Large herbivore assemblages in a changing climate: incorporating water dependence and thermoregulation.

The coexistence of different species of large herbivores (ungulates) in grasslands and savannas has fascinated ecologists for decades. However, changes in climate, land-use and trophic structure of ecosystems increasingly jeopardise the persistence of such diverse assemblages. Body size has been used successfully to explain ungulate niche differentiation with regard to food requirements and predation sensitivity. But this single trait axis insufficiently captures interspecific differences in water requirements and thermoregulatory capacity and thus sensitivity to climate change. Here, we develop a two-dimensional trait space of body size and minimum dung moisture content that characterises the combined food and water requirements of large herbivores. From this, we predict that increased spatial homogeneity in water availability in drylands reduces the number of ungulate species that will coexist. But we also predict that extreme droughts will cause the larger, water-dependent grazers as wildebeest, zebra and buffalo-dominant species in savanna ecosystems - to be replaced by smaller, less water-dependent species. Subsequently, we explore how other constraints such as predation risk and thermoregulation are connected to this two-dimensional framework. Our novel framework integrates multiple simultaneous stressors for herbivores and yields an extensive set of testable hypotheses about the expected changes in large herbivore community composition following climate change.

CMOS based image cytometry for detection of phytoplankton in ballast water.

We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

Occupational exposure to sevoflurane during cardiopulmonary bypass.

Volatile anaesthetic agents are widely used for maintenance of anaesthesia in all kinds of surgical procedures. Despite the implementation of measures such as adequate ventilation of the operating room and the use of efficient scavenging systems, concern remains about the risks for occupational exposure, especially in situations associated with an increased risk of anaesthetic gas waste, such as with the use of volatile anaesthetic agents on cardiopulmonary bypass. The present contribution reports the results of a preliminary safety assessment involving measurements of sevoflurane concentrations in the ambient air of a cardiac surgery operating room. In 22 cardiac surgical procedures with cardiopulmonary bypass (11 with open and 11 with closed venous reservoir), measurements of trace concentrations were obtained every 10 min at the following sites: at the outlet of the oxygenator, at the outlet of the cardiotomy reservoir, in the breathing zone of the perfusionist and above the surgical field. The concentrations were measured on-line using a photoacoustic infrared spectrometer. Mean sevoflurane waste concentrations remained consistently below the recommended target value of 4.68 ppm throughout the observation period at the different measurement sites. These results indicate that, with the use of sevoflurane on cardiopulmonary bypass, the recommended levels for occupational exposure are not exceeded, provided adequate operation room ventilation and waste gas scavenging is performed.

Treatment of ballast water; how to test a system with a modular concept?

A variety of methods were successfully applied to examine the efficacy of a modular ballast water system according to the standards as adopted by the International Maritime Organization. The ballast water treatment system had a capacity of 530 m3 h(-1) consisted of a pump system, a hydrocyclone, a 50 microm mesh-size self-cleaning filter and an installation for the addition of a chemical disinfectant (PERACLEAN Ocean). The land-based testing facility used natural sea water of high turbidity during the spring phytoplankton bloom. The mesozooplankton fraction was inspected with a standard binocular. Larger zooplankton were effectively removed with the filter; the smaller sized fraction containing larvae and nauplia were killed after chemical treatment. The phytoplankton component was monitored using flow cytometry. The huge colonies of the phytoplankton Phaeocystis globosa were disrupted in the hydrocyclone liberating the colony cells which passed as single cells through the filter. These cells remained viable but were finally killed in the secondary (chemical) step. Bacteria also passed all mechanical treatment steps unharmed but were killed in the final step. Viability tests with SYTOX Green, which were specifically designed for phytoplankton, showed that mechanical treatment did not affect the percentage of viable cells a short-term, but after several hours the viable cell counts dropped down to 70%. Phytoplankton cells recovered within a single day and formed a new dense bloom rapidly. The bacteriostatic component of the chemical disinfectant (H2O2) remained present for several days preventing regrowth of bacteria for up to 15 days after addition. In conclusion, the IMO standards were met using the modular ballast water treatment unit and the applied instruments and assays were effective and rapid tools to qualify and quantify the organisms present as well as their viability.

Effect of trace metal availability on coccolithophorid calcification.

