Feasibility of quantitative environmental surveillance in poliovirus eradication strategies

The progress of the Global Polio Eradication Initiative is monitored by acute flaccid paralysis (AFP) surveillance supplemented with environmental surveillance in selected areas. To assess the sensitivity of environmental surveillance, stools from (re)vaccinated elderly persons with a low seroprevalence and from wastewater were concurrently collected and analyzed in the Netherlands over a prolonged period of time. A total number of 228 healthy individuals with different levels of immunity were challenged with monovalent oral polio vaccine serotype 1 or 3. Poliovirus concentrations were determined by the titration of fecal suspensions on poliovirus-sensitive L20B cells and of sewage concentrates by L20B monolayer plaque assay. Almost half of the individuals (45%) shed poliovirus on day 3 after challenge, which peaked (57%) on day 8 with an average poliovirus excretion of 1.3 × 10(5) TCID(50) per g of feces and gradually decreased to less than 5% on day 42. The virus concentrations in sewage peaked on days 6 to 8 at approximately 100 PFU per liter, remained high until day 14, and subsequently decreased to less than 10 PFU per liter on day 29. The estimated poliovirus concentration in sewage approximated the measured initial virus excretion in feces, within 1 log(10) variation, resulting in a sensitivity of detection of 100 infected but mostly asymptomatic individuals in tens of thousands of individuals. An additional second peak observed in sewage may indicate secondary transmission missed by enterovirus or AFP surveillance in patients. This enables the detection of circulating poliovirus by environmental surveillance, supporting its feasibility as an early warning system.     

Low ambient temperature increases food intake and dropping production, leading to incorrect estimates of hormone metabolite concentrations in European stonechats

Non-invasive methods to measure steroid hormone metabolites in bird droppings or mammalian feces have become very popular. However, the accuracy of these measurements may be affected by many factors. Here, we use the stonechat (Saxicola torquata) as a passerine bird model to test whether differences in ambient temperature affect food intake and dropping production and whether these changes lead to measurement artefacts in hormone metabolite concentrations. In addition, we tested for diurnal patterns in hormone metabolites. We held European stonechats in climate chambers and subjected them to two different long-term ambient temperature regimes, +5 degrees C and +22 degrees C. As expected, food intake and dropping production was higher at +5 degrees C than at +22 degrees C. Plasma concentrations of corticosterone and testosterone did not differ between different ambient temperature regimes. However, corticosterone and testosterone metabolite concentrations (in ng/g) were significantly lower at +5 degrees C than at +22 degrees C. When we measured the rate of hormone metabolite excretion (in picogram per hour) instead of the concentration, there was no difference between treatment groups. Thus, the measurement of hormone metabolite concentrations can be flawed because, depending on the treatment, similar amounts of hormone metabolites can be excreted into very different amounts of droppings. In conclusion, hormone metabolite concentration measurements are sensitive to changes in ambient temperature and probably any other factor that alters metabolic rates. Any study involving systematic changes in metabolism--i.e., during molt, migration, hibernation, egg production, or seasonal comparisons--needs to take these caveats into account.     

Oxygen poisoning in Drosophila

Fruit flies live longer at the partial pressure of oxygen found in air than at either larger or smaller partial pressures. Flies exposed to 1 atm of oxygen for 8 hr every day do not recover completely in the remaining 16 hr. In general, intermittent exposures to 1 atm of oxygen are better tolerated than continuous exposure to the same average oxygen concentration per day, but exposures to higher pressures of 2–5 atm of oxygen for as little as a half hour every two days markedly shorten the life-span. Older flies consume more oxygen per minute and are more sensitive to oxygen poisoning than young flies, and the rate of dying in 6 atm of O₂, or the reciprocal of the survival time, is a linear function of the age. The oxygen pressure-time curve can be well expressed by the general empirical equation (P_OO2)² x time = 120 where P is in atmosphere and survival time in hours. The progress of oxygen poisoning appears to be linear with time rather than exponential.     

Partition of PGE between renal venous plasma and urine during renal ischemia

Radioimmunoassay was used to study the effects of renal ischemia on the distribution of PGE-like material between renal venous plasma and urine in anesthetized dogs. Renal venous and urinary concentrations of these substances were equal during control, ischemia and recovery periods. This relationship obtained despite significant increases in the concentration of PGE of both compartments during the ischemic insult. The renal secretion rates of PGE, calculated as the product of renal plasma flow and renal venous concentrations, was reduced during ischemia while urinary excretion, calculated as the product of urine flow and concentration, was unchanged. The evidence suggests that the increased PGE concentrations observed in both compartments during renal ischemia are primarily due to a dilutional factor rather than an increased synthesis. Furthermore, the data suggest that the net secretion of renal PG's per unit time may, in fact, be reduced during renal ischemia.     

