Erdal Erol

Liver fatty acid binding protein is required for high rates of hepatic fatty acid oxidation but not for the action of PPARα in fasting mice

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 α (PPAR‐α), 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‐α 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.

Beta-hemolytic Streptococcus spp. from horses a retrospective study (2000–2010)

The goal of this retrospective study was to have a comprehensive picture of the β-hemolytic streptococci of horses including tissue/organ distributions and susceptibility patterns against specific antimicrobials between January 1, 2000 and December 31, 2010. A total of 2,497 β-hemolytic streptococci were isolated from 2,391 cases, of which Streptococcus equi subsp. zooepidemicus was the most frequent isolate (72.0%). Other species isolated were Streptococcus dysgalactia subsp. equisimilis (21.3%), Streptococcus equi subsp. equi (5.8%), and unidentified β-hemolytic streptococci (0.9%). As expected, S. equi was mostly isolated from lymph node abscesses and the respiratory tract in foals and adult horses. Streptococcus equi subsp. zooepidemicus and S. equisimilis were mostly isolated from placenta, fetal tissues, and genital tract of horses; S. zooepidemicus and S. equisimilis were also recovered in significant numbers from a number of other organs including lung, liver, brain, kidney, and joints, indicating a much broader tissue tropism than S. equi. In addition, more than 1 Streptococcus spp. was recovered in 106 cases, indicating the co-existence of these bacteria in some horses. This data also suggested that S. equisimilis is a major bacterial agent of horses, contrary to present knowledge. Based on Kirby-Bauer antimicrobial susceptibility data, streptococci were found to be generally susceptible to cephalothin, erythromycin, nitrofurantoin, penicillin, and ticarcillin and clavulanate. Resistance to antimicrobials has not developed over the years, except for gentamicin and tetracycline against S. equisimilis.

FABPs as determinants of myocellular and hepatic fuel metabolism

In vitro experiments and expression patterns have long suggested important roles for the genetically related cytosolic fatty acid binding proteins (FABPs) in lipid metabolism. However, evidence for such roles in vivo has become available only recently from genetic manipulation of FABP expression in mice. Here, we summarize the fuel-metabolic phenotypes of mice lacking the genes encoding heart-type FABP (H−/− mice) or liver-type FABP (L−/− mice). Cytosolic extracts from H−/− heart and skeletal muscle and from L−/− liver showed massively reduced binding of long chain fatty acids (LCFA) and, in case of L−/− liver, also of LCFA-CoA. Uptake, oxidation, and esterification LCFA, when measured in vivo and/or ex vivo, were markedly reduced in H−/− heart and muscle and in L−/− liver. The reduced LCFA oxidation in H−/− heart and L−/− liver was not due to reduced activity of PPARa, a fatty acid-sensitive transcription factor that determines the lipid-oxidative capacity in these organs. In H−/− mice, mechanisms of compensation were partially studied and included a redistribution of muscle mitochondria as well as increases of cardiac and skeletal muscle glucose uptakes and of hepatic ketogenesis. In skeletal muscle, the altered glucose uptake included decreased basal but increased insulin-dependent components. Metabolic compensation was only partial, however, since the H−/− mice showed decreased exercise tolerance. In conclusion, the recent studies established H- and L-FABP as major determinants of regional LCFA utilization; therefore the H−/− and L−/− mice are attractive models for studying principles of fuel selection and metabolic homeostasis.

