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ABSTRACT: To evaluate effects of high incubation temperatures on results of protozoal culture and real-time PCR testing for Tritrichomonas foetus inoculated in a commercially available self-contained culture media system.
In vitro experimental study.
2 strains of T foetus (1 field isolate from the University of California-Davis and 1 field isolate from the Texas Veterinary Medical Diagnostic Laboratory).
2 sets of 36 dual-chamber media pouches were inoculated with T foetus (36 sample pouches/strain) and incubated at temperatures of 37.0°C (98.6°F), 46.1°C (115.0°F), or 54.4°C (130.0°F) for 1, 3, 6, or 24 hours. Six uninoculated media samples in pouches stored at 37.0°C for the entire treatment period were used as negative controls. Pouches were removed from incubators and stored at 22.2°C (72.0°F) until all treatments were complete. Samples were submitted to a diagnostic laboratory for protozoal culture and real-time PCR testing.
T foetus was detectable microscopically in inoculated pouches incubated at 37.0°C regardless of exposure time, whereas those incubated at 46.1°C yielded T foetus after 1 and 3 hours only, and those incubated at 54.4°C yielded T foetus after 1 hour only. Testing via real-time PCR assay yielded positive results for all inoculated media samples and negative results for all uninoculated control samples.
Samples collected into the self-contained culture media system for T foetus testing via culture alone should be protected from high temperatures. Realtime PCR amplification may be a more reliable method for identification of the organism if storage and transport temperatures cannot be controlled.
Journal of the American Veterinary Medical Association 12/2011; 239(12):1589-93. · 1.79 Impact Factor
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ABSTRACT: Bovine trichomoniasis is a sexually transmitted disease in cattle that causes considerable economic loss due to abortions and infertility. In vitro culture of the organisms is the traditional method for diagnosis. However, culture cannot differentiate Tritrichomonas foetus from other, closely related nonpathogenic protozoa. Recently, a quantitative real-time polymerase chain reaction (qPCR) was developed for the differential diagnosis of trichomoniasis. The objective of the current work was to evaluate the effect of different simulated transport conditions on samples containing T. foetus for the diagnosis of trichomoniasis using culture and qPCR. Results indicate that transport temperatures of 4-20°C for 1-3 days before culture will reduce or temporarily inhibit parasite replication but maintain viability. Testing of samples by either culture or qPCR would be expected to give positive results. However, diagnosis of trichomonads by both methods was negatively affected when specimens were maintained at transport temperatures of 42°C for 24 hr or more. The current study stresses the importance of ensuring that clinical samples arrive to the diagnostic laboratory within 24-48 hr and of avoiding temperature transport conditions above 37°C in order to achieve an accurate diagnosis of trichomoniasis in cattle.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 09/2011; 23(5):982-5. · 1.21 Impact Factor
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ABSTRACT: To estimate the sensitivity (Se) and specificity (Sp) for an enhanced direct-fecal PCR procedure, bacterial culture of feces (BCF), and a serum ELISA for detecting Mycobacterium avium subsp paratuberculosis (MAP) infection in adult dairy cattle.
Fecal and serum samples were collected from 669 adult cattle randomly selected from a 4,000-cow dairy herd known to contain animals infected with MAP.
Serum samples were evaluated for MAP-specific antibodies via ELISA. Fecal samples were evaluated by BCF and enhanced PCR methods (both gel-based [GB]-PCR and quantitative real-time [qRT]-PCR assays). Fecal samples also were pooled (5:1) and then subjected to GB-PCR assay. Bayesian statistical methods were used to estimate Se and Sp for each diagnostic test without knowledge concerning true MAP infection status.
Adjusting for Se conditional dependence between serum ELISA and BCR, overall Se and Sp were estimated at 33.7% and 95.9%, 51.3% and 99.0%, and 32.2% and 100% for serum ELISA, qRT-PCR, and BCF, respectively.The GB-PCR assay yielded positive results for 38.3% of the pools known to contain feces from at least 1 cow that had positive GBPCR results.