Distribution of Bacteria in Feces of Swine

A new technique is described for evaluating bacterial cell distribution in fecal samples. Spatial relationships of cells within an area rather than number of cells per unit volume or weight are measured by this technique. Measurements of cell distribution by this method indicated that bacteria occurred in freshly voided swine feces as pure, discrete colonies rather than as single cells distributed randomly or uniformly throughout the sample.     

Distribution of Escherichia coli O157 in bovine fecal pats and its impact on estimates of the prevalence of fecal shedding

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g(-1) between samples from the same fecal pat. The density in most positive samples was <100 CFU g(-1), the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.     

Reliability of Polyethyleneglycol as an Indicator for Digestion Studies with Swine

A new method for determining a small amount of polyethyleneglycol (mol. wt. about 4,000) in feces or ingesta was described. The rates of flow and excretion along the swine digestive tract were compared between polyethyleneglycol and chromic oxide used as an indicator for digestion studies.

The former was removed from stomach more rapidly than the latter, and would be delayed in its passage along large intestine owing to absorption of water therein. There was a general agreement between the excretion variations of both indicators. Recoveries during feces collection periods were lower for polyethyleneglycol than for chromic oxide. However, there was no essential difference between both indicators in retention time in the digestive tract, calculated following the idea of Castle.     

Partition of PGE between renal venous plasma and urine during renal ischemia.

Radioimmunoassay was used to study the effects of renal ischemia on the distribution of PGE-like material between renal venous plasma and urine in anesthetized dogs. Renal venous and urinary concentrations of these substances were equal during control, ischemia and recovery periods. This relationship obtained despite significant increases in the concentration of PGE of both compartments during the ischemic insult. The renal secretion rates of PGE, calculated as the product of renal plasma flow and renal venous concentrations, was reduced during ischemia while urinary excretion, was unchanged. The evidence suggests that the increased PGE concentrations observed in both compartments during renal ischemia are primarily due to a dilutional factor rather than an increased synthesis. Furthermore, the data suggest that the net secretion of renal PG's per unit time may, in fact, be reduced during renal ischemia.     

Vectorial entry and release of hepatitis A virus in polarized human hepatocytes

Hepatitis A virus (HAV) is an enterically transmitted virus that replicates predominantly in hepatocytes within the liver before excretion via bile through feces. Hepatocytes are polarized epithelial cells, and it has been assumed that the virus load in bile results from direct export of HAV via the apical domain of polarized hepatocytes. We have developed a subclone of hepatocyte-derived HepG2 cells (clone N6) that maintains functional characteristics of polarized hepatocytes but displays morphology typical of columnar epithelial cells, rather than the complex morphology that is typical of hepatocytes. N6 cells form microcolonies of polarized cells when grown on glass and confluent monolayers of polarized cells on semipermeable membranes. When N6 microcolonies were exposed to HAV, infection was restricted to peripheral cells of polarized colonies, whereas all cells could be infected in colonies of nonpolarized HepG2 cells (clone C11) or following disruption of tight junctions in N6 colonies with EGTA. This suggests that viral entry occurs predominantly via the basolateral plasma membrane, consistent with uptake of virus from the bloodstream after enteric exposure, as expected. Viral export was also found to be markedly vectorial in N6 but not C11 cells. However, rather than being exported from the apical domain as expected, more than 95% of HAV was exported via the basolateral domain of N6 cells, suggesting that virus is first excreted from infected hepatocytes into the bloodstream rather than to the biliary tree. Enteric excretion of HAV may therefore rely on reuptake and transcytosis of progeny HAV across hepatocytes into the bile. These studies provide the first example of the interactions between viruses and polarized hepatocytes.     

