Rumen microbial sequestration of [2-(13)C]acetate in cattle

To investigate the impact of rumen microbial sequestration of VFA carbon on estimates of acetate availability based on intraruminal infusion of [2-(13)C] acetate, three nonlactating or low-yielding dairy cows were continuously intraruminally infused with [2-(13)C]acetate for 26 h. The 13C content of ruminal VFA, duodenal carbon, and fatty acids (FA) and AA isolated from liquid-associated ruminal microbes and duodenal DM was measured by an isotope ratio mass spectrometer interfaced to an elemental analyzer or a gas-liquid chromatograph. The ruminal gross production of acetate was 38 +/- 4 mol/d and could account for about 38% of the DE intake. Of the intraruminally infused 13C in [2-(13)C]acetate, 7.6 +/- 0.9% was recovered at the duodenum. The 13C content of ruminal propionate, butyrate, and valerate increased (P < 0.05) with intraruminal infusion of [2-(13)C]acetate. It was estimated that about 28% of the 13C intraruminally infused in [2-(13)C]acetate could be accounted for by duodenal 13C flow and absorption of non-acetate VFA. A number of FA isolated from liquid-associated ruminal microbes (C6, C12, C14, anteiso C15, and iso C15) were enriched with 13C (P < 0.05) at a level comparable to the enrichment of ruminal butyrate. Any absorption of these FA from the rumen would further contribute to non-acetate 13C uptake. A maximum of 72% of the ruminal gross production of acetate represented acetate absorption from the rumen in the present study. Consequently, previously used models using intraruminal isotope dilution techniques seem not to be appropriate for measuring acetate availability in ruminants. The number of metabolites exchanging carbon with acetate was found to be so high that assessments of the entire range of inter conversions seem to be practically impossible. Portal absorption studies are discussed as an alternative method of estimating VFA availability to the metabolism in ruminants.     

CONTRAST-ENHANCED ULTRASONOGRAPHY IN NORMAL CANINE LIVER. EVALUATION OF IMAGING AND SAFETY PARAMETERS

Contrast-enhanced ultrasonography, a new imaging modality in veterinary medicine, can provide data on tissue perfusion. The objective of this study was to use the ultrasonographic contrast agent SonoVue to evaluate various transit time indices in the normal canine liver, to examine the effect of anesthesia on these parameters, and to evaluate the safety of this agent in dogs. The liver of 11 healthy dogs was studied by ultrasound during an intravenous bolus injection of SonoVue. Each dog underwent the examination twice, first with and later without the use of anesthesia. A time-intensity curve was generated from a selected region of interest within the liver from each scanning session. Ratios derived from peak enhancement, time to peak enhancement, up-slope and full-width half-maximum (FWHM) of the curve were calculated from the time-intensity curves, and are reported. There were no statistically significant differences (P > 0.05) in peak enhancement, up-slope and FWHM between dogs that were anesthetized and dogs that were not. Time to peak enhancement, however, was significantly shorter when the dogs were anesthetized than when they were nonanesthetized (P < 0.05). There were no biologically significant changes in clinical laboratory findings. This study indicates that contrast-enhanced ultrasound using SonoVue gives reproducible liver perfusion data, and appears to be a safe and well-tolerated agent for use in dogs. When considering normal values, the use of anesthetic drugs has to be considered.     

Decreased platelet function in Cavalier King Charles Spaniels with mitral valve regurgitation

With aggregometry, increased platelet activity has been reported in Cavalier King Charles Spaniels (CKCS) without mitral regurgitation (MR). In contrast, dogs with MR have been found to have decreased platelet activity. The purpose of this study was to test an easy bedside test of platelet function (the Platelet Function Analyzer [PFA-100]) to see if it could detect an increase in platelet activity in CKCS without MR and a decrease in platelet activity in CKCS with MR. This study included 101 clinically healthy dogs > 1 year of age: 15 control dogs of different breeds and 86 CKCS. None of the dogs received medication or had a history of bleeding. The PFA-100 evaluates platelet function in anticoagulated whole blood under high shear stress. Results are given as closure times (CT): the time it takes before a platelet plug occludes a hole in a membrane coated by agonists. The CT with collagen and adenosine-diphosphate as agonists was similar in control dogs (median 62 seconds; interquartile interval 55-66 seconds) and CKCS with no or minimal MR (55; 52-64 seconds). The CT was higher in CKCS with mild MR (regurgitant jet occupying 15-50% of the left atrial area) (75; 60-84 seconds; P = .0007) and in CKCS with moderate to severe MR (jet > 50%) (87: 66-102 seconds; P < .0001). CKCS with mild, moderate, and severe, clinically inapparent MR have decreased platelet function. The previous finding of increased platelet reactivity in nonthrombocytopenic CKCS without MR could not be reproduced with the PFA-100 device.     