Estimated Se values for the serum ELISA and BCF were slightly lower than those reported elsewhere. The enhanced qRT-PCR method offered relative improvements in Se of 52% and 59% over serum ELISA and microbial culture, respectively. Pooling of fecal samples and testing with the GB-PCR assay are not recommended. Additional studies with qRT-PCR and fecal pools are required.
American Journal of Veterinary Research 04/2007; 68(3):236-45. · 1.27 Impact Factor
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ABSTRACT: Heart-type fatty acid-binding protein (H-FABP) is a major fatty acid-binding factor in skeletal muscles. Genetic lack of H-FABP severely impairs the esterification and oxidation of exogenous fatty acids in soleus muscles isolated from chow-fed mice (CHOW-solei) and high fat diet-fed mice (HFD-solei), and prevents the HFD-induced accumulation of muscle triacylglycerols (TAGs). Here, we examined the impact of H-FABP deficiency on the relationship between fatty acid utilization and glucose oxidation. Glucose oxidation was measured in isolated soleus muscles in the presence or absence of 1 mM palmitate (simple protocol) or in the absence of fatty acid after preincubation with 1 mM palmitate (complex protocol). With the simple protocol, the mutation slightly reduced glucose oxidation in CHOW-muscles, but markedly increased it in HFD-muscles; unexpectedly, this pattern was not altered by the addition of palmitate, which reduced glucose oxidation in both CHOW- and HFD-solei irrespective of the mutation. In the complex protocol, the mutation first inhibited the synthesis and accumulation of TAGs and then their mobilization; with this protocol, the mutation increased glucose oxidation in both CHOW- and HFD-solei. We conclude: (i) H-FABP mediates a non-acute inhibition of muscle glucose oxidation by fatty acids, likely by enabling both the accumulation and mobilization of a critical mass of muscle TAGs; (ii) H-FABP does not mediate the acute inhibitory effect of extracellular fatty acids on muscle glucose oxidation; (iii) H-FABP affects muscle glucose oxidation in opposing ways, with inhibition prevailing at high muscle TAG contents.
Molecular and Cellular Biochemistry 03/2007; 296(1-2):59-67. · 2.06 Impact Factor
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ABSTRACT: Heart-type fatty acid-binding protein (H-FABP) is required for high rates of skeletal muscle long-chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild-type and H-FABP null mice were fed a standard chow or high-fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% (P < 0.01) at 0.02 mU/ml of insulin but did not cause a significant effect at 2 mU/ml of insulin; skeletal muscle triglyceride and long-chain acyl-CoA (LCA-CoA) levels remained normal. With the high-fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% (P < 0.01) at 2 mU/ml of insulin, thus partially preventing insulin resistance, and it completely prevented the threefold (P < 0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCA-CoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulin-independent) soleus muscle deoxyglucose uptake by 28% (P < 0.05). These results establish a close relation between FABP-dependent lipid pools and insulin sensitivity and indicate the existence of a nonacute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake.
AJP Endocrinology and Metabolism 11/2004; 287(5):E977-82. · 4.75 Impact Factor
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ABSTRACT: Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of long-chain fatty acids (LCFA) for oxidation and for peroxisome proliferator-activated receptor alpha (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced beta-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. During standard diet, mitochondrial HMG CoA synthase mRNA was selectively reduced in L-FABP null liver. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism, whereas L-FABP can activate ketogenic gene expression in fed mice. Thus, the mechanisms whereby L-FABP affects fatty acid oxidation may vary with physiological condition.