A specialist herbivore (Neotoma stephensi) absorbs fewer plant toxins than does a generalist (Neotoma albigula)

Detoxification capacity of enzymes in the liver is thought to be the primary factor governing dietary toxin intake by mammalian herbivores. Recently, toxin absorption in the gut was proposed as an alternative process that also influences toxin intake. We examined the role of the gut in regulating toxin absorption by quantifying excretion of a plant secondary compound in the feces. We hypothesized that specialists have a greater capacity to reduce intestinal absorption of toxins than do generalists. To test this hypothesis, we compared fecal excretion of alpha-pinene in specialist (Neotoma stephensi) and generalist (Neotoma albigula) woodrats. Alpha-pinene is the most abundant monoterpene in Juniperus monosperma, which occurs in the natural diet of both woodrat species. Woodrats were fed alpha-pinene in diets containing juniper foliage for 3 wk and, in a separate experiment, were given a single oral dose of alpha-pinene. Feces were collected from animals at the end of each experiment and analyzed for alpha-pinene concentration using gas chromatography. Both woodrat species excreted unchanged alpha-pinene in the feces. However, specialist woodrats excreted 40% more alpha-pinene per unit ingested from a juniper diet and excreted nearly four times a greater percentage of an oral dose of alpha-pinene compared with generalists.     

Excretion of multiple urinary biomarkers for radical induced damage in rats treated with three different nephrotoxic compounds

In the present study the urinary excretion of seven aldehydes, acetone and coproporphyrin III as non-invasive in vivo biomarkers of free radical damage was measured in rats after treatment with three nephrotoxic compounds: cisplatin, mercuric chloride (HgCl2) and N -acetyl- S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-Nac). A clear difference between the different nephrotoxic compounds was found in the time interval between dosage and maximal toxicity, as measured by clinical chemical parameters in urine. In rats treated with TFE-Nac and HgCl2 this was fast: 12 h and 24 h after treatment, respectively. In the rats treated with cisplatin, however, nephrotoxicity occurred later: 96 h-108 h after treatment. Urinary creatinine excretion was decreased in all treatments. Therefore, the excretion of the proposed biomarkers was expressed as amount excreted per 12 h urine fraction as well as amount excreted per mol creatinine in each 12 h urine fraction. Urinary excretion of coproporphyrin III was decreased in almost all 12 h urine fractions with all treatments, however, when expressed per mol creatinine, increases were found in urine of rats treated with cisplatin and HgCl2. In cisplatin-treated rats an increase was found in the excretion of formaldehyde per 12 h, but acetaldehyde, propanal and MDA levels were decreased. Expressed per mol creatinine, MDA levels were decreased, but other aldehydes were increased. In HgCl2-treated rats urinary aldehyde excretion expressed per mol creatinine was increased. In TFE-Nac treated animals the urinary levels of acetaldehyde per 12 h were increased and per mol creatinine the levels of some aldehydes were only slightly increased. With none of the treatments did the increase in the biomarkers expressed per mol creatinine exceed the decrease in creatinine excretion. Similar time intervals were found between dosage and maximal excretion of biomarkers as for the time intervals between dosage and maximal toxicity. With all treatments significant increases in the excretion of acetone were found both per 12 h and per mol creatinine, probably related to the increased glucose excretion. It was concluded that no convincing evidence for free radical damage was found in the present study with the employed biomarkers.     

Excretion of multiple urinary biomarkers for radical induced damage in rats treated with three different nephrotoxic compounds

In the present study the urinary excretion of seven aldehydes, acetone and coproporphyrin III as non-invasive in vivo biomarkers of free radical damage was measured in rats after treatment with three nephrotoxic compounds: cisplatin, mercuric chloride (HgCl2) and N -acetyl- S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-Nac). A clear difference between the different nephrotoxic compounds was found in the time interval between dosage and maximal toxicity, as measured by clinical chemical parameters in urine. In rats treated with TFE-Nac and HgCl2 this was fast: 12 h and 24 h after treatment, respectively. In the rats treated with cisplatin, however, nephrotoxicity occurred later: 96 h-108 h after treatment. Urinary creatinine excretion was decreased in all treatments. Therefore, the excretion of the proposed biomarkers was expressed as amount excreted per 12 h urine fraction as well as amount excreted per mol creatinine in each 12 h urine fraction. Urinary excretion of coproporphyrin III was decreased in almost all 12 h urine fractions with all treatments, however, when expressed per mol creatinine, increases were found in urine of rats treated with cisplatin and HgCl2. In cisplatin-treated rats an increase was found in the excretion of formaldehyde per 12 h, but acetaldehyde, propanal and MDA levels were decreased. Expressed per mol creatinine, MDA levels were decreased, but other aldehydes were increased. In HgCl2-treated rats urinary aldehyde excretion expressed per mol creatinine was increased. In TFE-Nac treated animals the urinary levels of acetaldehyde per 12 h were increased and per mol creatinine the levels of some aldehydes were only slightly increased. With none of the treatments did the increase in the biomarkers expressed per mol creatinine exceed the decrease in creatinine excretion. Similar time intervals were found between dosage and maximal excretion of biomarkers as for the time intervals between dosage and maximal toxicity. With all treatments significant increases in the excretion of acetone were found both per 12 h and per mol creatinine, probably related to the increased glucose excretion. It was concluded that no convincing evidence for free radical damage was found in the present study with the employed biomarkers.     

