Three methods of determining glomerular filtration rate (GFR) were performed in adult ferrets, 9 months to 7 years old. Endogenous creatinine clearance was determined, using serum and urine creatinine values obtained during 24- and 48-hour collection periods from 27 ferrets housed in metabolic cages. Creatinine and radiolabeled inulin were administered to 12 female ferrets by constant IV infusion during isoflurane-induced anesthesia. Serial 20-minute urine collections, together with serum samples obtained at the midpoint of urine collection, provided measures for clearance calculations of these substances. Mean +/- SD endogenous creatinine clearance in ferrets for metabolic cage collections was 2.50 +/- 0.93 ml/min/ kg of body weight. There were no significant differences between the 24- and 48-hour clearance rates. Mean inulin clearance was 3.02 +/- 1.78, and mean exogenous creatinine clearance was 3.32 +/- 2.16 ml/ min/kg. Analysis of variance, using least-squared means adjustment, did not yield any significant differences between inulin and exogenous creatinine clearance rates. Exogenous creatinine clearance-to-inulin clearance ratio was 0.99 +/- 0.46, and there was significant correlation between the 2 methods (r = 0.82, P = 0.0001). Significant body temperature effects on inulin or exogenous creatinine clearance were not found. Infused inulin clearance, the generally preferred method for GFR calculation in mammalian species, was significantly (P = 0.0069) higher in younger (3.65 ml/min/kg) vs older ferrets (2.29 ml/min/kg). Results of this study indicate that inulin clearance is an adequate measure of GFR in ferrets as it is in other species. Compared with inulin clearance, exogenous creatinine clearance also provides a reliable estimate of GFR in ferrets.

Cardiorespiratory effects of abdominal insufflation were evaluated in 8 dogs during isoflurane anesthesia. Each dog was studied 3 times, in 1 of the following orders of insufflation pressures: 10-20-30, 20-30-10, 30-20-10, 10-30-20, 20-10-30, and 30-10-20 mm of Hg. Anesthesia was induced by use of a mask, dogs were intubated, and anesthesia was maintained by isoflurane in 100% oxygen. After instrumentation, baseline values were recorded (time 0), and the abdomen was insufflated with nitrous oxide. Data were recorded at 5, 10, 15, 20, 25, and 30 minutes after insufflation. The abdomen was then desufflated, with recording of data continuing at 35 and 40 minutes. Mean arterial pressure increased at 5 minutes during 20 mm of Hg insufflation pressure, and from 20 to 30 minutes during 30 mm of Hg pressure. Tidal volume decreased from 5 to 30 minutes during 10 and 20 mm of Hg pressures, and from 5 to 40 minutes during 30 mm of Hg pressure. Minute ventilation decreased at 10 and 20 minutes during 20 mm of Hg pressure. End-tidal CO2 concentration increased from 5 to 30 minutes during 20 and 30 mm of Hg pressure. The PaCO2 decreased at 40 minutes during 10 mm of Hg pressure, at 30 minutes during 20 mm of Hg pressure, and from 10 to 40 minutes during 30 mm of Hg pressure. Values for pH decreased from 10 to 30 minutes during 20 and 30 mm of Hg pressures. The PaO2 decreased from 20 to 40 minutes during 10 mm of Hg pressure, at 30 minutes during 20 mm of Hg pressure, and from 10 to 40 minutes during 30 mm of Hg pressure. Percentage decrease in tidal volume was greater at 5 and 15 minutes with 30 mm of Hg pressure. Differences in percentage increase in end tidal CO2 concentration were observed among the 3 pressures from 5 to 30 minutes. Although significant, these changes do not preclude use of laparoscopy if insufflation pressure > 20 mm of Hg is avoided.

