Objective-To investigate the epidemiologic and financial impacts of targeted sampling of subpopulations of cows, compared with random sampling of all cows, for classification of dairy herd infection status for paratuberculosis. Animals-All cows from 4 infected herds with a low-to-moderate prevalence of paratuberculosis and from 1 noninfected herd in California. Procedure-The infection status of each cow was classified on the basis of results of an ELISA or combined ELISA and fecal culture results. Thirteen sampling schemes designed to randomly sample cows on the basis of lactation number, stage of lactation, and milk production were evaluated. Sampling without replacement was used to obtain a probability of herd detection of paratuberculosis for each evaluated sampling method and for simulated sample sizes between 30 and 150 cows. Marginal cost-effectiveness analysis was used to determine the cost increase relative to the increase in detection probability. Results-Sampling cows in the third or higher lactation and greater than or equal to 200 days into lactation yielded the highest detection probability in most instances, resulting in a detection probability that was 1.4 to 2.5 times that obtained by sampling 30 cows in the second or higher lactation. Costs of testing via the alternative method with a 95% detection probability were approximately $300 lower in a high-prevalence herd (31 %) and $800 lower in a low-prevalence herd (9%), compared with use of the reference method. Conclusions and Clinical Relevance-Detection of herds with paratuberculosis could be improved, and costs of testing substantially reduced by sampling targeted groups of cows.

Objective-To determine the pharmacokinetic disposition of IV administered caffeine in healthy Lama spp camelids. Animals-4 adult male alpacas and 4 adult female llamas. Procedures-Caffeine (3 mg/kg) was administered as an IV bolus. Plasma caffeine concentrations were determined by use of high-performance liquid chromatography in 6 animals and by use of liquid chromatography-mass spectrometry in 2 llamas. Results-Median elimination half-life was 11 hours (range, 9.3 to 29.8 hours) in alpacas and 16 hours (range, 5.4 to 17 hours) in llamas. The volume of distribution at steady state was 0.60 L/kg (range, 0.45 to 0.93 L/kg) in alpacas and 0.75 L/kg (range, 0.68 to 1.15 L/kg) in llamas. Total plasma clearance was 44 mL/h/kg (range, 24 to 56 mL/h/kg) in alpacas and 42 mL/h/kg (range, 30 to 109 mL/h/kg) in llamas. Conclusions and Clinical Relevance-High-performance liquid chromatography and liquid chromatography-mass spectrometry were suitable methods for determination of plasma caffeine concentrations in alpacas and llamas. Plasma caffeine concentration-time curves were best described by a 2-compartment model. Elimination half-lives, plasma clearance, volume of distribution at steady state, and mean residence time were not significantly different between alpacas and llamas. Intravenous administration of caffeine at a dose of 3 mg/kg did not induce clinical signs of excitement.

Objective-To quantitatively assess distraction-induced bone formation in a crural lengthening model in dogs by use of delayed-image bone scintigraphy. Animals-12 mature Labrador Retrievers. Procedure-Dogs were randomly allocated to 1 of 3 groups. A circular external skeletal fixation system was mounted on the right crus of each dog. Osteotomy of the distal portion of the tibia and fibula was performed in groups 1 and 2 and was followed by a lengthening procedure of 10 mm in the first group only. The third group served as sham-operated controls. Delayed-image bone scintigraphy with technetium-99m hydroxy methylene diphosphonate was performed 2, 4, and 6 weeks after surgery. Delayed-image-to-region-of-interest, delayed-image-to-crural, and delayed-image-to-femoral scintigraphic activity ratios were calculated. New bone formation was quantified by use of densitometric image analysis, and values for the scintigraphic ratios were compared. Results-In the distraction and osteotomy groups, delayed-image-to-region-of-interest and delayed-image-to-crural ratios increased significantly. Although densitometric image analysis revealed increased bone formation after distraction, the region-of-interest ratios and crural ratios were similar in both groups. All dogs had increased delayed-image-to-femoral ratios. Conclusions and Clinical Relevance-Delayed-image bone scintigraphy ratios were not effective at differentiating between the amounts of distraction-induced bone and osteotomy-induced bone. Metabolic bone activity in the adjacent femur was increased as a consequence of circular external skeletal fixator placement. Delayed-image bone scintigraphy was not adequately sensitive to quantitatively monitor bone formation but may be useful as an early predictor of bone healing.

Objective-To examine the accuracy and precision of isoflurane and sevoflurane anesthetic vaporizers. Sample Population-5 identcal isoflurane and 5 identical sevoflurane vaporizers. Procedures-Oxygen flow rates from 0.02 to 10 L/min were used with different vaporizer dial settings. Agent concentrations were measured at the common gas outlet by use of a refractometer. Accuracy was defined as the difference between measured agent concentrations, and dial settings were expressed as a percentage of the applied dial settings. Precision was defined as SD of the measured agent concentrations for each combination of dial setting and flow. Results-Isoflurane values were generally greater than the dial settings. Accuracy of the isoflurane vaporizer was > 20% when 0.6% and 1% was dialed. Accuracy of the sevoflurane vaporizer was always within +/- 20% but decreased at 0.02 L/min flow and at combinations of high flow and high dial settings. Overall precision of the isoflurane vaporizer was better than that of the sevoflurane vaporizer. Conclusions and Clinical Relevance-A possible explanation for the inaccuracy of the isoflurane vaporizer may be that it was manufactured to be accurate with air but not oxygen, which must be accounted for when using the vaporizer with oxygen, especially with nonrebreathing systems. The sevoflurane vaporizer may not deliver accurate agent concentrations at high flow and high dial settings. Both vaporizers are suitable for clinical use with a wide range of oxygen flow rates if these precautions are properly addressed.

Objective-To compare and correlate B-mode and color Doppler ultrasonographic characteristics with histopathologic findings of benign and malignant superficial lymph nodes in dogs. Study Population-50 superficial lymph nodes that were normal, abnormally large on physical examination, or represented regional lymph nodes draining an area of suspected primary malignancy in 30 dogs. Procedures-Before excision, lymph nodes were evaluated via B-mode and color Doppler ultrasonography to assess size, echogenicity, presence of a hilus, acoustic transmission, and vascular flow. Formalin-fixed, paraffin-embedded tissue sections of excised lymph nodes were stained with H&E and examined for the presence and extent of necrosis, fibrosis, fat, metastases, and tissue heterogeneity. To assess vascularity, the number and distribution of vessels stained by the Verhoeff van Gieson technique were recorded. Results-In superficial lymph nodes, a varied echogenicity corresponded to tissue heterogeneity. The ultrasonographic detection of a hilus was associated with the presence of fibrous tissue, fat, or both in the hilar region. Acoustic enhancement corresponded to presence of areas of intranodal necrosis. There was significant correlation between both the distribution and the number of vessels detected via ultrasonography and that detected by histopathology. The amount of flow estimated via ultrasonography was typically higher than that estimated via histologic examination. Conclusions and Clinical Relevance-Results indicated that histopathologic changes in canine lymph nodes have associated ultrasonographic changes and suggest that lymph node ultrasonography has an important role in the evaluation of lymph nodes in dogs in general and in dogs with neoplastic disease in particular.

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.