Characterization of C-strain "Riems" TAV-epitope escape variants obtained through selective antibody pressure in cell culture

ABSTRACT: Classical swine fever virus (CSFV) C-strain "Riems" escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo.It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.     

Cytotoxic ellagitannins from Reaumuria vermiculata

Three ellagitannins and one disulfated flavonol were isolated from the aerial parts of Reaumuria vermiculata L. Besides that, 16 known compounds were characterized as well. The structures of all compounds were elucidated on the basis of spectroscopic data including 1D and 2D NMR and ESI HR-FTMS. The in vivo antioxidant activity using the oxygen radical absorbance capacity (ORAC) method, of the extract, its column fractions and two of the isolated ellagitannins was accomplished. In addition, a possible cytotoxicity of the extract and two of the new ellagitannins on HaCaT human keratinocytes and the activity of both compounds against the prostate cancer cell line (PC-3) were also assessed, whereby a potent cytotoxicity with IC(50) less than 1μg/ml was determined for both compounds. Besides, the extract exhibited a potential cytotoxic effect against four different solid tumor cell lines, namely liver (Huh-7), colorectal (HCT-116), breast (MCF-7) and prostate (PC-3). The IC(50)s were found to be substantially low (ranged from 1.3±0.15 to 2.4±0.22μg/ml) with relatively low resistance possibility reaching to 0% in the case of Huh-7 cell.     

A longitudinal study of Campylobacter distribution in a turkey production chain

Background

Campylobacter is the most common cause of bacterial enteritis worldwide. Handling and eating of contaminated poultry meat has considered as one of the risk factors for human campylobacteriosis.Campylobacter contamination can occur at all stages of a poultry production cycle. The objective of this study was to determine the occurrence of Campylobacter during a complete turkey production cycle which lasts for 1,5 years of time. For detection of Campylobacter, a conventional culture method was compared with a PCR method. Campylobacter isolates from different types of samples have been identified to the species level by a multiplex PCR assay.

Methods

Samples (N = 456) were regularly collected from one turkey parent flock, the hatchery, six different commercial turkey farms and from 11 different stages at the slaughterhouse. For the detection of Campylobacter, a conventional culture and a PCR method were used. Campylobacter isolates (n = 143) were identified to species level by a multiplex PCR assay.

Results

No Campylobacter were detected in either the samples from the turkey parent flock or from hatchery samples using the culture method. PCR detected Campylobacter DNA in five faecal samples and one fluff and eggshell sample. Six flocks out of 12 commercial turkey flocks where found negative at the farm level but only two were negative at the slaughterhouse.

Conclusion

During the brooding period Campylobacter might have contact with the birds without spreading of the contamination within the flock. Contamination of working surfaces and equipment during slaughter of a Campylobacter positive turkey flock can persist and lead to possible contamination of negative flocks even after the end of the day''s cleaning and desinfection. Reduction of contamination at farm by a high level of biosecurity control and hygiene may be one of the most efficient ways to reduce the amount of contaminated poultry meat in Finland. Due to the low numbers of Campylobacter in the Finnish turkey production chain, enrichment PCR seems to be the optimal detection method here.     

Carbon stocks of soil and vegetation on Danubian floodplains

Riparian forests are assumed to play a crucial role in the global carbon cycle. However, little data are available on C stocks of floodplains in comparison to other terrestrial ecosystems. In this study, we quantified the C stocks of aboveground biomass and soils of riparian vegetation types at 76 sampling sites in the Donau‐Auen National Park in Austria. Based on our results and a remotely sensed vegetation map, we estimated total C stocks. Carbon stocks in soils (up to 354 t ha^–1 within 1 m below surface) were huge compared to other terrestrial ecosystems. As expected, soils of different vegetation types showed different texture with a higher percentage of sandy soils at the softwood sites, while loamy soils prevailed at hardwood sites. Total C stocks of vegetation types were significantly different, but reflect differences in woody plant biomass rather than in soil C stocks. Mature hardwood and cottonwood forests proved to have significantly higher total C stocks (474 and 403 t ha^–1, respectively) than young reforestations (217 t ha^–1) and meadows (212 t ha^–1). The C pools of softwood forests (356 t ha^–1) ranged between those of hardwood/cottonwood forests and of reforestations/meadows. Our study proves the relevance of floodplains as possible C sinks, which should be increasingly taken into account for river management. Furthermore, we conclude that plant‐species distribution does not indicate the conditions of sedimentation and soil C sequestration over the time span of interest for the development of soil C stocks.     

