Validation of a multiplex-tandem RT-PCR for the detection of bovine respiratory disease complex using Scottish bovine lung samples.

The welfare and economic impact of bovine respiratory disease complex (BRDC), and its associated antibiotic usage, are major challenges to cattle rearing and beef cattle finishing industries. Accurate pathogen diagnosis is important to undertake appropriate treatment and long-term management strategies, such as vaccine selection. Conventional diagnostic approaches have several limitations including high costs, long turnaround times and difficulty in test interpretation, which could delay treatment decisions and lead to unnecessary animal losses. We describe the validation of a multiplex-tandem (MT) reverse transcription-polymerase chain reaction (RT-PCR) for the detection of seven common pathogens associated with BRDC. This test has the potential to advance pathogen identification and to overcome many of the limitations of current testing methods. It requires a single sample and results are obtained quickly and not influenced by prior antimicrobial therapy or overgrowth of contaminating organisms. We demonstrated a test specificity of 100% and sensitivity ranging from 93.5% to 100% for these seven common pathogens. This test will be a useful addition to advance BRDC investigation and diagnosis.

Identification of oxidant-sensitive proteins: TNF-alpha induces protein glutathiolation.

Reactive oxygen species are thought to play a role in a variety of physiologic and pathophysiological processes. One possible mediator of oxidant effects at the molecular level is a subset of proteins containing reactive cysteine thiols that can be readily oxidized. The transient incorporation of glutathione into cellular proteins is an established response to oxidant stress and could provide a mechanism for reversible covalent modification in response to reactive oxygen species. To better understand the function of protein S-glutathiolation in vivo, a biotinylated membrane-permeant analogue of glutathione, biotinylated glutathione ethyl ester, was developed and used to detect proteins into which glutathione is incorporated under oxidant stress. Oxidant stress from exogenous hydrogen peroxide or generated in response to TNF-alpha was found to increase incorporation of biotinylated glutathione ethyl ester into several HeLa cell proteins. The identity of two of these proteins was determined by peptide sequencing and mass spectrometric peptide mapping. A 23 kDa S-glutathiolated protein was identified as thioredoxin peroxidase II, a member of the peroxiredoxin family of peroxidases known to play a role in redox-dependent growth factor and cytokine signal transduction. A second, 36 kDa, protein was identified as annexin II. Further investigation revealed a single reactive cysteine in the annexin II tail domain. Deletion of the identified cysteine was found to abolish S-glutathiolation of annexin II. These findings demonstrate a specific posttranslational modification associated with an endogenously generated oxidant stress and suggest a mechanism by which TNF-alpha might selectively regulate protein function in a redox-dependent fashion.

Amniotic fluid composition responds to changes in maternal dietary carbohydrate and is related to metabolic status in term fetal rats.

The objective of this study was twofold: 1) to determine whether amniotic fluid composition responded to differences in the level or source (glucose vs. fructose) of maternal dietary carbohydrate, and 2) to establish whether any dietary-induced changes in amniotic fluid composition correlated with maternal or fetal metabolic status at term. Pregnant rat dams were fed graded levels (0, 4, 12 and 60%) of glucose or fructose in a triglyceride-based diet (Experiment 1) or isoenergetic low carbohydrate diets having 4% glucose equivalents as glucose, fructose, or lipid-glycerol (Experiment 2) throughout pregnancy. Amniotic fluid and maternal and fetal samples were collected at term (d21). Results demonstrated a significant increase in amniotic fluid glucose and a significant decrease in amniotic fluid uric acid as the level of carbohydrate increased in the maternal diet. Pearson correlation coefficients showed amniotic fluid glucose to be positively associated with maternal and fetal liver glycogen and fetal weight; amniotic fluid uric acid and urea nitrogen were negatively correlated with these same measures. Regression analysis indicated that amniotic fluid glucose was predictive of fetal body weight and fetal liver glycogen at term. The findings show that amniotic fluid can be modified by maternal diet and suggest that composition of amniotic fluid might be used as an accessible nutritional indicator of carbohydrate status in the developing fetus.

Comparison of effects of dietary glucose versus fructose during pregnancy on fetal growth and development in rats.

Dietary carbohydrate during pregnancy is essential. Whether this requirement is specific to glucose was investigated. We examined whether fructose at low, intermediate and high levels can substitute for an isoenergetic amount of glucose by feeding graded levels of both carbohydrates (0, 4, 12, 60%) in a triglyceride-based diet throughout pregnancy. It was concluded that the carbohydrate requirement for the rat during pregnancy is not specific to glucose and that the level, not the type, of carbohydrate was critical (experiment 1). A second aspect of the study (experiment 2) was the comparison of isoenergetic, low carbohydrate diets containing different sources of 4% glucose equivalents: glucose, fructose or lipid-glycerol. Fructose and lipid-glycerol were not equivalent substitutes for glucose in the pregnant rat dam at these very low intakes.

AIDS in Colombia.

Between January 1984 and December 1987 a total of 178 AIDS cases were reported to the Colombian Ministry of Health. The location of these cases suggests that the human immunodeficiency virus (HIV) is widely distributed in Colombia. Most of those afflicted (97%) have been adult males. HIV seroprevalence studies of selected population groups revealed the highest antibody prevalence (5.65% in females, 22.5% in males) among individuals involved in high-risk behaviors who participated in a free AIDS testing program. High prevalences (from 0.6% to 3.9% in females, and 14.6% to 15.9% in males) were also found in patients (primarily female prostitutes and male homosexuals) attending clinics for sexually transmitted diseases in several urban areas. The number of AIDS cases in Colombia has doubled or tripled annually since reporting began in 1984, a pattern similar to that observed worldwide.

Measurement of factor VIIIRAg-1. A method for use in the Hyland laser nephelometer.

Factor VIIIRAg was measured by laser nephelometry using a new diluent containing a citrate-carbonate buffer, pluronic polyol P94 and triton X100. No pre-treatment of the samples was required, the incubation time was 0.5 to 1.5 hours depending on the antiserum used, and the tests could be read during the following 4 hours (plasma) or 1.5 hours (intermediate purity F VIII). The CV for the assay was less than 10% for plasma or dilutions of plasma in which the minimum detectable amount of factor VIIIRAg was about 0.04 u/ml. A significant correlation was obtained between the nephelometric method and the Laurell rocket method using plasma, but agreement between the methods when therapeutic products were assayed was poor.

Prediction of the outcome of rhesus haemolytic disease of the newborn: additional information using an ADCC assay.

An in vitro test system is described which measures the ability of anti-D sera to lyse Rh(D)-positive red cells. This test was applied to anti-D sera from 11 cases of HDN selected in Glasgow and tested 'blind' in Edinburgh. Evidence is presented to support the view that the ADCC (antibody-dependent cell-mediated cytotoxicity ) assay can correctly identify those cases where there is a satisfactory clinical outcome despite a high level of anti-D suggesting otherwise. Amniocentesis might therefore be avoided in this group.