Emission of nitrous oxide from hydrocarbon contaminated soil amended with waste water sludge and earthworms

Soils in Mexico are often contaminated with hydrocarbons and addition of waste water sludge and earthworms accelerates their removal. However, little is known how contamination and subsequent bioremediation affects emissions of N₂O and CO₂. A laboratory study was done to investigate the effect of waste water sludge and the earthworm Eisenia fetida on emission of N₂O and CO₂ in a sandy loam soil contaminated with the polycyclic aromatic hydrocarbons (PAHs): phenanthrene, anthracene and benzo(a)pyrene. Emissions of N₂O and CO₂, and concentrations of inorganic N (ammonium (NH₄⁺), nitrite (NO₂^?) nitrate (NO₃^?)) were monitored after 0, 5, 24, 72 and 168 h. Adding E. fetida to the PAHs contaminated soil increased CO₂ production rate significantly 2.0 times independent of the addition of sludge. The N₂O emission rate from unamended soil expressed on a daily base was 5 μg N kg^?1 d^?1 for the first 2 h and increased to a maximum of 325 μg N kg^?1 d^?1 after 48 h and then decreased to 10 μg N kg^?1 d^?1 after 168 h. Addition of PAHs, E. fetida or PAHs + E. fetida had no significant effect on the N₂O emission rate. Adding sludge to the soil sharply increased the N₂O emission rate to >400 μg N kg^?1 d^?1 for the entire incubation with a maximum of 1134 μg N kg^?1 d^?1 after 48 h. Addition of E. fetida, PAHs or PAHs + E. fetida to the sludge-amended soil reduced the N₂O emission rate significantly compared to soil amended with sludge after 24 h. It was found that contaminating soil with PAHs and adding earthworms had no effect on emissions of N₂O. Emission of N₂O, however, increased in sludge-amended soil, but addition of earthworms to this soil and contamination reduced it.     

Progress in the control of bovine tuberculosis in Spanish wildlife

Despite the compulsory test and slaughter campaigns in cattle, bovine tuberculosis (bTB) is still present in Spain, and the role of wildlife reservoirs is increasingly recognized. We provide an update on recent progress made in bTB control in Spanish wildlife, including aspects of epidemiology, surveillance, host-pathogen interaction and wildlife vaccination. At the high densities and in the particular circumstances of Mediterranean environments, wild ungulates, mainly Eurasian wild boar and red deer, are able to maintain Mycobacterium bovis circulation even in absence of domestic livestock. Infection is widespread among wild ungulates in the south of the country, local infection prevalence being as high as 52% in wild boar and 27% in red deer. Risk factors identified include host genetic susceptibility, abundance, spatial aggregation at feeders and waterholes, scavenging, and social behaviour. An increasing trend of bTB compatible lesions was reported among wild boar and red deer inspected between 1992 and 2004 in Southwestern Spain. Sporadic cases of badger TB have been detected, further complicating the picture. Gene expression profiles were characterized in European wild boar and Iberian red deer naturally infected with M. bovis. The comparative analysis of gene expression profiles in wildlife hosts in response to infection advanced our understanding of the molecular mechanisms of infection and pathogenesis, revealed common and distinctive host responses to infection and identified candidate genes associated with resistance to bTB and for the characterization of host response to infection and vaccination. Ongoing research is producing valuable knowledge on vaccine delivery, safety and efficacy issues. Baits for the oral delivery of BCG vaccine preparations to wild boar piglets were developed and evaluated. The use of selective feeders during the summer was found to be a potentially reliable bait-deployment strategy. Safety experiments yielded no isolation of M. bovis BCG from faeces, internal organs at necropsy and the environment, even after oral delivery of very high doses. Finally, preliminary vaccination and challenge experiments suggested that a single oral BCG vaccination may protect wild boar from infection by a virulent M. bovis field strain.     