The FASEB Journal 03/2004; 18(2):347-9. · 5.71 Impact Factor
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ABSTRACT: The low-molecular-mass, cytosolic heart-type fatty acid-binding protein (H-FABP) is thought to be required for shuttling FA through the cytosol. Therefore, we examined the effects of an H-FABP-null mutation on FA and carbohydrate metabolism in isolated soleus muscle at rest and during a period of increased metabolic demand (30-min contraction). There were lower concentrations of creatine phosphate (-41%), ATP (-22%), glycogen (-34%), and lactate (-31%) (P < 0.05) in H-FABP-null soleus muscles, but no differences in citrate synthase and beta-3-hydroxyacyl-CoA dehydrogenase activities or in the intramuscular triacylglycerol (TAG) depots. There was a 43% increase in subsarcolemmal mitochondria in H-FABP-null solei. FA transport was reduced by 30% despite normal content of sarcolemmal long-chain fatty acid transporters fatty acid translocase/CD36 and plasma membrane-associated FABP transport proteins. Compared with wild-type soleus muscles, the H-FABP-null muscles at rest hydrolyzed less TAG (-22%), esterified less TAG (-49%), and oxidized less palmitate (-71%). The H-FABP-null soleus muscles retained a substantial capacity to increase FA metabolism during contraction (TAG esterification by +72%, CO2 production by +120%), although these rates remained lower (TAG esterification -26% and CO2 production -64%) than in contracting wild-type soleus muscles. Glycogen utilization during 30 min of contraction did not differ, whereas glucose oxidation was lower at rest (-24%) and during contraction (-32%) in H-FABP-null solei. Although these studies demonstrate that the absence of H-FABP alters rates of FA metabolism, it is also apparent that glucose oxidation is downregulated. The substantial increase in FA metabolism in contracting H-FABP-null muscle may indicate that other FABPs are also present, a possibility that we were not able to completely eliminate.
AJP Endocrinology and Metabolism 10/2003; 285(3):E481-9. · 4.75 Impact Factor
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ABSTRACT: Although liver fatty acid-binding protein (L-FABP) is an important binding site for various hydrophobic ligands in hepatocytes, its in vivo significance is not understood. We have therefore created L-FABP null mice and report here their initial analysis, focusing on the impact of this mutation on hepatic fatty acid binding capacity, lipid composition, and expression of other lipid-binding proteins. Gel-filtered cytosol from L-FABP null liver lacked the main fatty acid binding peak in the fraction that normally comprises both L-FABP and sterol carrier protein-2 (SCP-2). The binding capacity for cis-parinaric acid was decreased >80% in this region. Molar ratios of cholesterol/cholesterol ester, cholesteryl ester/triglyceride, and cholesterol/phospholipid were 2- to 3-fold greater, reflecting up to 3-fold absolute increases in specific lipid classes in the order cholesterol > cholesterol esters > phospholipids. In contrast, the liver pool sizes of nonesterified fatty acids and triglycerides were not altered. However, hepatic deposition of a bolus of intravenously injected [14C]oleate was markedly reduced, showing altered lipid pool turnover. An increase of approximately 75% of soluble SCP-2 but little or no change of other soluble (glutathione S-transferase, albumin) and membrane (fatty acid transport protein, CD36, aspartate aminotransferase, caveolin) fatty acid transporters was measured. These results (i) provide for the first time a quantitative assessment of the contribution of L-FABP to cytosolic fatty acid binding capacity, (ii) establish L-FABP as an important determinant of hepatic lipid composition and turnover, and (iii) suggest that SCP-2 contributes to the accumulation of cholesterol in L-FABP null liver.
Journal of Biological Chemistry 06/2003; 278(24):21429-38. · 4.77 Impact Factor
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Erdal Erol
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ABSTRACT: Heart-type Fatty Acid-Binding Protein (H-FABP) is required for high rates of skeletal muscle long chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild type and H-FABP null mice were fed a standard chow or high fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% (P<0.01) at 0.02 mU/ml of insulin, but did not cause a significant effect at 2 mU/ml insulin; skeletal muscle triglyceride and long chain acyl-CoA (LCACoA) levels remained normal. With the fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% (P<0.01) at 2 mU/ml insulin, thus partially preventing insulin resistance, and completely prevented the threefold (P<0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCACoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulinindependent) soleus muscle deoxyglucose uptake by 28% (P<0.05). These results establish a close relationship of FABP-dependent lipid pools with insulin sensitivity, and indicate the existence of a non-acute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake. Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of chain LCFA for oxidation and for peroxisome proliferator-activated receptor (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced β-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism.
