Effect of Tween 80 and monensin on ruminal fermentation of the diet containing 70% wheat straw treated by white-rot fungus in artificial rumen

The objective of this study was to investigate the effect of Tween 80 and monensin on rumen fermentation of the diet containing 70% wheat straw treated by white-rot fungus Pleurotus tuber-regium (TWS-PT) and 30% barley in artificial rumen (RUSITEC). The RUSITEC consisted of four fermentation vessels (V1, V2, V3, V4): V1 was without additives (control), V2 received daily 10 mg of monensin, V3 received daily 0.5% Tween 80 (vol.wt-1) and V4 involved the combination of 10 mg of monensin with 0.5% Tween 80 (vol.wt-1). After an adaptation period (6 days) the fermentation parameters were determined for six consecutive days. Tween 80 did not affect the rumen fermentation of the diet consisting 70% TWS-PT and 30% barley in RUSITEC. Monensin affected the rumen fermentation of the diet by the decreased degradability of DM, neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose, cellulose (p < 0.001), the decrease of methane production (p < 0.001) and the higher proportion of propionate within the volatile fatty acids (p < 0.001) in comparison to control. Tween 80 did not improve the potency of monensin. Only some indices of the increase mol% of propionate (about 3.4%) and the decrease of methane production (about 0.47 mmol.day-1) were found by using Tween 80 plus monensin in comparison to use of monensin alone.     

Mycoplasma gallisepticum hemagglutinin V1hA, pyruvate dehydrogenase PdhA, lactate dehydrogenase, and elongation factor Tu share epitopes with Mycoplasma imitans homologues

Mycoplasma gallisepticum is a major pathogen of poultry. Mycoplasma imitans is genetically and antigenically closely related to M. gallisepticum, but so far, only a few proteins of M. imitans have been identified as sharing epitopes with M. gallisepticum. In this study, we identified three proteins of M. gallisepticum that share with M. imitans epitopes defined by monoclonal antibodies (MAbs). MAb 9D4 reacted with the 67-kD hemagglutinin V1hA (previously termed pMGA) of M. gallisepticum and with its continuously expressed 40-kD protein. This MAb also reacted with a 40-kD protein of M. imitans, but not with its putative V1hA. Two-dimensional (2D) immunoblots of M. gallisepticum strains showed that their 40-kD proteins reacting with MAb 9D4 are expressed as major forms with isoelectric points (pI) around 6, and also as less-abundant forms differing in pI. In M. imitans, major forms of 40-kD proteins recognized by MAb 9D4 had pI around 6, whereas minor forms had pI between 5.5 and 5.8. The N-terminal sequence of the M. gallisepticum 40-kD protein recognized by MAb 9D4 strongly indicates that this protein is pyruvate dehydrogenase E1, subunit alpha (PdhA protein, also termed AcoA). The position of elongation factor Tu (EF-Tu), detected by the reference MAb GB8, was very similar in the 2D proteome maps of M. gallisepticum and M. imitans (MW of about 45 kD; pI - 5.6). In both M. gallisepticum and M. imitans, MAb 7G1 reacted with proteins of about 36 kD with similar charges (major forms with pI of about 8). The position of this protein in the proteome map of M. gallisepticum and its N-terminal sequence strongly suggest that MAb 7G1 recognizes lactate (malate) dehydrogenase (Ldh or Mdh). Comparison of 2D proteomes of 10 M. gallisepticum strains indicated that positions of EF-Tu, PdhA, and Ldh proteins are rather consistent and can be used as reference points in further analyses of the M. gallisepticum proteome.     

The image of exocytosis during neutrophils and macrophages phagocytic activities in inflammation of mammary gland triggered by experimental Staphylococcus aureus infection

The experiments were carried out in five clinically normal virgin heifers. Before the experimental infection and at 24, 48, 72 and 168 h after the infection, respective mammary glands were rinsed with phosphate buffered saline. Neutrophils as well as macrophages underwent a classic exocytosis accompanied by translocation of lysosomal granules. The granules filled the protuberances of the plasmalemma and after the protuberances separated from the cell, they entered the extracellular space in the shape of round bodies of different sizes. After exocytosis, neutrophils displayed a smaller nucleus/cytoplasm ratio, a greater chromatin density of the nucleus, and an overall smaller size. Macrophages phagocytosed bacteria and/or neutrophils with and without signs of apoptosis (early and late apoptotic respectively) and neutrophils after exocytosis. Macrophages underwent cytolysis that was accompanied by extrusion of granules, phagosomes and phagolysosomes containing phagocytosed bacteria or neutrophils. Confluences were formed in which the process of digestion continued. Apoptosis of neutrophils gradually appeared and intensified in resolution of inflammation. The macrophages contributed to the inactivation of bacterial noxa as well as of histotoxic contents of neutrophils. Nevertheless, macrophages often underwent cytolysis at the site of inflammation.     

Identification of major immunogenic proteins of Mycoplasma synoviae isolates

Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively.     

Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland lymphocytes

The aim of this study was to determine whether lymphocyte apoptosis is modulated by infections caused by Staphylococcus aureus and Streptococcus uberis. Samples of cell populations were obtained by lavage of the mammary glands at 4 intervals (24, 48, 72 and 168 h) following infection. The percentage of apoptotic lymphocytes peaked at 168 h after challenge with S. aureus or S. uberis. Subsequent experiments focused on in vitro cultivation of mammary gland lymphocytes with S. aureus and S. uberis. These experiments showed a lower percentage of apoptotic lymphocytes following 3 h of cultivating cells with bacteria than after cultivation without bacteria. The results demonstrate that during both experimental infection of bovine mammary glands with S. aureus or S. uberis and during in vitro cultivation of lymphocytes with S. aureus or S. uberis, apoptosis of lymphocytes is delayed.     