The deposition of atmospheric dust into the ocean has varied considerably over geological time. Because some of the trace metals contained in dust are essential plant nutrients which can limit phytoplankton growth in parts of the ocean, it has been suggested that variations in dust supply to the surface ocean might influence primary production. Whereas the role of trace metal availability in photosynthetic carbon fixation has received considerable attention, its effect on biogenic calcification is virtually unknown. The production of both particulate organic carbon and calcium carbonate (CaCO3) drives the ocean's biological carbon pump. The ratio of particulate organic carbon to CaCO3 export, the so-called rain ratio, is one of the factors determining CO2 sequestration in the deep ocean. Here we investigate the influence of the essential trace metals iron and zinc on the prominent CaCO3-producing microalga Emiliania huxleyi. We show that whereas at low iron concentrations growth and calcification are equally reduced, low zinc concentrations result in a de-coupling of the two processes. Despite the reduced growth rate of zinc-limited cells, CaCO3 production rates per cell remain unaffected, thus leading to highly calcified cells. These results suggest that changes in dust deposition can affect biogenic calcification in oceanic regions characterized by trace metal limitation, with possible consequences for CO2 partitioning between the atmosphere and the ocean.

Transmission of bovine herpesvirus 1 within and between herds on an island with a BHV1 control programme.

Transmission of bovine herpesvirus 1 (BHV1) within and between herds was studied on the island of Ameland, The Netherlands. There were 50 herds with 3300 head of cattle on the island. Herds were divided into three groups: (1) only containing seronegative cattle, (2) containing seronegative cattle and vaccinated seropositive cattle, and (3) containing only vaccinated cattle. All 23 herds in groups 1 and 2 were monitored. Three major outbreaks of BHV1 infections were observed due to the introduction of infectious cattle. Another major outbreak was most likely induced by reactivation of latent BHV1 in seropositive cattle. The basic reproduction ratio within these herds was estimated at least 4. Only one of these outbreaks led to three secondary outbreaks in susceptible herds in which all cattle were seronegative. These outbreaks were most likely due to respectively, direct animal contact, human transmission, and aerogenic transmission. The basic reproduction ratio between herds in this study was estimated to be 0.6.

Severity of episodes of care assessed by family physicians and patients: the DUSOI/WONCA as an extension of the International Classification of Primary Care (ICPC).

OBJECTIVES: The DUSOI/WONCA is included in the second edition of the International Classification of Primary Care (ICPC-2), as an extension to assess the severity of episodes of care. We studied (i) family physician's (FPs') assessment of three DUSOI/WONCA parameters per episode of care; (ii) how these relate to patient and episode of care characteristics, and to the interventions that occur; and (iii) how FPs' and patients' assessment of severity compare. METHODS: Twelve FPs participated and coded patient and encounter data with ICPC. Also, they answered three DUSOI/WONCA questions, that were also answered (after the consultation) by 300 patients. Odds ratios were calculated for the relationships of the severity elements to patient and episode characteristics, and interventions. The relative agreement between FPs' and patients' ratings of severity was assessed. RESULTS: In 2033 consultations, 2860 episodes of care were documented, with a subset of 411 with a paired assessment by patient and FP. Patients appeared to be less hindered by symptoms/ complaints than the FPs thought, and less optimistic about the prognosis without care than the FP. Clear relationships existed between the FPs' assessment of severity and the patient, encounter and episode of care characteristics. Substantial agreement existed between FPs' and patients' assessment of severity. CONCLUSIONS: This study confirms the feasibility for FPs routinely to code the separate elements of severity for episodes of care, simultaneously using ICPC and DUSOI/WONCA. The studied elements of severity all provide relevant information: the interventions that occurred all related to them in a logical fashion. The FP-patient agreement on severity is satisfactory, also in the sense that it seems realistic to include these elements of severity as a topic in the communication with the patient.

Diel patterns of UVBR-induced DNA damage in picoplankton size fractions from the Gulf of Aqaba, Red Sea.

