Comparison of binding and effects of Escherichia coli Shiga toxin 1 on bovine and ovine granulocytes

Granulocytes play a pivotal role in the pathogenesis of Shiga toxin (Stx)-producing Escherichia coli (STEC) related diseases in humans. Granulocytes are attracted and activated by Stxs in the enteric mucosa and are believed to thereby contribute to the intestinal inflammation. Mature ruminants, the main reservoir hosts of STEC, do not develop pathological changes that can be attributed to the Stxs. To prove whether the latter phenomenon correlates with the inability of the Stxs to affect granulocytes of ruminants, we investigated the ability of Stx1 to bind to granulocytes of cattle and sheep and analysed the effects of Stx1 on viability, phagocytosis, and oxidative burst activity. Bovine granulocytes from blood and milk did not express Stx1-binding sites even after activation of the cells and also were resistant to Stx1. In contrast to bovine granulocytes, granulocytes of sheep constitutively expressed Stx1-receptors of the Gb(3)/CD77 type ex vivo and bound the recombinant B-subunit of Stx1 (rStxB1). Stx1 holotoxin induced apoptosis in ovine granulocytes after prolonged incubation (18h) but Stx1 only slightly altered the phagocytosis and oxidative burst activities. The rStxB1 had no effect on granulocytes of either species. While arguing in favour of our initial hypothesis, that granulocytes of both, cattle and sheep are not activated by Stxs, the results of our study are the first evidences for differences in the cellular distribution of Stx-receptors in species equally regarded as STEC carriers.     

Coccidial infections in housed lambs: oocyst excretion, antibody levels and genetic influences on the infection

Faecal Eimeria oocyst excretion, body weights, humoral antibodies against E. ovinoidalis sporozoite antigen and related heritabilities were determined in housed Merinoland sheep lambs throughout a period of 100 days after birth in Germany. Altogether 10-11 Eimeria spp. were found. Cumulative incidences of E. ovinoidalis and E. weybridgensis/crandallis increased rapidly resulting in almost 100% incidence in 8 weeks old lambs. In the other species, the cumulative incidence increased more continuously. Except for E. granulosa oocysts of all species had been excreted at last once until day 30. By far the highest oocyst counts (OpG) were observed with E. ovinoidalis, followed by E. weybridgensis/crandallis. High counts were limited to the period of 5-8 weeks after birth. In the other Eimeria species oocyst counts persisted at comparatively low levels until the end of the observation period although their proportion of the total counts increased with age of the lambs. Time courses of oocyst excretion suggest an early onset of effective immunity to the major Eimeria spp., which differed for the minor species. Mean and maximum oocyst counts and body weights of the lambs were inversely correlated suggesting negative effects of the infection on the lamb's performance. High mean antibody levels on day 7 after birth dropped until day 40 and increased subsequently again. There were no indications that maternal antibodies were protective. Antibody levels on day 40 after birth were positively correlated with oocyst counts in the faeces whereas those determined on day 80 were independent of infection parameters. Heritabilities of log(10)OpG were not significantly different from 0 up to an age of 60 days. Later estimated values were between 0.54 and 0.79 suggesting that immune protective effects rather than innate effects determining disease susceptibility are under genetic influence.     

Root turnover and root necromass accumulation of Norway spruce (Picea abies) are affected by soil acidity