16alpha-Bromo-epiandrosterone therapy modulates experimental feline immunodeficiency virus viremia: initial enhancement leading to long-term suppression

16alpha-Bromo-epiandrosterone (epiBr), a synthetic derivative of the natural hormone dehyroepiandrosterone (DHEA), was evaluated for its effects on feline immunodeficiency virus (FIV) infection in experimental cats. The rationale for this study was based on the ability of DHEA to significantly reduce the mortality to viral infections in mice. DHEA and epiBr also have demonstrable in vitro anti-viral activity for both HIV-1 and FIV. Preliminary pharmacokinetic studies in cats demonstrated that subcutaneously injected epiBr was rapidly absorbed, completely metabolized, and nontoxic. Metabolites were excreted in both urine and feces, with the latter having the most complex pattern of breakdown products. Cats were then divided into four groups; two groups were infected with FIV and two uninfected. Two groups, one infected and one uninfected were treated on 5 consecutive days of weeks 0, 4, 8, 12 and 16 with epiBr. The remaining two groups were mock treated with the drug vehicle alone. Treatment started 1 week prior to infection and extended for 4 weeks after infection. Cats were observed for 20 weeks post-FIV infection. Infected cats had identical decreases in blood neutrophil and lymphocyte counts following, regardless of whether they were treated with epiBr or vehicle alone. The CD4/CD8 T-cell ratio was decreased following FIV exposure, but was significantly more decreased for the epiBr treated animals from week 2 post-infection onward. CD4+ T cells were decreased in FIV-infected cats treated with epiBr compared to their untreated cohort, while CD8+ T cells tended to be higher in treated animals. FIV infected cats that were treated with epiBr had over one-log higher virus loads at week 2 post-infection than non-epiBr treated cohorts. In spite of this enhanced initial viremia, the subsequent levels of virus in the blood were significantly lower in epiBr treated versus untreated animals. EpiBr treated cats had significantly higher FIV-p24 antibody responses than control cats receiving vehicle alone, although primary and secondary antibody responses to a T-cell dependent non-FIV antigen, keyhole limpet hemocyanin (KLH), were unaffected. EpiBr treatment significantly decreased the expected FIV-induced suppression of IL-12 p40 mRNA levels in peripheral blood mononuclear cells (PBMCs) observed at weeks 4, 5, 8, 9 and 16 post-infection, but had no influence on FIV-induced changes in IL-4, IL-6, IL-10, IFN-gamma, MIP-1alpha and RANTES.     

A Bayesian approach to the accuracy of clinical observations

The accuracy of clinical observations was estimated using Bayesian latent-class models with two or more independent tests. Four veterinarians carried out systematic independent clinical examinations on 155 pigs in three herds. Based on the results of binary recordings of clinical observations on dullness, poor body condition (PBC), skin lesions, lameness, respiratory disease, and diarrhea, a latent disease state for each clinical disease was estimated using Gibbs sampling.

The accuracy of the clinical observations differed for the four observers and for different clinical signs. Population parameters were estimated from a Bayesian hierarchical model, and the accuracy of a random observer was calculated. We concluded that the accuracy of the veterinarians in this study substantiated the need to pursue more-precise definitions of the clinical findings and that larger sample sizes would be needed to provide reasonable variance estimates. Finally, we concluded that the uncertainty in the clinical decision-making process (starting with the clinical examination) needs to be represented fully.     

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class I) were cloned and sequenced for two haplotypes (H4 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs. The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered single-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA-I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J. Gen. Virol. 76 (1995) 3039). Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide-occupied forms of SLA-I. It remains to be seen whether the rapid and technically very simple in vitro refold assay described here will prove generally applicable for the screening of virus-derived peptides for SLA-I binding.