This study focuses on the impact of natural levels of UVBR (ultraviolet-B radiation: 280 to 315 nm) on bacterio- and phytoplankton (<10 microm) from the Gulf of Aqaba, Red Sea. Incident biologically effective doses (BEDs) and attenuation of biologically effective radiation in the water column were measured using a DNA biodosimeter. UVBR-induced DNA damage was measured as cyclobutane pyrimidine dimers (CPDs), using an antibody directed to CPDs followed by chemiluminescent detection. Depth profiles of DNA damage were determined in two plankton size fractions (0.2 to 0.8 microm and 0.8 to 10 microm) collected down to 50 m depth. Furthermore, accumulation and removal of CPDs were monitored in surface plankton samples during several daily cycles. Small plankton (plankton <10 microm) composition was determined by flow cytometry. The plankton community in the Gulf of Aqaba was dominated by nonphototrophic bacteria and the free-living prochlorophyte Prochlorococcus spp. (<0.8 microm). In general, no DNA damage could be detected in dosimeter DNA below 15 m. In contrast, DNA damage (up to 124 CPD Mnucl-1) could be detected in all bacterio- and phytoplankton samples. DNA damage accumulated throughout the day, indicating that plankton in the Gulf of Aqaba undergo UVBR stress via CPD induction. Although the numbers of CPDs decreased during darkness, both size fractions showed some residual DNA damage at the end of the night. This suggests that dark repair processes did not remove all CPDs, or that part of the plankton community was incapable of repair at all. CPD levels in the two size fractions showed no significant differences in situ. During full solar radiation exposures (samples incubated in bags), more CPDs were detected in the smaller (0.2 to 0.8 microm) size fraction as compared to the larger (0.8 to 10 microm) size fraction. In these experiments, initial plankton composition was significantly different from the field samples. This implies that a shift in the population structure or irradiance conditions can lead to a significant change in UVBR sensitivity. In conclusion, the results show that the picoplankton-dominated phyto- and bacterioplankton communities in the clear surface waters from the Gulf of Aqaba undergo UVBR stress. Repair pathways are not sufficient to eliminate damage during or after UVBR exposure hours, suggesting photomortality as a potential loss parameter of the plankton community.

Presence of bovine herpesvirus 1 gB-seropositive but gE-seronegative Dutch cattle with no apparent virus exposure.

Two hundred and thirty-seven of 2052 cattle which had not been vaccinated against bovine herpesvirus 1 (BHV-1) were seropositive in a glycoprotein B (gB)-blocking ELISA, but seronegative in a glycoprotein E (gE)-blocking ELISA. In order to detect whether they were latently infected with BHV-1, 10 of them were treated with corticosteroids in an attempt to reactivate putatively latent virus. After successive treatments with dexamethasone and prednisolone, no virus excretion was detected and they showed no increase in antibody titres. In contrast, one gE-seropositive animal re-excreted BHV-1 and had a four-fold increase in antibody titre after the corticosteroid treatments. After slaughter, no BHV-1 DNA could be detected with a sensitive PCR in samples of the trigeminal, cervical and sacral ganglia and spinal cords of the gE-seronegative cattle.

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains.

Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex.

Epitopes on glycoprotein C of bovine herpesvirus-1 (BHV-1) that allow differentiation between BHV-1.1 and BHV-1.2 strains.

In cattle, bovine herpesvirus-1 (BHV-1) can cause a mild genital disease known as infectious pustular vulvovaginitis (IPV) and a more severe respiratory disease known as infectious bovine rhinotracheitis (IBR). On the basis of epidemiological data, it has been proposed that these diseases are caused by strains with different genotypes (IBR by BHV-1.1 and IPV by BHV-1.2 strains). By using a panel of 237 BHV-1 isolates, a monoclonal antibody (MAb 71) was found that failed to react with all 54 putative IPV strains in the panel, and another MAb (77) was found that did not react with 16 of these 54 IPV strains. Because MAbs 71 and 77 also failed to react with a BHV-1.1 glycoprotein C (gC)-deletion mutant, it was hypothesized that both MAbs recognize BHV-1.1 gC. By marker-rescue experiments and by expressing fragments of the BHV-1.1 gC gene in recombinant baculoviruses, it was shown that both MAbs indeed recognize BHV-1.1 gC. MAb 71 recognizes the N-terminal half and MAb 77 recognizes the C-terminal half of BHV-1.1 gC. In a PEPSCAN analysis with 12-mer oligopeptides, MAb 71 reacted with overlapping peptides containing gC amino acid residues 75-80 and MAb 77 did not react in this analysis. The differences in gC found in this study may contribute to the biological differences between BHV-1.1 and BHV-1.2.

Strains of bovine herpesvirus 1 that do not express an epitope on glycoprotein E in cell culture still induce antibodies that can be detected in a gE-blocking ELISA.