Bartonella bovis isolated from a cow with endocarditis

A 7-year-old pregnant Angus cow was found dead in the field. At necropsy, the aortic valve was expanded by moderate fibrous connective tissue and acidophilic coagulum containing multifocal marked bacteria, mineral, neutrophils, and red blood cells. Numerous tiny grayish, opaque bacterial colonies were detected on blood agar plates at 7 days after inoculation with a swab of the heart valve of the cow. The bacterium was a Gram-negative, very small coccobacillus that was catalase, oxidase, and urease negative, and did not change litmus milk, triple sugar iron agar, and sulfide-indole-motility medium. The bacterium was negative for esculin hydrolysis, phenylalanine deaminase, nitrate reduction, and gelatin hydrolysis. The isolate did not produce acid from glycerol, inulin, lactose, maltose, mannose, raffinose, salicin, sorbitol, sucrose, trehalose, glycogen, ribose, or starch. Polymerase chain reaction tests for the gltA, ssrA, ftsZ, ribC, rpoB, and 16S ribosomal RNA genes of Bartonella species were positive for the isolate. Amplicons were sequenced, and the gltA, ribC, ssrA, and 16S ribosomal RNA gene sequences were found to have 100% homology to the type strain of Bartonella bovis, whereas the fts and rpoB sequences showed 99.9% and 99.6% homology, respectively, to the type strain of Bartonella bovis. Diagnosticians should be aware of slow-growing microorganisms, and culture media should be incubated beyond the standard period to enhance the recovery of Bartonella species.

MICs of 32 antimicrobial agents for Rhodococcus equi isolates of animal origin

OBJECTIVES The aim of this study was to determine the MICs of 32 antimicrobial agents for 200 isolates of Rhodococcus equi of animal origin by applying a recently described broth microdilution protocol, and to investigate isolates with distinctly elevated rifampicin MICs for the genetic basis of rifampicin resistance. METHODS The study included 200 R. equi isolates, including 160 isolates from horses and 40 isolates from other animal sources, from the USA and Europe. MIC testing of 32 antimicrobial agents or combinations thereof followed a recently published protocol. A novel PCR protocol for the joint amplification of the three rpoB regions in which rifampicin resistance-mediating mutations have been reported was applied to isolates with elevated rifampicin MICs. The amplicons were sequenced and screened for mutations. RESULTS Susceptibility testing revealed a rather uniform distribution of MICs for most of the antimicrobial agents tested. The lowest MICs were seen for clarithromycin, rifampicin and imipenem. Six isolates (3%) exhibited distinctly higher MICs of rifampicin than the remaining 194 isolates. In five of these six isolates, single bp exchanges, which resulted in the amino acid exchanges Gln513Leu, Asp516Val, His526Asp or Ser531Leu, were detected in the rifampicin resistance-determining region 1 of the rpoB gene, with Gln513Leu representing a novel substitution for R. equi. CONCLUSIONS This study shows the MIC distribution of 32 antimicrobial agents for a large collection of R. equi isolates of animal origin from two continents. Isolates that exhibited distinctly elevated MICs of rifampicin were only rarely detected.

Leptospirosis in Turkey.

The bird was discharged with advice to confine it to a small well-bedded area for two weeks and then to gradually increase the size of the pen if progress was satisfactory. Radiographs were taken four and eight weeks postoperatively. The screws were found to have bent four weeks later and by eight weeks they had broken. The bird was last examined seven months postoperatively. It walked with a mild limp but was mobile and in good health. Two months later the bird died after being attacked by another turkey. Its body was available for postmortem examination and the right femur was removed for radiography and further examination. Complete fracture healing was documented radiographically (Fig 1c) and, although all four screws had broken, they had not migrated from the bone. A literature search failed to reveal either any reports of femoral fracture repair in the turkey or the use of the interlocking nail in avian fractures. Intramedullary pinning is a very common means of fracture repair in birds but a major disadvantage is the lack of rotational stability it affords (Levitt 1989). Interlocking nailing has the ability to resist rotation and collapse of the fracture. Avian bones have thin, brittle cortices and there is a common belief that they will not provide sufficient holding power for bone screws (Roush 1980). In one study, in which semitubular plates were used on avian leg fractures, the cortical bone screws did not strip or loosen, although there was a problem with bending of the plates (Howard 1990). In the present case, the screws did not loosen but they were not sufficiently strong to withstand the forces of weight bearing, resulting in them bending and breaking. Screw breakage may not always be a deleterious situation; in some fractures in human beings, interlocking nail screws are deliberately removed from the construct to allow axial compression a process known as dynamisation (Bucholz and Jones 1991). However, a short 6-0 mm nail used with 2-7 mm screws rather than the 4-7 mm nail would probably have been more suitable for the turkey. Unfortunately, the interlocking nail system is designed for dogs and cats and a short 6-0 mm nail is not available. Further advantages of interlocking nail placement over plate fixation include less soft tissue damage and disruption to the vascular supply and less need for implant removal. Its position in the intramedullary canal is advantageous biomechanically, being the neutral axis of the bone. In conclusion, the use of an interlocking nail was successful in the management of a fractured femur in a turkey with the bird restored to ambulatory function, and able to preen, display and lead a normal life. The use of interlocking nails in large birds with leg fractures requires further investigation.

