Characterizing pharmaceutical, personal care product, and hormone contamination in a karst aquifer of southwestern Illinois, USA, using water quality and stream flow parameters.

Karst aquifers are drinking water sources for 25% of the global population. However, the unique geology of karst areas facilitates rapid transfer of surficial chemicals to groundwater, potentially contaminating drinking water. Contamination of karst aquifers by nitrate, chloride, and bacteria have been previously observed, but little knowledge is available on the presence of contaminants of emerging concern (CECs), such as pharmaceuticals. Over a 17-month period, 58 water samples were collected from 13 sites in the Salem Plateau, a karst region in southwestern Illinois, United States. Water was analyzed for 12 pharmaceutical and personal care products (PPCPs), 7 natural and synthetic hormones, and 49 typical water quality parameters (e.g., nutrients and bacteria). Hormones were detected in only 23% of samples, with concentrations of 2.2-9.1ng/L. In contrast, PPCPs were quantified in 89% of groundwater samples. The two most commonly detected PPCPs were the antimicrobial triclocarban, in 81% of samples, and the cardiovascular drug gemfibrozil, in 57%. Analytical results were combined with data of local stream flow, weather, and land use to 1) characterize the extent of aquifer contamination by CECs, 2) cluster sites with similar PPCP contamination profiles, and 3) develop models to describe PPCP contamination. Median detection in karst groundwater was 3 PPCPs at a summed concentration of 4.6ng/L. Sites clustered into 3 subsets with unique contamination models. PPCP contamination in Cluster I sites was related to stream height, manganese, boron, and heterotrophic bacteria. Cluster II sites were characterized by groundwater temperature, specific conductivity, sodium, and calcium. Cluster III sites were characterized by dissolved oxygen and barium. Across all sites, no single or small set of water quality factors was significantly predictive of PPCP contamination, although gemfibrozil concentrations were strongly related to the sum of PPCPs in karst groundwater.

Elemental analysis of a single-wall carbon nanotube candidate reference material.

A material containing single-wall carbon nanotubes (SWCNTs) with other carbon species, catalyst residues, and trace element contaminants has been prepared by the National Institute of Standards and Technology for characterization and distribution as Standard Reference Material SRM 2483 Carbon Nanotube Soot. Neutron activation analysis (NAA) and inductively coupled plasma mass spectrometry (ICP-MS) were selected to characterize the elemental composition. Catalyst residues at percentage mass fraction level were determined with independent NAA procedures and a number of trace elements, including selected rare earth elements, were determined with NAA and ICP-MS procedures. The results of the investigated materials agreed well among the NAA and ICP-MS procedures and good agreement of measured values with certified values was found in selected SRMs included in the analyses. Based on this work mass fraction values for catalyst and trace elements were assigned to the candidate SRM.

Estimating background and threshold nitrate concentrations using probability graphs.

Because of the ubiquitous nature of anthropogenic nitrate (NO3(-)) in many parts of the world, determining background concentrations of NO3(-) in shallow ground water from natural sources is probably impossible in most environments. Present-day background must now include diffuse sources of NO3(-) such as disruption of soils and oxidation of organic matter, and atmospheric inputs from products of combustion and evaporation of ammonia from fertilizer and livestock waste. Anomalies can be defined as NO3(-) derived from nitrogen (N) inputs to the environment from anthropogenic activities, including synthetic fertilizers, livestock waste, and septic effluent. Cumulative probability graphs were used to identify threshold concentrations separating background and anomalous NO(3)-N concentrations and to assist in the determination of sources of N contamination for 232 spring water samples and 200 well water samples from karst aquifers. Thresholds were 0.4, 2.5, and 6.7 mg/L for spring water samples, and 0.1, 2.1, and 17 mg/L for well water samples. The 0.4 and 0.1 mg/L values are assumed to represent thresholds for present-day precipitation. Thresholds at 2.5 and 2.1 mg/L are interpreted to represent present-day background concentrations of NO(3)-N. The population of spring water samples with concentrations between 2.5 and 6.7 mg/L represents an amalgam of all sources of NO3(-) in the ground water basins that feed each spring; concentrations > 6.7 mg/L were typically samples collected soon after springtime application of synthetic fertilizer. The 17 mg/L threshold (adjusted to 15 mg/L) for well water samples is interpreted as the level above which livestock wastes dominate the N sources.