Acceptance and palatability for domestic and wildlife hosts of baits designed to deliver a tuberculosis vaccine to wild boar piglets

Tuberculosis (TB) caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, is an important health problem worldwide. The control of TB through vaccination of wildlife reservoirs may potentially have advantages over other management strategies. The most practical approach to deliver vaccines to wildlife is using oral baits that are stable under field conditions and effective in reaching the target species. Baits were developed in our laboratory to deliver oral vaccines to wild boar piglets. However, these baits were well accepted by other wild species. Therefore, bait consumption by different M. bovis hosts was evaluated herein. The results showed that the baits were well accepted by cattle, feral pigs, and adult red deer whereas small mammals like badgers and possums showed varying bait acceptance. Bait acceptance by different species has the advantage of targeting more than one wildlife reservoir when they coexist in the same area and need to be vaccinated for TB control. However, bait delivery methods such as the use of selective feeders to target the desired species should be developed to avoid bait consumption by other species.     

Influence of thermal treatments simulating cooking processes on the polyphenol content in virgin olive oil

Virgin olive oils were subjected to simulated common domestic processing, including frying, microwave heating, and boiling with water in a pressure cooker. The impact of these processes on polyphenol content and physicochemical characteristics of oils was assessed. Thermal oxidation of oils at 180 degrees C caused a significant decrease in hydroxytyrosol- and tyrosol-like substances. In contrast, oils heated for 25 h still retained a high proportion of the lignans 1-acetoxypinoresinol and pinoresinol. Thermal oxidation also resulted in a rapid degradation of alpha-tocopherol and the glyceridic fraction of oils. Microwave heating of oils for 10 min caused only minor losses in polyphenols, and the oil degradation was lower than that in thermoxidation assays. Again, lignans were the least affected polyphenols and did not change during microwave heating. Boiling a mixture of virgin olive oil and water in a pressure cooker for 30 min provoked the hydrolysis of the secoiridoid aglycons and the diffusion of the free phenolics hydroxytyrosol and tyrosol from the oil to the water phase. Losses of polyphenols were detected only at pH lower than 6. Moreover, alpha-tocopherol and the glyceridic fraction of oils were not modified during this process. It is worth noting that all the heating methods assayed resulted in more severe polyphenols losses and oil degradation for Arbequina than for Picual oil, which could be related to the lower content in polyunsaturated fatty acids of the latter olive cultivar. These findings may be relevant to the choice of cooking method and olive oil cultivar to increase the intake of olive polyphenols.     

Evaluation of baits for oral vaccination of European wild boar piglets

The objective of this study was to develop and evaluate new baits for the oral delivery of vaccine preparations to 2–4 month-old wild boar piglets. Baits were prepared using a matrix composed of wild boar feed, wheat flour, paraffin, sacarose and cinnamon-truffle powder attractant with polyethylene capsules dipped into the matrix to introduce vaccine formulation. Physical stability studies demonstrated that baits were stable for at least three days at temperatures as high as 42 °C. Recombinant Escherichia coli expressing the membrane-displayed BM95-MSP1a fusion protein were used to test bacterial viability in the baits and the antibody response in orally immunized wild boar. The E. coli viability was not significantly affected after bait incubation at 25 and 37 °C for 96 h. Bait acceptance studies using artificial feeders in the field showed that baits were accepted by 2–3 month-old animals, the preferred age for vaccination. Orally immunized wild boar piglets excreted recombinant E. coli in the feces and developed antibody titers to recombinant BM95-MSP1a protein, thus confirming that vaccine composition was released and reached the wild boar gastrointestinal track. The results of these experiments support the use of these baits for oral delivery of vaccine formulations to 2–4 month-old wild boar piglets.     

Serotypes,phage types and virulence genes of shiga-producing Escherichia coli isolated from sheep in Spain

PROBLEM ADDRESSED: Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. OBJECTIVE: The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. METHODS AND APPROACH: Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. RESULTS: STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx(1) genes, 10 (4%) possessed stx(2) genes and 133 (53%) both stx(1) and stx(2). Enterohaemolysin (ehxA) and intimin (eae) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H-, O117:H-, O128:H-, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type gamma1, and two strains of serotype O156:H- had the new intimin zeta. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). CONCLUSIONS: This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.     

