance liquid chromatography. The concentration of enrofloxacin (mean +/- SD) was 0.33 +/- 0.14 microgram/mL in plasma, 3.34 +/- 2.4 micrograms/mL in AM and 4.79 +/- 5.0 micrograms/mL in ELF. The concentration of ciprofloxacin was 0.42 +/- 0.26 microgram/mL in plasma, 1.15 +/- 1.03 micrograms/mL in AM and 0.26 +/- 0.26 microgram/mL in ELF. Mean concentrations of both drugs in AM were greater than in plasma (AM to plasma ratio, 10.3 for enrofloxacin and 4.7 for ciprofloxacin). Mean concentrations of enrofloxacin, but not ciprofloxacin, in ELF were greater than in plasma (ELF to plasma ratio, 13.5 for enrofloxacin and 0.52 for ciprofloxacin). Enrofloxacin concentrations in AM and ELF largely exceeded the MICs of the major bacterial pathogens and surpassed by about two times the breakpoint MIC of that drug, and ciprofloxacin concentrations in AM surpassed the MIC of many susceptible organisms. These results suggest that sufficient antimicrobial activity is present in AM and ELF of dogs following oral administration of enrofloxacin to be effective in the treatment of lower respiratory tract infections involving susceptible organisms.

Quantitative comparison of the stress response of striped bass (Morone saxatilis) and hybrid striped bass (Morone saxatilis x Morone chrysops and Morone saxatilis x Morone americana).

Striped bass (Morone saxatilis) exposed to a standardized confinement stress had markedly different clinical and endocrinologic responses, compared with hybrid striped bass exposed to the same stress. Plasma cortisol concentration increased at a faster rate and appeared to reach a higher value in striped bass than in hybrid bass. Mean plasma cortisol concentration was 742 +/- 43 ng/ml in striped bass, compared with 490 +/- 37 and 531 +/- 40 ng/ml in striped bass x white perch (M americana) and striped bass x white bass (M chrysops) hybrids, respectively, after a 45-minute net confinement. Plasma cortisol concentration also remained significantly (P = 0.003) higher in striped bass for at least 48 hours after the net confinement. These hormonal differences were associated with a markedly lower survival and resistance to infection in striped bass, compared with the hybrids.

Rapid high-performance liquid chromatographic method for the determination of ketamine and its metabolite dehydronorketamine in equine serum.

A simple, rapid and sensitive high-performance liquid chromatographic procedure has been developed for the determination of ketamine and dehydronorketamine in equine serum. Sample preparation consisted of mixing equal volumes of serum and acetonitrile-phosphoric acid (85%)-water (20:2:78, v/v/v), followed by ultrafiltration through a 10,000 molecular mass cut-off filter. Separation of these two analytes in the ultrafiltrate was accomplished on a reversed-phase phenyl column eluted with methanol-acetonitrile-phosphate buffer solution. Ketamine and dehydronorketamine were detected by a variable photometric UV-Vis detector set at 215 nm, and confirmed by a photodiode array detector operated in the 200-320 nm range. The limit of detection for ketamine was 5-15 ng/ml in equine serum. Additionally, the dehydronorketamine peak identity was tentatively confirmed by thermospray liquid chromatography-mass spectrometry.

Pharmacokinetics of ibuprofen in lactating dairy cows.

The pharmacokinetics of ibuprofen were studied in 6 adult lactating dairy cows after a single IV or oral administration of ibuprofen (25 mg/kg of body weight). Ibuprofen concentrations in milk and serum were analyzed by use of high-performance liquid chromatography. The lower limit of detection of the ibuprofen assay was 50 ng/ml. Serum ibuprofen concentration-time curves after IV administration best fit an open two-compartment model. Harmonic mean volume of distribution at steady state was 0.14 (range, 0.12 to 0.17) L/kg, elimination half-life was 1.55 (range, 1.33 to 1.73) hours, and total clearance was 86.2 (range, 68.8 to 106.2) ml/kg/h. Harmonic mean oral bioavailability was 99% (range, 79 to 112). Adverse effects were not observed in cows given ibuprofen.

Comparison of the disposition of carbimazole and methimazole in clinically normal cats.

The oral disposition of the antithyroid drugs methimazole and carbimazole were compared in nine clinically normal cats. After the administration of 5 mg of methimazole, serum concentrations of methimazole increased in all the cats, with mean drug concentrations reaching peak values (1.37 micrograms ml-1) at 30 minutes. After administration of 5 mg carbimazole, serum concentrations of carbimazole remained low, but serum methimazole became readily measurable, with mean drug concentrations reaching peak values (0.79 microgram ml-1) at 120 minutes. When serum concentrations of methimazole attained after administration of the two antithyroid drugs were compared, the mean maximum serum methimazole concentration achieved after administration of methimazole was approximately twofold higher than peak concentrations measured after administration of carbimazole. In addition, the mean area under the serum concentration curve (AUC) after administration of methimazole was approximately twofold higher than the mean AUC determined after administration of carbimazole. When the differences in molecular weight between the two drugs was taken into consideration, however, these methimazole:carbimazole ratios of 2:1 were nearly equivalent to the molar ratio of the 5 mg doses of the drugs given (1.63). Results of this study indicate that carbimazole is nearly totally converted to methimazole after oral administration to cats, similarly to the findings in man. The finding of less available serum methimazole after administration of a 5 mg tablet of carbimazole than after methimazole is also consistent with published antithyroid drug dosages needed to control hyperthyroidism in cats.