Two bovine herpesvirus 1 (BHV1) field strains that do not express an epitope on glycoprotein E (gE) in cell culture were inoculated into calves to examine whether their sera became positive in a gE-blocking ELISA that detects antibodies against gE. This gE-blocking ELISA uses one monoclonal antibody that is directed against the above mentioned epitope. All calves, except one, infected with these gE-epitope negative BHV1 strains, became positive in this gE-blocking ELISA, about two weeks later than in another gE-ELISA and a gB-ELISA. However, cattle infected with BHVI strains that do express this particular gE-epitope showed a similar type of antibody responses. These findings demonstrate that BHV1 strains that do not express a particular gE-epitope in cell culture, still can induce antibodies that are detected in a blocking ELISA that measures antibodies against that epitope.

Reactivation of latent bovine herpesvirus 1 in cattle seronegative to glycoproteins gB and gE.

Six heifers were vaccinated intranasally with the live bovine herpesvirus 1 (BHV1) temperature-sensitive (ts) vaccine strain RBL106 within 3 weeks of birth. These calves most likely still had maternal antibodies against BHV1. Thereafter, these heifers were vaccinated several times with an experimental BHV1 glycoprotein-D (gD) subunit vaccine. At the age of 3 years these 6 heifers were seronegative in the BHV1 gB and gE blocking ELISAs, but had neutralizing antibodies against BHV1, probably induced by the vaccinations with the gD subunit vaccine. Five of these 6 heifers excreted BHV1 after treatment with dexamethasone. Restriction enzyme analysis of the genome of the excreted viruses revealed that all 5 isolates had a BHV1.1 genotype and that isolates of 3 heifers were not obviously different from the ts-vaccine strain. The restriction enzyme fragment pattern of the isolate of 1 heifer was clearly different from the pattern of the ts-vaccine strain. It is concluded that cattle can be seronegative against BHV1 gB and gE but can still carry BHV1 in a latent form. This finding strongly suggests that there are completely BHV1 seronegative cattle that are latently infected with BHV1. The impact of this finding on BHV1 eradication programmes is discussed.

Comparison of DNA application methods to reduce BRSV shedding in cattle.

We compared the protection afforded by three different DNA application methods against bovine respiratory syncytial virus (BRSV) infection in cattle. A synthetic gene that codes for the G protein of BRSV was inserted into a eukaryotic vector and was used in the vaccine. Intradermal (i.d.) application with a needleless injector (NI), the Pigjet, reduced BRSV excretion significantly better after BRSV challenge than intramuscular (i.m.) or i.d. vaccination with a needle. Serum antibodies against the G protein were consistently the highest and showed less variation in Calves vaccinated with the NI compared with those in i.m. and i.d. vaccinated calves. After BRSV challenge, secondary serum and mucosal antibody responses were also the highest in NI vaccinated calves. We conclude that DNA application with the needleless injector is substantially better than i.m. or i.d. application, and is capable to prime the immune response at the respiratory mucosa.

An inactivated gE-negative marker vaccine and an experimental gD-subunit vaccine reduce the incidence of bovine herpesvirus 1 infections in the field.

An inactivated glycoprotein E-negative vaccine and an experimental glycoprotein D-subunit vaccine against bovine herpesvirus 1 (V1) were examined for their effectiveness in a randomized, double-bline, placebo-controlled field trial comprising 130 dairy farms. The use of these marker vaccines enabled us to monitor the incidence of infections in vaccinated populations. The aims of this trial were to evaluate whether these vaccines: (1) reduce the proportion of outbreaks in dairy herds; and (2) reduced virus transmission within dairy herds and to what extent. Vaccination with either of the two vaccines significantly reduced the proportion of herds wherein an outbreak occurred as well as the virus transmission within herds, as compared to placebo-treated herds. The estimated number of secondary cases caused by one infectious animal, expressed as the reproduction ratio R, was for both vaccines significantly > 1. This indicates that when BHV1 is introduced into vaccinated herds, major outbreaks may still occur.

Deliberate departures from good general practice: a study of motives among Dutch general practitioners.

BACKGROUND: When general practitioners (GPs) act contrary to their own standards of good practice, they usually cite patient demands as the main reason. However, up until now, studies have relied on doctors' recollections of departures from their own norms, which may be unreliable. AIM: To systematically explore GPs' motives for deliberate departures from their own conception of good practice. METHOD: Forty-nine GPs, over five days, registered to what extent they had deviated from their own norms, and recorded the motives underlying any deviation. RESULTS: Of the 6087 consultations registered, 10% contained some departure from 'good' general practice, the majority (75%) of which was perceived by the doctor concerned as 'slight'. Doctors underpinned their departures mostly by referring to the doctor-patient relationship: the wish to be nice was used, on average, in 42% of deviations, and the wish to prevent a conflict in 30%. The most important non-relational motive was clinical uncertainty, which doctors used in 11% of their cases. DISCUSSION: Contrary to common belief, GPs often comply with patient requests because they wish to, and not because they feel forced to. Whether or not this behaviour affects the quality of care is largely dependent on the model of 'good' general practice used.