Pathotypes of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> causing damage to oilseed rape in the Czech Republic and Poland

Winter oilseed rape (Brassica napus) is an important crop in the Czech Republic and Poland. Clubroot disease caused by the pathogen Plasmodiophora brassicae is a serious and still-growing problem for oilseed rape growers in both countries. The aim of this study was to evaluate the pathotype composition of P. brassicae populations from the Czech Republic and Poland, according to the three evaluation systems, and to determine soil inoculum loads for representative fields via traditional end-point PCR as well as quantitative PCR analysis. There were considerable differences between the populations of P. brassicae from both countries, and the number of pathotypes varied depending on the evaluation system and the threshold used to distinguish susceptible vs. resistant plant reactions. This is the first study comparing the effect of different thresholds. Using an index of disease (ID) of 25 % to distinguish susceptible vs. resistant reactions, there was a total of seven pathotypes identified based on the differentials of Williams, five with the system of Somé et al., and 18 with the European Clubroot Differential (ECD) set. However, based on a threshold of 50 %, there were nine pathotypes according to the evaluation system by Williams, four based on the differentials of Somé et al., and 15 with the ECD set. Changing of the thresholds led to the reclassification of some pathotypes. Several pathotypes were common in both countries. High amounts of pathogen DNA were found in many of the field soils analysed by quantitative PCR. There was a weak correlation between soil pH and infestation of P. brassicae for the Polish soils.     

Simultaneous detection and genetic variability of stone fruit viroids in the Czech Republic

In this paper, we report a large-scale survey for the incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit collections and commercial orchards in the Czech Republic. From the 645 samples analysed, PLMVd was detected in 80 (26.6%) of peaches and the HSVd in 3 (1.3%) of apricot and 1 (0.33%) of peach trees. Sixty-seven accession of peach (44.6%) from the Czech Clonal GeneBank were infected by PLMVd. In addition, we used naturally infected trees to standardise the simultaneous detection of PLMVd and HSVd plus host mRNA as the control by means of one-step multiplex RTC-PCR. Eleven PLMVd and two HSVd isolates were sequenced and analysed. All the PLMVd variants were highly homologous (97–100%) to previously reported PLMVd variants from Tunisian peach and almond trees, and clustered together in the previously reported phylogenetic group III. The HSVd variants obtained from apricot and peach trees were included in the previously proposed recombinant group PH/cit3.     

Dead wood in European beech (Fagus sylvatica) forest reserves

Data were analysed on the volume of dead wood in 86 beech forest reserves, covering most of the range of European beech forests. The mean volume was 130 m³/ha and the variation among reserves was high, ranging from almost nil to 550 m³/ha. The volume depended significantly on forest type, age since reserve establishment and volume of living wood. More dead wood was found in montane (rather than lowland/submontane) reserves, longer-established reserves (time since designation) and reserves with higher volumes of living wood.

On average, fallen dead wood contributed more to the total dead wood volume than standing dead wood. The percentage of dead wood that was standing was almost twice as high in montane than in lowland/submontane forest reserves (45% versus 25%). The volume of dead wood at selected sites changed considerably over time. The fluctuations were significantly higher in lowland/submontane than montane reserves, possibly connected with differences in the disturbance regimes and especially damage caused by windstorms. In NW Europe, the blow down of formerly managed, even-aged stands led to extraordinary high volumes of dead wood shortly after reserve establishment.

The implications for forest management and biodiversity conservation are discussed. An increase in dead wood volumes must be carried out in accordance with the local/regional forest type and disturbance regime. Thus, in order to fulfil the requirements of as many wood-depending organisms as possible, it is important to preserve not only larger amounts of dead wood, but also dead wood of different types and dimensions as well as securing a long-term continuity of dead wood.     

Neutrophil apoptosis during experimentally induced Staphylococcus aureus mastitis

The objective of this study was to determine whether neutrophil apoptosis and their consequent elimination by macrophages from the mammary gland is modulated by an infection caused by Staphylococcus aureus (S. aureus). The study was performed on twenty mammary glands of 5 virgin heifers. A buffered physiological solution (PBS) was administered as a means of control into the mammary glands of the heifers and after 168 h, the glands were inoculated with S. aureus. The samples of cell populations were obtained by lavages of the mammary glands in 4 intervals (24, 48, 72 and 168 h) after the experimental infection. Flow cytometry was used for determination of Annexin-V positivity and propidium iodide (PI) negativity of neutrophils. Light microscopy was used for determination of neutrophil karyopyknosis. Cytochemistry was used for the detection of myeloperoxidase-positive (MPO+) macrophages. Instillation of S. aureus resulted in an intramammary infection which persisted during the following experimental period. The total number of both Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils peaked at 24 h after both of PBS and S. aureus administration. The highest percentages of Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils were detected 48 and 168 h after PBS and S. aureus administration, respectively. The total number of MPO+ macrophages was the highest 24 h and 48 h after PBS and S. aureus administration, respectively; the percentage of MPO+ macrophages was the highest at 72 h in both cases. The dynamics of resolution of mastitis caused by S. aureus was very similar to the resolution of inflammatory response of the mammary gland after PBS administration. Mechanisms of cell pathogen elimination as well as inflammation resolution were very intensively involved; nevertheless, the mammary gland infection persisted. An early inclusion of the mechanisms of an acute inflammatory resolution thus paradoxically led to chronic infection.