Fine root distribution and turnover were investigated in ca. 40-year-old pure Norway spruce (Picea abies Karst.) stands in Germany, growing on four sites that differed in soil acidity (Eberg?tzen < Barbis < Fichtelgebirge = Harz). The density of fine root biomass and necromass in different soil horizons differed among the sites. At one of the most acidic sites (Harz), fine root density in the humus layer was more than twice that at the least acidic site (Eberg?tzen). At the two most acidic sites, Fichtelgebirge and Harz, the ratio of biomass to necromass was significantly lower than at Eberg?tzen and Barbis, particularly in the subsoil layer. In each stand, clear vertical gradients in fine root length density and root tip density were observed. Most of the roots and the root tips were in the humus layer and in the first mineral soil horizon (0-10 cm). There was a significantly different decrease in specific root length (cm gDM (-1)) and specific root tip density (root tips gDM (-1)) in the more acidified stands Fichtelgebirge and Harz compared with Eberg?tzen and Barbis. Fine root production estimated by ingrowth cores and a net method was approximately twice as high in the more acidic stands Fichtelgebirge and Harz compared with Eberg?tzen and Barbis. Rates of living fine root biomass turnover were higher at the Fichtelgebirge and Harz sites than at the Eberg?tzen site. Rates of necromass turnover were similar at all sites. The results suggest that the accumulation of necromass was not due to a slower disappearance at the more acid sites, but to earlier root death. Roots contributed 46% to root + needle litter and 32% to root + total aboveground litter at the Harz site in 1997.     

Comparative use of faecal egg count reduction test,egg hatch assay and beta-tubulin codon 200 genotyping in small strongyles (cyathostominae) before and after benzimidazole treatment

A survey on benzimidazole (BZ) resistance in small strongyles was performed on three farms in the tenth region in Chile. Samples from a total of 100 horses were tested using the faecal egg count reduction test (FECRT), the egg hatch assay (EHA) and an allele-specific PCR for the detection of beta-tubulin isotype 1 genes coding for phenylalanine (phe) or tyrosine (tyr) at codon 200. In the past, BZ-type drugs have been used within anthelmintic campaigns on all the three farms. This has predictably led to a high degree of BZ resistance at the Valdivia and Ri?ihue farms and to a lesser degree at the Frutillar farm, as demonstrated by all the three tests. The FECRT indicated resistance in every farm by faecal egg count reductions (FECR) of 27% (S.D. +/- 33), 26.5% (S.D. +/- 26.9) and 83.9% (S.D. +/- 22.8) for the Valdivia, Ri?ihue and Frutillar farms, respectively. With the EHA, the following mean LD(50) values were found before and after treatment with fenbendazole (FBZ): 0.093, 0.141 and 0.066 microg TBZ/ml and 0.149, 0.158 and 0.091 microg TBZ/ml, respectively, for the Valdivia, Ri?ihue and Frutillar samples. The corresponding LD(96) values were 0.222, 0.263 and 0.188 microg TBZ/ml before treatment and 0.316, 0.322 and 0.221 microg TBZ/ml after treatment, indicating BZ resistance in all the cases. Genotyping was performed on more than 1700 single larvae, at least 10 per faecal sample, for 98 pre- and 66 post-treatment samples. Despite a general trend toward higher percentages of phe/tyr and tyr/tyr individuals following treatment, no statistically significant difference was found between these two and the phe/phe genotype percentages. However, a significantly negative correlation was detected between the LD(50) values and the phe/phe percentages and there was a positive correlation between the FECRT results and the phe/phe percentages. Thus, there seems to be a difference in the significance of the codon 200 polymorphism in the mechanisms of BZ resistance in small strongyles of the horse and sheep trichostrongyles.     

Lack of detectable West Nile virus RNA in brains and kidneys of dogs and cats with immunohistological precipitates using virus-specific antibodies

Five dogs and four cats from Germany suffering from encephalitis revealed positive immunoreactivity using two West Nile virus (WNV) specific monoclonal antibodies in brain and in kidney. However, WNV-infection could not be confirmed by additional PCR analyses. This study indicated that positive immunoreactivity for WNV in dogs and cats must be interpreted cautiously and should be confirmed by a second virus-specific technique.     