The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep.

The pathological findings in sheep with peracute experimental Clostridium perfringens type D enterotoxemia are described. Of 16 animals inoculated intraduodenally with a whole culture of this microorganism and a starch solution in the abomasum, 12 developed clinical signs including increased respiratory efforts, recumbency, paddling, bleating, convulsions, blindness, and opisthotonus. Diarrhea was not observed in any of the animals. The time lapse between the beginning of intraduodenal infusion and onset of clinical signs varied between 30 minutes and 26 hours, and the clinical course varied between 1 and 9 hours. Gross postmortem changes were observed in these 12 animals and included pulmonary edema; excess pericardial, peritoneal, or pleural fluid with or without strands of fibrin; liquid small intestinal contents; leptomeningeal edema; cerebellar coning; and subcapsular petechiae on kidneys. Histological changes consisted of severe edema of pleura and interlobular septa and around blood vessels and airways and acidophilic, homogeneous, proteinaceous perivascular edema in the brain. Five of 12 animals (42%) with clinical signs consistent with enterotoxemia lacked specific histological lesions in the brain. None of the intoxicated or control animals developed nephrosis. Glucose was detected in the urine of 3 of 6 animals that were tested for this analyte. These results stress the importance of the use of histological examination of the brain, coupled with epsilon toxin detection, for a definitive diagnosis of C. perfringens type D enterotoxemia in sheep.

Comparison of four techniques for the detection of Clostridium perfringens type D epsilon toxin in intestinal contents and other body fluids of sheep and goats.

Polyclonal capture enzyme-linked immunosorbent assay (PC-ELISA), monoclonal capture ELISA (MC-ELISA), mouse neutralization test (MNT), and counterimmunoelectrophoresis (CIEP), were compared for their ability to detect epsilon toxin in intestinal contents and body fluids of sheep and goats. When used to evaluate intestinal contents of sheep artificially spiked with epsilon prototoxin, PC-ELISA detected 0.075 mouse lethal dose (MLD)50/ml, whereas the MNT, MC-ELISA, and CIEP detected 6, 25, and 50 MLD50/ml, respectively. Amounts of epsilon toxin detected by PC-ELISA, MC-ELISA, MNT, and CIEP in sheep pericardial fluid artificially spiked with epsilon prototoxin were 0.075, 0.75, 6, and 200 MLD50/ml, respectively. For assaying epsilon toxin in aqueous humor, PC-ELISA and MC-ELISA detected 0.075 MLD50/ml, whereas CIEP detected 200 MLD50/ml (MNT was not evaluated). When 51 samples of intestinal contents of sheep and goats (32 positive and 19 negative to MNT) were analyzed by the other 3 techniques, the relative sensitivity of PC-ELISA, MC-ELISA, and CIEP was 93.75, 84.37, and 37.50%, respectively. The specificity of PC-ELISA, MC-ELISA, and CIEP was 31.57, 57.89, and 84.21%, respectively. The absolute sensitivity of PC-ELISA, MC-ELISA, CIEP, and MNT was 90.90, 69.69, 15.15, and 54.54%. The absolute specificity of the 4 techniques was 100%. These results show that there is a marked inconsistency among techniques routinely used to detect Clostridium perfringens epsilon toxin. Until more consistent results are achieved, the diagnosis of enterotoxemia should not only be based solely on epsilon toxin detection, but also on clinical and pathological data.

Assessment of organoleptic postmortem inspection techniques for bovine offal.

OBJECTIVE: To evaluate organoleptic postmortem inspection techniques for bovine livers and kidneys. STUDY DESIGN: At Australian export abattoirs, bovine liver and kidneys are assessed and graded by qualified meat inspectors during normal operations. Over a 12-month period at a large abattoir in eastern Australia during 1997 and 1998, a sample of these organs was reassessed independently using a range of pathological and microbiological methods. Agreement between routine inspection and independent assessment was evaluated using methods of inter-rater agreement. RESULTS: A total of 944 livers and 1374 kidneys were included in the study. All of these organs had been classified during routine inspection. The authors examined 363 livers and 329 kidneys both grossly and histologically, including 36 livers and 14 kidneys that were also subjected to microbiological examination. All other organs were only examined grossly. There was only a moderate level of agreement between the routine and independent assessment methods. For livers, the percentage agreement was 80.2%, McNemar's test of symmetry 55.2 (3 degrees of freedom, P < 0.001) and kappa 0.63. For kidneys, the percentage agreement was 67.8%, McNemar's test of symmetry 9.9 (1 df, P = 0.002) and kappa 0.35. CONCLUSIONS: The results reinforce concerns from a number of authors about organoleptic postmortem inspection. Risk assessment methodologies offer the opportunity to modify inspection techniques in a manner that is most relevant to current public health concerns.