Investigation of serological prevalence and risk factors of paratuberculosis in dairy farms in the state of Mecklenburg-Westpommerania, Germany

A total of 2,997 serum samples were randomly taken at 59 pre-selected dairy farms covering all districts with an average of 334 animals kept per farm. Samples were analysed for antibodies against Mycobacterium avium subspecies paratuberculosis (M.a.p.) using Svanovir-ELISA. Risk factors on farm characteristics and animal health were recorded at all participating farms by use of a questionnaire. Fifty out of 59 farms (84.7%) showed at least one reactor. The average within herd prevalence was 12.2%. In the univariate analysis of risk factors no significant differences towards farm prevalences were found. Results are discussed with special emphasis on the need for a common national strategy to control the disease in Germany.     

Screening for antibodies against zoonotic agents among employees of the Zoological Garden of Vienna, Schönbrunn, Austria

The aim of this study was to investigate the prevalence of antibodies against zoonotic agents in employees of the zoological garden of Vienna, Sch?nbrunn, Austria. Sixty out of 120 employees participated in the study. In 97% of them antibodies to at least one zoonotic agent were identified. Only two participants were free of antibodies to the zoonotic agents tested. The following seroprevalences (in brackets) were obtained: Viral zoonotic (and potentially zoonotic) agents: Influenzavirus A/H1N1 (58%), Influenzavirus A/H3N2 (85%), Lymphocytic choriomeningitis virus (13%), Encephalomyocarditis virus (5%), Orthopox- (Cowpox-) virus and Hantavirus type Puumala (3%). Hantavirus type Hantaan and Borna disease virus (all negative). Bacterial zoonotic agents: Bartonella henselae (65 %), Borrelia burgdorferi (10%), Leptospira interrogans serovar copenhageni and serovar icterohaemorrhagiae as well as Chlamydophila psittoci (2% each). Brucella spp., Coxiella bumetii, and Francisella tularensis (all negative). Parasitic zoonotic agents: Toxoplasma gondii (53%), Toxocara spp. (21%), Capillaria hepatica (2%), Fasciola hepatica, Schistosoma mansoni, E. multilocularis, and E. granulosus (all negative). The remarkably high seroprevalence to the causative agent of cat scratch disease, Bartonella henselae, is probably due to the private contact of the employees to cats. Regarding viral zoonotic agents it has to be mentioned that Influenzavirus vaccination and/or human-to-human transmission of especially A/H3N2 Influenzaviruses probably attributed significantly to the very high seroprevalence to both Influenzavirus types A/H1N1 and A/H3N2. When investigating parasitic zoonotic agents, high prevalence rates were found against Toxoplasma gondii and Toxocara spp., however, it was not possible to establish a causal link between seropositivity and the professional activity in the zoo. Interestingly, in the case of antibodies to T. gondii, the typical correlation with age was not found in this study, while in the case of the Toxocara spp. positive subjects a correlation was identified with both age and duration of employment in the zoo. Regarding the later two zoonotic parasites, employees of the zoological garden showed significantly higher seroprevalences than the average Austrian population. Antibodies to Capillaria hepatica, a hepatic-parasite in rodents which is diagnosed in humans rarely, were identified in one employee and another one showed a questionable positive result. Further investigations did not exhibit clinical infestations with the parasite in these two individuals so far.     

