Biosensor assay for determination of haptoglobin in bovine milk

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3.6 and 8.6% at concentrations between 4.0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed.     

Factors contributing to the occurrence of antimicrobial drug residues in Kenyan milk

The study investigated factors contributing to the occurrence of antimicrobial drug residues in milk within four major milk production districts in Kenya. The frequency of contamination was studied among small- and large-scale dairy producers to determine if there were differences between the two types of producers. Field samples (n = 1,600) were analyzed with the improved Dutch tube diffusion test, a microbial inhibitor test (Bacillus stearothermophilus). In total, 144 and 64 samples from small- and large-scale producers, respectively, were found to contain beta-lactam antibiotics at levels exceeding the established Codex maximum residue level for penicillin G (4 microg/kg). The difference in results between the two categories of producers was found to be significant (P < 0.001). To explain the higher frequency of antibiotic contamination of milk from small-scale producers, a questionnaire was constructed and used with 220 randomly selected smallholders in the selected districts. The results suggested (i) lack of understanding of risks related to antibiotic contamination of food, (ii) poor or no treatment records, and (iii) lack of a monitoring system as major risks for contamination. It was concluded that intensification of the education among small-scale dairy producers would greatly reduce the occurrence of antimicrobial residues in milk.     

Nodule detection in digital chest radiography: effect of system noise

Apart from the image content that is the reproduction of anatomy and possible lesions, an X-ray image also contains system noise due to the limited number of photons and other internal noise sources in the system (image plate artefacts, electronic noise, etc.). The aim of this study was to determine the extent to which the system noise influences the detection of subtle lung nodules in five different regions of the chest. This was done by conducting a receiver operating characteristic (ROC) study with five observers on two different sets of images; clinical chest X-ray images and images of a LucAl phantom at similar dose levels found in the different regions of the chest. In both image types, mathematically simulated nodules (with a full-width-at-fifth-maximum of 10 mm) were added to the images at varying contrast levels. As a measure of the influence of system noise on the detection of subtle lung nodules, the ratio between the contrast needed to obtain an area under the ROC curve of 0.80 in the system noise images to that needed in the clinical images was used. The contrast ratio between system noise images and clinical images ranged from approximately 0.02 (in the hilar region) to 0.18 (in the lower mediastinal region). The maximum difference in contrast needed for the corresponding system noise images, collected at the lowest and the highest dose represented in the anatomical image, was a factor of 2. These results indicate that probably no region in a chest X-ray image is limited by the number of quanta to the detector for the detection of 10 mm lung nodules when a radiation dose corresponding to a system with speed class 200 (leading to a detector dose of approximately 9 muGy behind the parenchyma) is used.     

Sodium silicate as alternative to liming-reduced aluminium toxicity for Atlantic salmon (Salmo salar L.) in unstable mixing zones

When acid aluminium (Al) rich water is limed, unstable mixing zones are formed until equilibrium is reached. In such mixing zones transient high molecular mass positively charged Al-species (HMM Al(i)) being extremely gill reactive are produced, causing toxic effects in fish. The transient HMM Al(i)-species are formed due to hydrolysis and polymerization of low molecular positively charged Al-species (LMM Al(i)), e.g. initiated by liming and the subsequent increase in pH. To counteract the toxicity of transient Al polymers in such mixing zones, sodium silicate, forming non-toxic hydroxyaluminosilicate (HAS) complexes, can be used as alternative to liming. In the present work the effect of sodium silicate on polymerization of LMM Al(i) in unstable mixing zones and subsequent gill reactivity and mortality of fish was compared to results obtained from liming. Diluted sodium silicate (<1.5 g l(-1)) and lime slurry (Ca(OH)(2)), respectively, were continually added to acidified Al-rich water in six different channel-tank systems, to obtain mixing zones with pH 5.9, 6.0 and 6.4, respectively. Utilising in situ size and charge fractionation techniques and following the exposure of Atlantic presmolt (Salmo salar L.) kept in cages at defined stations along the channel-tank systems, changes of Al-species in the mixing zones, the gill reactivity of Al-species and thus Al toxicity could be followed downstream the confluences (time of reaction after mixing: 1-100 min). By increasing the pH of the acid water to 6.0 or 6.4 by sodium silicate, the detoxification of Al was faster than using lime. Using sodium silicate, the transformation of LMM Al(i), the formation of HMM Al(i), the Al deposition in fish gills and fish mortality were lower than using lime. The formation of neutral LMM Al-species (Al(o)) was, however, higher and the formation of colloidal Al-species (Al(c)) lower in the presence of silicate compared to lime. Furthermore, the Al deposition in fish gills and fish mortality decreased by increasing concentration of sodium silicate dosed. Thus, sodium silicate is a good alternative to liming, and under certain circumstances when aging of water may represent a problem (e.g. aquaculture) sodium silicate should be the preferred agent.     