A study of "white spotted kidneys" in cattle.

A study was performed at an abattoir in Australia, in an attempt to correlate focal chronic interstitial nephritis (FCIN) producing the so-called "white spotted kidney", with Leptospira spp. and other pathogens in cattle. Samples of kidneys, urine and blood were collected immediately after slaughter from 46 two-year-old heifers, and 72 cows and bulls with gross lesions consistent with FCIN. The same samples were also collected from nine heifers and 12 cows with no gross kidney lesions. Aqueous humour was also collected from the eye of 17 of the adult animals. The sera were processed by a microscopic agglutination test for leptospira antibodies, while all the other samples were cultured for Leptospira spp. and also processed for routine aerobic and anaerobic culture for other pathogens. Sub-samples from all the kidneys were fixed in 10% buffered formalin and processed histologically. Antibody titers of 1:400 or higher for Lepstospira borgpeterseni serovar hardjo were found in six adult animals with FCIN and in one adult animal with no gross kidney changes, while antibody titers of 1:400 to L. borgpeterseni serovar tarassovi were found in only one animal with FCIN. L. borgpeterseni serovar hardjo was isolated from the urine and kidney of one adult animal and from the urine of another adult animal, both with FCIN. No pathogens were isolated from any of the other samples. The histological lesions were consistent in most cases with FCIN. The results suggest that neither Leptospira spp. nor active infection by other bacteria are associated with the so-called "white spotted kidneys".

Effects of intravenous injection of Clostridium perfringens type D epsilon toxin in calves.

In cattle, a neurological lesion similar to that produced in sheep and goats by Clostridium perfringens type D enterotoxaemia has been reported. However, no causal relationship has been established between this disease and the lesion in cattle. The effects of single and multiple intravenous injections of epsilon toxin in three calves aged 6 months were studied. A further calf was inoculated intravenously with saline solution and used as a control. Epsilon toxin invariably produced neurological signs within 2-60 min of the end of the injection process. Clinical signs consisted of loss of consciousness, recumbency, convulsions, paddling, opisthotonus, hyperaesthesia and dyspnoea. Gross changes consisted of severe acute pulmonary oedema, which was particularly marked in the interlobular septa. The histological lesions consisted of intra-alveolar and interstitial oedema of the lung and variable degrees of perivascular proteinaceous oedema in the internal capsule, thalamus and cerebellar white matter. No clinical or post-mortem changes were observed in the control calf. These results show that calves are susceptible to the intravenous injection of epsilon toxin, and that they can show at least some of the histological lesions produced in sheep and goats by this toxin.

An alternative method for the certification of the sulfur mass fraction in coal Standard Reference Materials.

The S mass fractions of coal SRMs 2682b, 2684b, and 2685b are certified by direct comparison with coal SRMs 2682a, 2684a, and 2685a, respectively, using high-temperature combustion analysis with infrared (IR) absorption detection. The S mass fractions of the "a" materials used for calibration were previously determined by means of isotope-dilution thermal-ionization mass spectrometry (ID-TIMS). Therefore, the comparisons performed with the combustion-IR absorption method establish direct traceability links to accurate and precise ID-TIMS measurements. The expanded uncertainties associated with the certified S mass fractions are of approximately the same magnitude as would be expected for the ID-TIMS methodology. An important aspect of these certifications is that each "b" material is essentially identical with the corresponding "a" material, because both were produced from the same bulk, homogenized coal. As a test of the efficacy of the new certification approach when calibrant and unknown are not identical, the S mass fraction of coal SRM 2683b has been determined by direct comparison to coal SRM 2683a. These two coals, which have both previously been analyzed with ID-TIMS, are different in terms of S content and other properties. Whereas the S mass fraction for SRM 2683b determined with the new methodology agrees statistically with the ID-TIMS value, there is reason for caution in such cases. In addition to the usefulness of the alternative approach for certification activities within NIST, this approach might also be an excellent way of establishing NIST traceability during the value assignment process for reference materials not issued by NIST. Further research is needed, however, to understand better the scope of applicability.