Pharmacokinetics of ibuprofen in lactating dairy goats.

Pharmacokinetic variables of ibuprofen were studied in 6 adult lactating dairy goats after single administration of the drug (14 and 25 mg/kg of body weight, IV, and 50 and 100 mg/kg, PO). Each of the goats was given all doses, with a minimum of 1 week between doses. Ibuprofen concentration in serum was analyzed by use of high-performance liquid chromatography. The lower limit of detection for the ibuprofen assay was 50 ng/ml. Ibuprofen pharmacokinetic variables after IV administration best fit an open two-compartment model. Geometric mean (range) volume of distribution at steady state was 0.16 (0.11 to 0.19) and 0.17 (0.15 to 0.19) L/kg, and terminal half-life was 1.08 (0.79 to 1.70) and 1.27 (1.03 to 1.88) hours, for ibuprofen dosages of 14 and 25 mg/kg, respectively. After 50 and 100 mg/kg administered orally, bioavailability was 90.8 and 106%, respectively. Area under the curve increased linearly with dose administered. Adverse effects were not observed in goats given ibuprofen.

Enrofloxacin treatment of long-tailed macaques with acute bacillary dysentery due to multiresistant Shigella flexneri IV.

Thirty-four cases of acute bacillary dysentery occurred within 90 days among macaques housed at the California Regional Primate Research Center. Cases were identified by depression, diarrhea with blood and leukocytic exudate, and/or leukocytosis with a left shift. Antimicrobial susceptibility testing of enteric isolates and plasmid profile analyses established an etiologic diagnosis of multiple antibiotic resistant Shigella flexneri IV infection. When standard therapies were invalidated by high frequencies of resistance among the isolates, therapy with enrofloxacin, a fluoroquinolone antimicrobial, was initiated to interrupt the epidemic. Serum concentrations of enrofloxacin and its primary metabolite ciprofloxacin were measured in selected cases. A serum concentration-time data analysis was performed to evaluate the oral enrofloxacin dose and dosing interval for nonfasted macaques. Once daily administration of 5 mg/kg enrofloxacin by gastric intubation produced 24-hour serum concentrations above the MICs for the Shigella isolates from this outbreak.

Determination of ceftazidime in dolphin serum by liquid chromatography with ultraviolet-visible detection and confirmation by thermospray liquid chromatography-mass spectrometry.

A simple and sensitive liquid chromatographic method has been developed for the determination of therapeutic levels of ceftazidime in dolphin serum. The method involved an ultrafiltration of diluted serum with an equal amount of acetonitrile-ethanol-water (40:40:20, v/v/v) through a 10,000 daltons molecular mass cut-off filter. Separation of ceftazidime from the other serum components was performed by ion-paired (dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile-water solution. The ultraviolet absorbance of the column effluent was monitored in the 200-340 nm range of a photodiode-array detector or at 258.8 nm on a variable-wavelength ultraviolet-visible detector. Recoveries of ceftazidime from dolphin serum spiked with 20 and 2 micrograms/ml were 92.9 and 91.1% with coefficients of variation of 5.5 and 5.7%, respectively. A correlation coefficient of 0.9994 occurred with ceftazidime in aqueous solutions (n = 6, in duplicates). The limit of detection for this antibiotic was estimated to be approximately 50 ppb (ng/ml). The unbound ceftazidime concentrations in dosed dolphin serum were determined to calculate the protein bindings of this antibiotic which yielded 32 +/- 2%. The ceftazidime peak identity in dosed dolphin serum was confirmed by thermospray liquid chromatography-mass spectrometry. The thermospray mass spectrum of ceftazidime exhibited only the fragment ions, involving the opening of the beta-lactam ring, at m/z 237, 255 and 315 when positive-ion detection mode was employed and the fragment ions at m/z 235, 253 and 313 when negative-ion detection mode was used.

Relation between pharmacokinetics of amikacin sulfate and sepsis score in clinically normal and hospitalized neonatal foals.

Pharmacokinetic values after IV administration of amikacin sulfate were determined for clinically normal and hospitalized foals during the first week of life. The relations between drug disposition and sepsis score and serum creatinine concentration also were studied. In clinically normal foals, differences in sepsis score, serum creatinine concentration, and pharmacokinetic variables of amikacin were not found between foals 1 to 3 and 4 to 7 days old. In hospitalized foals, sepsis score, serum creatinine concentration, area under the curve, area under the moment curve, and mean residence time were greater, and total clearance was decreased, compared with values in clinically normal foals. Sepsis score and serum creatinine concentration were inversely correlated to amikacin clearance and appeared to be useful indicators of altered drug disposition.

Pharmacokinetic disposition of cefazolin in serum and tissue during canine total hip replacement.