The effect of a synthetic steroid (trienbolone) on the rate of release and excretion of subcutaneously administered estradiol in calves (18).

With the objective of obtaining values for the rate of release and excretion of subcutaneously implanted estradiol and to relate them to the metabolic effect of the hormone, a study was carried out with young Jersey bull calves. After subcutaneous administration of lactose tablets (implants) containing tritiated estradiol (4 mCi in 20 mg estradiol) the activity was followed in plasma, urine and feces for 107 days. Three calves received implants containing 140 mg trienbolone in addition to the 20 mg estradiol. In the first group maximum plasma concentration of estradiol-17 beta was 3 nmol/1. In the other group it was only 0.33 nmol/1. In calves receiving estradiol as the only steroid, 95% of the activity was excreted within 20 days after implantation. In the other group collection of urine and feces had to be carried out for 107 days in order to account for all the implanted activity. No 3H could be detected in urine and feces samples collected from the estradiol group more than 31 days ater implantation. The feces and urine samples collected from calves in the estradiol-trienbolone group 107 days after implantation contained from 1.4 - 3 nCi per gram. The remarkably decreasing effect of trienbolone on the release of estradiol and its possible importance for the effect of subcutaneously administered estradiol are discussed.     

Comparative excretion and tissue distribution of selenium in mice and rats following treatment with the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate

In a previous preliminary investigation, we reported on the excretion, tissue disposition and metabolism of the chemopreventive agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) in the rat, but similar studies in the mouse have not been explored. Following the oral administration of p-XSC (50 micromol/kg body weight), selenium excretion in feces was comparable to that in urine in mice, but in rats, feces was the major route of excretion. Tetraselenocyclophane (TSC) was the major metabolite detected in mouse and rat feces. In both species, levels of selenium in exhaled air were negligible. At termination, in the mouse, the stomach had the highest selenium content followed by liver and blood, but lung and kidney contained negligible levels of selenium; in the rat, the selenium level in liver was the highest followed by kidney, stomach, blood and lung. The identification of TSC as a fecal metabolite in both species let us to postulate the following metabolic pathway: p-XSC-->glutathione conjugate (p-XSeSG)-->a selenol (p-XSeH)-->TSC. Since the glutathione conjugate appears to be the proximal precursor for the selenol metabolite that may be an important intermediate in cancer chemoprevention, we report for the first time the synthesis of p-XSeSG and its other potential metabolites, namely the cysteine- and N-acetylcysteine-conjugates of p-XSC. HPLC analysis of the urine and bile showed a few metabolites of p-XSC; none of which eluted with the synthetic standards described above. When we examined the conversion of p-XSC and p-XSeSG in vitro using rat cecal microflora, TSC was formed from p-XSeSG but not from p-XSC. The formation of TSC from p-XSC in vivo but not in vitro suggests that p-XSC needs to be metabolized to p-XSeSG or an intermediate derived from its further metabolism. Thus, p-XSeSG was given orally to rats and the results showed that the pattern of selenium excretion after p-XSeSG treatment was similar to that of p-XSC; TSC was also identified as a fecal metabolite of p-XSeSG. It may be that the conversion of p-XSeSG to TSC is too facile, or the mere conjugation of p-XSC with glutathione does not occur in rats and mice.     

Distribution of Escherichia coli O157 in Bovine Fecal Pats and Its Impact on Estimates of the Prevalence of Fecal Shedding

The distribution of Escherichia coli O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of E. coli O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of E. coli O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g⁻¹ between samples from the same fecal pat. The density in most positive samples was <100 CFU g⁻¹, the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of E. coli O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of E. coli O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.