Renal clearance procedures were performed on adult mixed-breed dogs with a wide range of renal function. Endogenous creatinine clearance was computed after analyzing plasma and urine for creatinine by use of 2 methods, PAP and kinetic Jaffe. For 20-minute clearance procedures, [14C]inulin clearance was measured simultaneously with endogenous creatinine clearance. For 111 twenty-minute clearance procedures performed on 24 dogs, [14C]inulin clearance was highly correlated with creatinine clearance for both methods of creatinine analysis (R2 = 0.979 for [14C]inulin-PAP; R2 = 0.943 for [14C]inulin-Jaffe). The absolute values for PAP and [14C]inulin clearance were nearly the same (PAP-to-[14C]inulin clearance ratio = 1.03 +/- 0.08), but those for Jaffe clearance were substantially less than those for [14C] inulin clearance Jaffe-to-[14C]inulin clearance ratio = 0.88 +/- 0.10). The Jaffe-to-[14C] inulin clearance ratio was inversely correlated with degree of renal function (R2 = 0.464), whereas the PAP-to-[14C]inulin clearance ratio was not correlated with degree of renal function (R2 = 0.060). Thus, Jaffe-determined creatinine clearance varied, in relation to [14C] inulin clearance, depending on degree of renal function. In 4 clinically normal dogs, 20-minute and 24-hour sample collections analyzed by use of the PAP method gave clearance values significantly greater, for both periods, than did Jaffe analyses. The PAP-determined creatinine clearance values were less than, but not significantly different from 20-minute exogenous creatinine clearance values determined 10 days after 24-hour collections. For 20-minute and 24-hour collections, the difference in clearance values between the PAP and Jaffe methods was attributable mostly to lower plasma creatinine values for the PAP method (mean +/- SEM, plasma PAP-to-Jaffe ratio = 0.798 +/- 0.053). However, urine creatinine values also were less by use of the PAP method.

A protocol was developed for preparation of platelet concentrates (PC) to support thrombocytopenic dogs. Four clinically normal dogs with platelet counts that ranged from 200 to 330 X 10(9) platelets/L were used as donors. One unit (450 ml) of blood was collected by venipuncture into a double blood bag. Whole blood (WB) was centrifuged for 4 minutes at 1,000 X g (braking time = 2 minutes, 30 seconds) to prepare platelet-rich plasma (PRP). The PRP was expressed into the satellite bag and was centrifuged for 10 minutes at 2,000 X g (braking time = 2 minutes, 36 seconds). The platelet-poor plasma was expressed, leaving 40 to 70 ml of plasma and the pelleted platelets in the satellite bag. The resulting PC was left undisturbed for 60 minutes to promote disaggregation, and the platelets were then resuspended by gentle manual agitation. Forty-eight PC were prepared. Mean (+/- SD) platelet yield from WB to PRP was 78 +/- 13)% (range, 35 to 97%); yield from PRP to PC was 94 (+/- 6) % (range, 75 to 100%); and overall yield (PC from WB) was 74 (+/- 13) % (range, 36 to 91%). Mean PC platelet count was 8.0 (+/- 3.0) X 10(10) platelets/PC (range, 2.3 to 13.4 X 10(10) platelets/PC). The WBC content was 0.1 to 2.3 X 10(9) platelets/PC, representing 3 to 74% of WBC in the WB. Hematocrit was 0.1 to 26.2%. Results of bacterial and fungal culturing were negative.

In genome sequencing project, we encounter the DNA regions that often contain stable secondary structure with high GC content. These regions are difficult to not only amplify by PCR for template preparations, but also deter mine the DNA sequences using standard Cycle sequencing (CS) method. Transcriptional sequencing (TS) is a unique DNA sequencing method using RNA polymerase, and is based on the principles of the chain-termination method, which is a powerful method to analyze GC-rich sequences. In this study, we examined the multiple displacement amplification (MDA) to overcome low efficiency of PCR amplification in GC-rich regions and subjected to TS reaction. Combination of MDA and TS (MDA-TS) was extremely successful with GC content ranging from 65 % to 85%, which are difficult to analyze with PCR and CS. We also report plasmid vector, pTS1, which has the stronger T7 and T3 promoters than those of conventional vectors, and the sequence that decreases transcriptional efficiency was removed from its multiple cloning sites. pTS1 resulted in the improved sequencing accuracy and reduced reaction time up to 5 min. These results showed that MDA-TS is a rapid and accurate method for the analysis of GC-rich templates.

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.