An apparent outbreak of cutaneous papillomatosis in merino sheep in patagonia, Argentina.

A retrospective study was performed on skin samples from an outbreak of cutaneous papillomatosis in Merino sheep that occurred in 1995. The samples were processed for routine histology, electron microscopy and immunocytochemistry for papilloma viruses. Particles of approximately 55 nm diameter were found in some nuclei of the stratum granulosum cells, while immunocytochemistry gave positive staining of cell nuclei in this layer. This study confirms that papillomas associated with papillomaviruses occur in sheep in Patagonia.

Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro.

Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD50/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD50/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD50/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro.

PCR detection of Clostridium perfringens type D in formalin-fixed, paraffin-embedded tissues of goats and sheep.

A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep. When pure cultures of Cl. perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers). When used to identify Cl. perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl. perfringens. No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique.

Comparison of the effects of Clostridium perfringens type D culture supernates in ligated intestinal loops of goats and sheep.

The effects of Clostridium perfringens type D culture supernates were compared in ligated loops of the small intestine (ileum) and colon of four goat kids and four lambs, the loops being examined histopathologically and electron microscopically 7 h after inoculation. No lesions were observed in the small intestine of any animal, or in control colonic loops. In the caprine and ovine colonic loops treated with culture supernates, most goblet cells were empty and the lumina contained a layer of mucus, polymorphonuclear leucocytes, bacteria and sloughed epithelial cells. The apical cytoplasm of the superficial epithelial cells was lost. Moderate oedema was observed in the submucosa and muscular layer. The colonic lesions were more severe in kids than in lambs. No changes were seen in vascular endothelial cells in any loop. 1999 W.B. Saunders and Company Ltd.

Antibody response in goats vaccinated with liposome-adjuvanted Clostridium perfringens type D epsilon toxoid.

A trial was performed using 20 goats to evaluate the antibody responses to a liposome-adjuvanted Clostridium perfringens epsilon toxoid vaccine (LIPV). The antibody response was compared with those produced by epsilon toxoid vaccines prepared using aluminium hydroxide (ALV) and incomplete Freud's adjuvant (FAV). The animals were allocated to four groups at the beginning of the trial. The animals in group 1 were vaccinated with ALV, while the animals in group 2 received FAV and those in groups 3 and 4 were vaccinated with LIPV. The animals in groups 1 to 3 received three doses of the corresponding vaccine at intervals of three weeks, while those in group 4 received only 1 dose of vaccine at the beginning of the trial. A blood sample was obtained from all the goats at the beginning of the trial and then weekly for 8 weeks. The samples were analysed for epsilon toxoid antibodies by an indirect ELISA technique. No major clinical abnormalities were observed in the animals after vaccination, with the exception of those that received the FAV, which experienced transient lameness. The highest antibody response was observed in the animals vaccinated with FAV, but they presented moderate to severe inflammatory tissue reactions at the injection site. Moderately high antibody responses were obtained with the ALV, with which only minor local reactions were observed. No significant antibody responses were obtained with the LIPV, nor were local reactions observed.

Variability of serum antibody responses of goat kids to a commercial Clostridium perfringens epsilon toxoid vaccine.

Twenty-nine Angora goats were used in a trial of a commercial enterotoxaemia (pulpy kidney disease) vaccine. The animals were allocated to four groups, of which three received an initial dose of vaccine, two also received a booster of the same vaccine either 28 or 42 days after the first vaccination, and the fourth remained as an unvaccinated control group. An indirect ELISA technique was used to measure the titres of Clostridium perfringens type D epsilon antitoxin in serum samples taken before vaccination and 17, 28, 42, 59, 70, 86, 98 and 128 days after vaccination. There was a wide range of antibody titres after vaccination, and the great majority of the vaccinated animals had titres below the protective level, arbitrarily set at 0.25 iu/ml, by day 98.