Intraoperative cefazolin concentrations were measured in serum, joint capsule, cancellous bone of the acetabulum, and proximal cancellous bone of the femur in 15 dogs undergoing total hip replacement. Cefazolin (22 mg/kg intravenously [IV]) was administered every hour for three doses. The mean peak serum concentrations (+/- SEM) were 387.79 +/- 27.56 micrograms/mL, 521.71 +/- 28.00 micrograms/mL, and 542.20 +/- 30.91 micrograms/mL, respectively. Mean serum concentrations just before administration of doses 2 and 3 were 51.77 +/- 2.39 micrograms/mL, and 64.84 +/- 3.46 micrograms/mL, respectively. The mean cefazolin concentrations in the joint capsule, cancellous bone of the acetabulum, and cancellous bone of the femur were 34.71 +/- 2.50 micrograms/g, 28.70 +/- 7.40 micrograms/g, and 36.20 +/- 3.80 micrograms/g, respectively. The minimum inhibitory concentration of cefazolin for 90% of the common contaminants (MIC90) in this clinic is less than or equal to 2 micrograms/mL or per gram of tissue. Serum concentrations never fell below 15 times the MIC90 (lowest trough, 35.93 micrograms/mL), and the lowest tissue concentration (6.57 micrograms/mL in cancellous bone from the acetabulum) was still more than 3 times the MIC90. The mean tissue concentration was 15 times the MIC90.

Pharmacokinetics of intravenous and oral methimazole following single- and multiple-dose administration in normal cats.

The pharmacokinetics of methimazole (MMI) administered intravenously and orally were determined in six adult domestic shorthaired cats. There was no significant difference between mean serum MMI concentrations after oral and i.v. administration by 30 min post-MMI administration, indicating relatively rapid and complete absorption of the drug. The bioavailability of MMI ranged from 27% to 100% (mean = 81.1 +/- 11.4%). The mean serum elimination half-life was 6.6 +/- 2.0 h, with a wide range of values (1.9 h to 15.1 h). After repeat i.v. administration of MMI following 2 weeks of oral administration of the drug, no significant difference was found between mean serum concentrations after single-dose and multiple-dose administration. No significant change in serum elimination half-life or total body clearance was found after multiple-dose administration of MMI. Two cats with the longest half-lives (9.9 h and 15.1 h), however, did exhibit markedly shorter t1/2 values (3.5 h and 3.3 h, respectively) after multiple-dose administration. Values for central and steady state volumes of distribution also decreased after multiple-dose administration, possibly indicating saturation of thyroid uptake of MMI with chronic administration. These results indicate that MMI has good oral bioavailability and has a longer mean serum elimination half-life than propylthiouracil, the other anti-thyroid drug that has been evaluated in cats. Although no significant change in mean values occurred after multiple-dose administration of MMI, drug-induced acceleration of metabolism may occur in some cats after long-term MMI administration.

Intramuscular and oral disposition of enrofloxacin in African grey parrots following single and multiple doses.

The intramuscular (IM) and oral (PO) disposition of enrofloxacin, a new fluoroquinolone antimicrobial drug, were evaluated in African grey parrots. Peak enrofloxacin concentration, mean (+/- SEM), at 1 h following a 15-mg/kg IM dose was 3.87 (+/- 0.27) micrograms/ml and declined with a mean residence time of 3.05 h. Peak enrofloxacin plasma concentrations at 2 to 4 h following oral doses of 3, 15, and 30 mg/kg were 0.31 (+/- 0.11), 1.12 (+/- 0.11), and 1.69 (+/- 0.23) micrograms/ml, respectively, and declined with a mean residence time of 3.44-5.28 h. The relative bioavailability of the 15-mg/kg oral dose was 48%. An equipotent metabolite, ciprofloxacin, was detected in plasma at concentrations ranging from 3 to 78% of those of enrofloxacin. Enrofloxacin concentrations and area under the curve were significantly lower, the mean residence time significantly shorter and the ciprofloxacin/enrofloxacin ratios higher, following 10 days of oral treatment at 30 mg/kg every 12 h. Following 10 days of treatment, no significant biochemical changes were noted; however, polydipsia and polyuria occurred in treated birds, but resolved quickly upon discontinuation of enrofloxacin administration. These studies indicate that a rational starting dose for enrofloxacin in psittacines (7.5-30 mg/kg BID) should be higher than those in other domestic animals.

Rapid high-performance liquid chromatographic method for the determination of propranolol levels in canine and feline plasma.

A sensitive high-performance liquid chromatographic method that does not require organic extraction has been developed for the determination of propranolol levels in canine and feline plasma. Equal volumes of plasma and a mixture of methanol-acetonitrile-0.1 M sodium hydroxide (3:3:4, v/v/v) were added to a microseparation unit with a 10,000 molecular mass cut-off filter. The ultrafiltrate was analyzed by reversed-phase liquid chromatography with fluorimetric detection. The consistency of the recoveries obtained eliminated the need for an internal standard (coefficients of variation less than 4%). Linear regressions for the standard curves (2.5-100 ng/ml) gave correlation coefficients above 0.9955. The detection limit was 1 ng/ml. The assay retains high sensitivity while eliminating laborious sample preparation.