A diagnostic evaluation of real-time PCR, fluorescent antibody and microscopic agglutination tests in cases of equine leptospiral abortion

REASONS FOR PERFORMING STUDY A comprehensive evaluation of the real-time PCR assay for leptospirosis in comparison with other diagnostic assays on a large-scale basis is fundamental in validating the assay and determining the causes of equine abortions. OBJECTIVES To compare and evaluate the diagnostic value of real-time PCR assay for leptospirosis with traditional methods in equine leptospiral abortions. STUDY DESIGN Cross-sectional observational study. METHODS A Leptospira spp. fluorescent antibody test (FAT), microscopic agglutination test (MAT) and real-time PCR (targeting the LipL32 gene) were compared and evaluated in equine fetal necropsy specimens (placenta, kidney, liver and heart blood) and maternal serum (when available) in 339 equine fetuses. RESULTS From a total of 339 equine fetuses necropsied, 21 cases (6.19%) were diagnosed as leptospiral abortion. The majority of leptospiral abortions occurred in January (8 cases) and February (5 cases). Real-time PCR detected 21 of 21 cases, whereas MAT and FAT detected 19 and 18 (including 2 suspicious cases) cases, respectively. Comparing tissues, placenta yielded somewhat similar cycle of threshold values by real-time PCR compared with kidney, whereas kidney was the best specimen for the diagnosis of leptospirosis by the FAT test. In all MAT positive cases, the predominant titre in fetal heart blood was to serovar Pomona (ranging 1:100 to 1:204,800) with little or no cross-reaction to serovar Grippotyphosa. CONCLUSIONS The results indicate that real-time PCR is an effective method for the diagnosis of leptospiral abortion in horses. However, MAT should continue to be used in clinical cases for serovar determination.

An investigation of a recent outbreak of nocardioform placentitis caused abortions in horses

Nocardioform placentitis associated with gram positive branching actinomycetes caused a record number of abortions in mares diagnosed by the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) affecting the 2011 foal crop (2011 foal crop: the cohort of foals conceived during the 2010 breeding season). The goal of the present study is to make a comprehensive analysis of this outbreak in terms of frequencies of the bacteria causing nocardioform placentitis mediated abortions and to investigate the ages of fetuses, abortion months and breeding times. In the present study, characteristic slow-growing, pungent/soil odor gram positive branching actinomycetes were recovered in high numbers in placental specimens in 76 abortion cases diagnosed as nocardioform placentitis by pathologists. To determine the type of actinomycetes responsible for the abortions, PCR assays were performed on the gram positive branching bacilli. The most prominent actinomycetes species were Amycolatopsis spp. (37 cases, 48.7%) and Crossiella equi (C. equi) (22 cases, 28.9%). Six cases (7.9%) contained both Amycolatopsis spp., and C. equi. 10 isolates were unidentified by PCR assays and shown to have high DNA sequence homology to Streptomyces species, Microbacterium species, Nocardia species and Allokutzneria species, as evidenced by 16 rRNA gene sequencing and phylogenetic analysis. Nocardioform placentitis related abortions occurred mostly between December 2010 and April 2011 happening exclusively in the last trimester. Breeding time of aborted pregnancies ranged from March 2010 to July 2010, suggesting that if transmission of the actinomycetes agents occurred during breeding, it was not related to a specific season.

The influence of temperature and simulated transport conditions of diagnostic samples on real-time polymerase chain reaction for the detection of Tritrichomonas foetus DNA.

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.

Evaluation of 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

OBJECTIVE 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. DESIGN In vitro experimental study. SAMPLE 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). PROCEDURES 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. RESULTS 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. CONCLUSIONS AND CLINICAL RELEVANCE 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.