Seroepidemiological studies of zoonotic infections in hunters in southeastern Austria--prevalences,risk factors,and preventive methods

The aim of this study was to investigate the seroprevalences to zoonotic pathogens in hunters, to propose preventive measures and to obtain more information about the occurrence of zoonotic pathogens in local wild animal populations. From 146 male and 3 female hunters originating from the south-eastern Austrian federal states of Styria and Burgenland blood samples were taken and anamnestic data were obtained using a questionnaire. The serological investigations included the following viral, bacterial and parasitic zoonotic agents or zoonoses, respectively (antibody-seroprevalences in brackets): encephalomyocarditis virus (EMCV, 15%), Puumala-Hantavirus (10%), Newcastle Disease virus (NDV, 4%), borreliosis (IgG 42%, IgM 7%), brucellosis (1%), chlamydiosis (3%), ehrlichiosis (IgG 15%, IgM 3%), leptospirosis (10%), tularaemia (3%), Q fever (0%), Echinococcus multilocularis/E. granulosus (5%/11%), toxocariasis (17%). Out of a control group of 50 persons (urban population, no hunters) only one person was found to be seropositive for Toxocara canis and NDV and four for EMCV, all other results were negative in the control group. The high seroprevalences especially to Borrelia burgdorferi s.l., Ehrlichia spp., Leptospira interrogans, E. granulosus, E. multilocularis, encephalomyocarditis virus and Puumala virus demonstrate that hunters are particularly exposed to zoonotic pathogens. It should also be noted that one hunter was seropositive for Brucella abortus and five exhibited antibodies to Francisella tularensis. In these cases, as well as in the cases of the 15 seropositives for Leptospira interrogans, the suspected source of infection may--besides rodents--also include wild boars and brown hares. The infections with NDV and Chlamydophila psittaci may be traced back to contact with certain species of birds (potential risk: aviaries). For Hantaviruses, rodents are considered to be the main source of human infections.     

Denitrification- and Nitrate Leaching-Losses in an Intensively Cropped Watershed

In continuation of former measurements about gaseous denitrification losses, these losses together with those by nitrate leaching were measured by different methods in a field cropped during two years by wheat. Furthermore, N-uptake by the plants of fertilizer- and soil-N as well as N-im-mobilization in soils during and after the cropping periods was determined by application of highly enriched¹⁵N-labeled fertilizer. Denitrification losses determined by the N₂O release from C₂H₂–treated undisturbed soil cores agreed reasonably well with losses obtained by ¹⁵N-balance measurements. They both amounted during the cropping periods 12–15 and 6–20 kg N ha^?1, respectively. Gaseous N-losses increased mainly during wet periods when the field was barren. Denitrifying enzyme activities and soil respiration (CO₂-release) was measured throughout one year. Leaching losses of NO₃ from soil-and fertilizer-N occurred only during fall until spring. Leached NO₃^? originated mostly from mineralized soil-N and very little from previously immobilized fertilizer-N.     

Adding dissolved organic carbon to simulate freeze‐thaw related N2O emissions from soil

It has been assumed that high winter N₂O emissions from soils are the result of increased amounts of microbially available organic C liberated during freezing and metabolized during subsequent thawing. In a laboratory experiment, we attempted to simulate freeze‐thaw events by adding dissolved organic C (DOC) to sieved soil of high water content (95% water‐filled pore space). In a full factorial design, CO₂ and N₂O emissions of a) soil samples provided with DOC extracted from frozen soil and b) soil samples frozen for 46 days and thawed were compared. Additionally, NO , DOC and microbial ATP contents of all treatments were repeatedly analyzed during the experiment. The addition of DOC to unfrozen soil (–F+C) resulted in a substantial (22‐fold) increase in N₂O emissions as compared to the control (–F–C). However, following thawing, the increase in N₂O emissions was much larger (828‐fold in +F–C and 1243‐fold in +F+C). Freezing, but not the addition of DOC led to increased CO₂ emissions. Neither treatment affected microbial adenylate content. By adding ¹⁵N‐labeled nitrate to the soil samples, the main process leading to elevated N₂O flux rates after both DOC addition and freeze‐thaw treatment was identified as denitrification. We conclude that the availability of C substrate plays an important role for freeze‐thaw‐related N₂O emissions. However, the fact that the simulated treatment and the freeze‐thaw treatment yielded significantly different amounts of N₂O suggests that both quantity and quality of available C differed between the treatments. The localization of the liberated substrate, i.e., the availability in situ, seems to be of major importance for the amount of N₂O produced.