Effects of chronic neuroleptic treatments on dopamine D1 and D2 receptors: homogenate binding and autoradiographic studies

The antipsychotic effects of neuroleptics are believed to be mediated via dopamine D2 receptor blockade; however, the anatomical and pharmacological targets of these drugs remain somewhat controversial. The purpose of this study was to examine the effects of chronic clozapine (CLZ) and haloperidol (HAL) treatments on the densities of DA D1 and D2 receptors. Adult male Sprague-Dawley rats (300-350 g) were treated for 21 days with either HAL (1 mg/kg/day, i.p.), CLZ (20 mg/kg/day, i.p.) or saline. Three days after ending the treatments, the brains were removed and used for biochemical assays of tissue DA and metabolites as well as for receptor studies. DA D1 and D2 receptors were labelled with [3H]SCH23390 and [3H]raclopride, respectively, and measured in the neostriatum by binding studies, and in autoradiograms of forebrain sections by quantitative densitometry. The autoradiographic measurements revealed significant increases in the densities of D2 receptors in nucleus accumbens, in the medio-ventral, latero-dorsal and latero-ventral quadrants of the rostral neostriatum, in caudal neostriatum and in globus pallidus of both HAL-(28-44%) and CLZ-treated (15-85%) animals. The HAL-induced up-regulation of D2 receptors in rostral and caudal neostriatum was homogenous, but CLZ produced a more uneven increase, with the highest absolute densities measured in latero-dorsal neostriatum, as well as with changes in the medio-dorsal rostral neostriatum. For D1 receptors, only CLZ and not HAL, produced significant increases in five regions, namely nucleus accumbens (43%) latero-dorsal rostral neostriatum (16%), caudal neostriatum (30%), globus pallidus (67%) and substantia nigra (12%). The observation that CLZ, contrary to HAL, also has an effect on D1 receptor densities may explain the greater therapeutic and selective efficacy with fewer side-effects of this agent, in comparison to other neuroleptics.     

Haptoglobin and serum amyloid A in relation to the somatic cell count in quarter, cow composite and bulk tank milk samples

Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0.3 mg/l and 1.0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC.     

Urinary 1-hydroxypyrene in children living in city and rural residences in Denmark

AIMS: The present study aims to assess the biological uptake in children of polycyclic aromatic hydrocarbons measured as 1-hydroxypyrene in urine from children living in city and rural residences. METHODS: 103 children living in Copenhagen and 101 children living in rural residences of Denmark collected urine samples Monday to Friday morning. Each day, the family filled in a printed diary that included questions about the time and activity patterns of the child. Multiple regression analyses were used to identify predictors of the excreted 1-hydroxypyrene level. RESULTS: During the week, the children excreted on average 0.07 [95% CI: 0.01-0.41] mumol urinary 1-hydroxypyrene per mol creatinine. Children living in urban residences excreted 0.02 [95% CI: 0.01-0.05] mumol more 1-hydroxypyrene than children living in rural residences. This was confirmed in the multiple regression analysis showing a 29% (95% CI: 2-64%) higher excretion among urban children than rural children. Moreover, the regression analysis showed that for each hour per day spent outside the children excreted 58% (1.58 [1.22-2.03]) more 1-hydroxypyrene in urine. CONCLUSION: The present study indicates that children living in urban residences are more exposed to PAH than children living in rural residences. Time spent outdoors increased the excretion of 1-hydroxypyrene, which was most evident among urban children. Higher concentrations of ambient air pollution in urban areas may explain this finding. No influence of environmental tobacco smoke, cooking habits, and heating facilities was detected.     

Transformation of iron species in mixing zones and accumulation on fish gills

The speciation of iron (Fe) strongly influences the deposition and accumulation on gills causing toxicitytoward fish. The impacts of ferric (Fe(III)) and ferrous (Fe(II)) species on gill accumulation were studied in parallel flow-through channel experiments where Atlantic salmon (Salmo salar L.) was kept in cages. Downstream of the pH 6.3 mixing point, where Fe(III) ions or Fe(ll) ions were added continuously to lake water, the molecular mass of Fe(III) increased within 0.5 min after mixing due to hydrolysis and polymerization, while the Fe(II) species remained as low molecular mass (LMM) species 20 min after mixing. For fish exposed to the Fe(lll) enriched water (0.5 mg L(-1)) the Fe accumulation on gills was high and decreased downstream, while low when Fe(II) was added to water. By adjusting the Fe(II) enriched water to pH 6.7, the oxidation of Fe(II) forming Fe(III) accelerated, the Fe accumulation on fish gills increased by a factor of 3, and high mortality (33%) was observed. Thus, input of Fe(ll) ions, oxidation of Fe(ll) at rates higher than 1.5 microg L(-1) min(-1), and continuous formation of LMM Fe(III) species accumulating on gills can induce toxicity toward fish present in circumneutral freshwaters a long distance downstream from the entry points.     

Urinary 1-hydroxypyrene in children living in city and rural residences in Denmark

AIMS: The present study aims to assess the biological uptake in children of polycyclic aromatic hydrocarbons measured as 1-hydroxypyrene in urine from children living in city and rural residences. METHODS: 103 children living in Copenhagen and 101 children living in rural residences of Denmark collected urine samples Monday to Friday morning. Each day, the family filled in a printed diary that included questions about the time and activity patterns of the child. Multiple regression analyses were used to identify predictors of the excreted 1-hydroxypyrene level. RESULTS: During the week, the children excreted on average 0.07 [95% CI: 0.01-0.41] micromol urinary 1-hydroxypyrene per mol creatinine. Children living in urban residences excreted 0.02 [95% CI: 0.01-0.05] micromol more 1-hydroxypyrene than children living in rural residences. This was confirmed in the multiple regression analysis showing a 29% (95% CI: 2-64%) higher excretion among urban children than rural children. Moreover, the regression analysis showed that for each hour per day spent outside the children excreted 58% (1.58 [1.22-2.03]) more 1-hydroxypyrene in urine. CONCLUSION: The present study indicates that children living in urban residences are more exposed to PAH than children living in rural residences. Time spent outdoors increased the excretion of 1-hydroxypyrene, which was most evident among urban children. Higher concentrations of ambient air pollution in urban areas may explain this finding. No influence of environmental tobacco smoke, cooking habits, and heating facilities was detected.