Immunization of cattle with a BHV1 vector vaccine or a DNA vaccine both coding for the G protein of BRSV.

A gE-negative bovine herpesvirus 1 (BHV1) vector vaccine carrying a gene coding for the G protein of bovine respiratory syncytial virus (BRSV) (BHV1/BRSV-G) induced the same high degree of protection in calves against BRSV infection and BHV1 infection as a multivalent commercial vaccine. A DNA plasmid vaccine, carrying the same gene as the BHV1/BRSV-G vaccine, significantly reduced BRSV shedding after BRSV infection compared with that in control calves, but less well than the BHV1/BRSV-G vaccine. Flow cytometric analysis showed a significant relative increase of gamma/delta+ T cells in peripheral blood after BRSV challenge-infection of the calves of the control group but not in the vaccinated groups. These results indicate that the G protein of BRSV can induce significant protection against BRSV infection in cattle, and that the BHV1/BRSV-G vaccine protects effectively against a subsequent BRSV and BHV1 infection.

An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle.

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and cattle that had been repeatedly vaccinated with gE-negative marker vaccines scored negative, whereas sera from cattle naturally or experimentally infected with BHV1 field strains scored positive in the gE-ELISA. Antibodies against gE appeared in the serum around 11 days after infection. Cattle that were first vaccinated and then challenged, thus having less virus replication, also became gE-seropositive. The sensitivity and specificity of the gE-ELISA is high, and therefore the gE-ELISA is suitable for differentiating between infected cattle and vaccinated cattle with a gE-negative vaccine.

An inactivated vaccine based on a glycoprotein E-negative strain of bovine herpesvirus 1 induces protective immunity and allows serological differentiation.

The bovine herpesvirus 1 (BHV1) strain Za is a conventionally attenuated strain with a 2.7 kb deletion that encompasses the complete coding region for glycoprotein E (gE). This gE-negative strain was used as whole-virus antigen in an inactivated virus vaccine. Three different antigen concentrations of this vaccine were evaluated for safety and efficacy in a vaccination-challenge experiment in calves. No adverse effects were observed in any of the calves vaccinated with the gE-negative vaccines. Calves given the vaccine with the highest antigen concentration were adequately protected against challenge; clinical symptoms were virtually absent and challenge virus shedding was significantly reduced as compared with unvaccinated calves. We developed a sensitive blocking enzyme-linked immunosorbent assay (ELISA) to detect antibodies against gE. After vaccination, calves did not produce antibodies against gE, but these antibodies were detectable within 2 weeks after challenge both in vaccinated and in unvaccinated calves. These results demonstrate the efficacy of a gE-negative inactivated BHV1 vaccine and the detectability of antibodies against gE after infection. The combined use of the marker vaccine and the gE-blocking ELISA makes it possible to differentiate between vaccinated animals and infected animals. This possibility may be very useful in BHV1 control programmes.

A simple, specific, and highly sensitive blocking enzyme-linked immunosorbent assay for detection of antibodies to bovine herpesvirus 1.

By using a monoclonal antibody directed against an epitope located on glycoprotein B of bovine herpesvirus 1 (BHV1), a simple, convenient blocking enzyme-linked immunosorbent assay (ELISA) which combines a high sensitivity with a low false-positive rate has been developed. The test can be performed at low variance on undiluted bovine serum samples. The epitope on glycoprotein B appears to be conserved, because it could be detected by immunostaining in all of 160 BHV1 isolates originating from 10 countries. In testing 215 anti-BHV1 antibody-negative and 179 anti-BHV1 antibody-positive serum samples, specificity and sensitivity were 0.96 and 0.99, respectively. This blocking ELISA is superior to a commercially available indirect ELISA and to the 24-h virus neutralization test in detecting low antibody levels in serum. In addition, this blocking ELISA is able to detect specific antibodies in serum as early as 7 days postinfection. To minimize any risk of introducing latent BHV1 carriers among noninfected cattle, this blocking ELISA would be, in our opinion, the test of choice.