Assessment of the leaching potential of 12 substituted phenylurea herbicides in two agricultural soils under laboratory conditions

In this study, the potential groundwater pollution of 12 substituted phenylurea herbicides (chlorbromuron, chlorotoluron, diuron, fenuron, fluometuron, isoproturon, linuron, metobromuron, metoxuron, monolinuron, Monuron, and neburon) was investigated under laboratory conditions. For this purpose, leaching studies were conducted using disturbed soil columns filled with two different agricultural soils, one hypercalcic calcisol (HC) and the other endoleptic phaeozem (EP). In the case of the HC, all of the studied herbicides were found in leachates, while for the EP only, chlorbromuron, chlorotoluron, isoproturon, monolinuron, and, especially, fenuron were recovered. For both soils, the groundwater ubiquity score (GUS) index was calculated for each herbicide on the basis of its persistence (as t(1/2)) and mobility (as K(OC)). The half-lives obtained were markedly higher in the EP (217-518 days) than in the HC (71-178 days). As a consequence, higher values of GUS indexes were observed for EP. The ratio of the GUS between the EP and the HC was about 1.3.     

Intracellular distribution of copper and zinc in the liver of copper-exposed cattle from northwest Spain

The intracellular distribution of copper (Cu) and zinc (Zn) in the livers of normal and moderately Cu-exposed cattle was investigated with the aim of improving understanding of the pathophysiology of Cu accumulation in cattle. At total liver Cu concentrations within the generally accepted normal range (25-100 mg/kg fresh weight) the large-granule fraction was the main subcellular compartment for Cu accumulation, followed by the cytosol and the nucleus, whereas in the microsomal fraction Cu accumulation was very low. With increasing Cu exposure, the capacity of the large-granule fraction to accumulate Cu decreased, proceeding towards a plateau (estimated at about 80 mg/kg at a projected total liver Cu concentrations of 450 mg/kg), accompanied by progressively greater Cu accumulation in the nucleus and cytosol. Total liver Cu concentration had little influence on subcellular Zn distribution, with hepatic Cu concentrations being only moderately correlated with Zn concentration in the large-granule fraction. There was a strong association between the proportion of total Cu bound to metallothionein (MT) and the proportion of total Zn in the large-granule fraction, suggesting that Zn displaced from MT is taken up by the lysosomes for excretion. This pattern of Cu accumulation, as in sheep, may be due to the limited capacity for metallothionein binding of Cu and excretion in bile.     

Aujeszky's disease virus infection patterns in European wild boar

Evidence of exposure (i.e. seroprevalence) to Aujeszky's disease virus (ADV) is high among wild boars from south-central Spain. This research aims to determine the presence of ADV by molecular detection, and to describe the patterns of ADV infection in wild boars. Tonsils (TN) and trigeminal ganglia (TG) for ADV molecular detection, and sera were collected from wild boars (n = 192) in 39 hunting estates from south-central Spain (2004/2005). A nested polymerase chain reaction (PCR) for a fragment of the ADV surface glycoprotein B was performed on collected tissues. Individual status of presence of viral DNA was tested against explanatory variables by means of a Generalized Linear Mixed Model (GLIMMIX) analysis. Viral detection prevalence was 30.6 ± 6.7%. Although there was an increasing pattern with age and females presented higher prevalences, no statistically significant influence of sex and age was found for viral presence. Molecular testing in TN and TG allowed classifying infection status into (i) ADV negative (in both TN and TG), (ii) only positive in TN, (iii) only positive in TG and (iv) positive in both TN and TG. ADV DNA was statistically more frequently evidenced in TN in females than in males. With the exception of one individual, all wild boars with presence of ADV DNA in TN and TG or only in TG reacted positive in the ELISA. In contrast, animals with only ADV DNA in TN serorreacted positively and negatively. Interestingly, 45% of the PCR positive wild boars (n = 59) were seronegative in the serological test, all of them with viral DNA only in TN. Our results provide evidence for latency of ADV in wild boars and stress the fact that antibody detection based tests may fail to detect a proportion of recently infected animals. This is of great concern since current management schemes in our study promote animal translocation for hunting purposes, with the associated risk of under-detecting ADV infected individuals when using serology to screen for ADV infection.