Base cations ameliorate Zn toxicity but not Cu toxicity in sugar beet (Beta vulgaris)

Seedlings of sugar beet (Beta vulgaris) were grown in nutrient solutions containing a range of Cu and Zn concentrations. Based on measurements of shoot and root length and dry weight, copper was found to be already toxic at 10 μM, while Zn became toxic at 100 μM. At Cu and Zn levels found to induce a similar level of growth inhibiton, the influence of increasing the supply of K, Ca and Mg was investigated. Increasing the concentration of both Ca and Mg in the nutrient solution attenuated the degree of inhibition of root growth by Zn, but not Cu. Potassium did not affect the toxicity of either Cu or Zn. An increase in Ca decreased the level of both Cu and Zn in roots. Magnesium ameliorated the toxicity effects of Zn without effecting the Zn concentration in the roots. Treatment with Zn significantly decreased the concentration of Mg in the roots. An increased supply of Mg lowered the percentage decrease in root Mg concentration due to Zn toxicity. The maintenance of an adequate Mg level in the roots may be critical to prevent Zn induced inhibition of root growth.     

Aluminium and lead tolerance in ectomycorrhizal fungi

Al and Pb tolerance of different ectomycorrhizal fungi was tested in Petri dishes divided into three compartments. One compartment contained nutrient solution supplemented with Al or Pb but without P to avoid precipitation of Al or Pb phosphates. Phosphate was supplied in the agar compartment while a third compartment served as control containing nutrient solution without P. The tested ectomycorrhizal fungal species and isolates varied greatly in their Al and Pb tolerance. A more than 50% growth reduction was observed at 2000 μM Al in Paxillus involutus MAI but already at 200 μM in P. involutus NAU and P. involutus 533. In contrast, growth of Pisolithus tinctorius 956 appeared to be stimulated by Al. Al tolerance therefore decreased in the following order: P. tinctorius 956 > P. involutus MAI > P. involutus NAU, P. involutus 533. To test the effect of agar on Al tolerance, P. involutus MAI was grown with agar in all three compartments. In presence of agar, even 2000 μM Al had no effect on growth. Lead at concentrations of 1 and 10 μM Pb had no significant effect on fungal growth. A more than 50% decrease of mycelial surface area occurred at 500 μM Pb in P. involutus MAI and P. involutus 533 and at 100 μM Pb in P. involutus NAU and P. tinctorius 956. Pb tolerance therefore decreased in the following order: P. involutus MAI, P. involutus 533 > P. involutus NAU, P. tinctorius 956. Divided Petri plates appear to be a valuable tool to test metal tolerance of fungi as complexation of the metals by agar or precipitation with P are avoided. The results indicate that some ectomycorrhizal fungi may be more sensitive to Al and Pb than previously reported.     

Relation between respiration,ATP content,and Adenylate Energy Charge (AEC) after incubation at different temperatures and after drying and rewetting

Temperature, drying, and rewetting are important climatic factors that control microbial properties. In the present study we looked at the respiration rates, adenosine 5′‐triphosphate (ATP) content, and adenylate energy charge (AEC) as a measure for energy status of microbial biomass in the upper 5 cm of mineral soils of three beech forests at different temperatures and after rewetting. The soils differed widely in pH (4.0 to 6.0), microbial biomass C (92 to 916 μg (g DW)^—1) and ATP content (2.17 to 7.29 nmol ATP (g DW)^—1). The soils were incubated for three weeks at 7 °C, 14 °C, and 21 °C. After three weeks the microbial properties were determined, retaining temperature conditions. The temperature treatment did not significantly affect AEC or ATP content, but respiration rates increased significantly with increasing temperature. In a second experiment the soils were dried for 12 hours at 40 °C. Afterwards the soils were rewetted and microbial properties were monitored for 72 hours. After the drying, respiration rates dropped below the detection limit, but within one hour after rewetting respiration rates increased above control level. Drying reduced AEC by 16 % to 44 % and ATP content by 47 % to 78 %, respectively. Rewetting increased AEC and ATP content significantly as compared to dry soil, but after 72 hours the level of the controls was still not reached. The level of AEC values indicated dormant cells, but ATP content increased. These results indicate that the microbial carbon turnover was not directly linked to microbial growth or microbial energy status. Furthermore our results indicate that AEC may describe an average energy status but does not reflect phases of growing, dormant, or dying cells in the complex microbial populations of soils.