In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife

Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.     

Chronic Social Isolation Compromises the Activity of Both Glutathione Peroxidase and Catalase in Hippocampus of Male Wistar Rats

Chronic neuroendocrine stress usually leads to the elevation of the stress hormones and increased metabolic rate, which is frequently accompanied by oxidative damage to the CNS. In the present study we hypothesized that chronic psychosocial isolation (CPSI) of male Wistar rats, characterized by decreased serum corticosterone (CORT), unaltered catecholamines (CTs), and low blood glucose (GLU), may also promote oxidative imbalance in the CNS, by targeting antioxidant defense system. To test it, we have examined the relation between these input signals and protein expression/activity of antioxidant enzymes (AOEs): superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GLR) in the hippocampus (HIPPO) of CPSI animals. We found that CPSI did not affect SODs or CAT, but decreased activity of GPx and compromised GLR, an enzyme highly dependent on blood GLU for its substrate precursor. Further, we have tested whether the CPSI experience altered AOEs response to a novelty stress, and found that it attenuated peroxide-metabolizing enzymes, CAT and GPx, and decreased GLR activity, even though blood GLU was restored. The altered ratios of hippocampal AOEs in CPSI animals, which were worsened under the combined stress conditions, may lead to the accumulation of peroxide products and oxidative imbalance. The mechanism by which CPSI generate oxidative imbalance in the HIPPO is most likely based on poor systemic energy conditions set by this stress. Such conditions may cause functional decline of CNS structures, such as HIPPO, and are likely to promote state linked to onset of many mood disorders.     

Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase,a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.The ergot fungus Claviceps purpurea is a phytopathogenic ascomycete which infects the ears of several grasses, replacing the ovary and producing a hibernation structure, the so-called sclerotium, in which the ergot alkaloids are formed. These substances show a high level of structural homology to some neurotransmitters like serotonin and dopamine and can therefore bind to the same receptors in the central nervous system (CNS), which is the basis for the application of ergot alkaloids in a variety of clinical conditions (15).The biochemistry of ergot alkaloid biosynthesis was first investigated by isolation of intermediates and postulation of a hypothetical pathway as well as enzymes needed for the successive biosynthetic steps of the production (Fig. ​(Fig.1).1). Most of the data were collected by pursuing the fate of radiolabeled precursors in feeding experiments (4). The first enzyme which could be assigned to alkaloid production was dimethylallyltryptophan synthetase (DMATS), which is the key enzyme of the pathway and is encoded by the gene dmaW (18). These analyses were performed with a Claviceps fusiformis strain, but a homolog of dmaW ({"type":"entrez-nucleotide","attrs":{"text":"AY259840","term_id":"32402653","term_text":"AY259840"}}AY259840) possessing a similar function could also be isolated in C. purpurea, as was confirmed by a knockout approach (N. Lorenz and P. Tudzynski, unpublished data). Using genome walking combined with cDNA screening, a 68.5-kb genomic region surrounding dmaW could be sequenced and revealed 14 open reading frames (ORFs) (putative genes) encoding, among others, nonribosomal peptide synthetases (NRPSs), a putative catalase, a CYP450-1 monooxygenase, a putative methyltransferase, and several oxidoreductases (6, 13, 19) (Fig. ​(Fig.2).2). Some of these genes were functionally and biochemically analyzed by a gene replacement approach which revealed their function within the pathway (2, 5, 7). However, there is still a deficit in functional analyses, especially with respect to the early steps within this pathway. The conversion from N-methyl-dimethylallyltryptophan (Me-DMAT) to agroclavine via chanoclavine I and chanoclavine I aldehyde includes successive oxidation and reduction steps mediated by a specific class of enzymes, the oxidoreductases (15) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Biosynthetic pathway of the ergot alkaloid biosynthesis of C. purpurea. Genes analyzed so far have been assigned to the corresponding enzyme at the corresponding position within the pathway. DMAPP, dimethylallyldiphosphate; DMAT, dimethylallyltryptophan; Me-DMAT, N-methyl-DMAT. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)Open in a separate windowFIG. 2.Alkaloid biosynthesis gene cluster of C. purpurea. Highlighted in white is the gene of interest ccsA. (Adapted from reference 7 with permission of Wiley-VCH Verlag GmbH & Co. KGaA.)These enzymes are involved in the biosynthesis of many fungal secondary metabolites. A prominent example is the family of the cytochrome P450 monooxygenases (named after the characteristic peak of 450 nm when complexed with carbon monoxide). Cytochrome P450 (CYP450) monooxygenases catalyze the transfer of one oxygen atom from molecular oxygen to various substrates, mostly accomplished by the involvement of NAD(P)H as an electron donor. The eas cluster of C. purpurea also includes a gene encoding a CYP450 monooxygenase: cloA is involved in the oxidation of elymoclavine, leading to the formation of paspalic acid (7).No further monooxygenase-encoding genes seem to be present in the eas cluster, but several genes code for putative oxidoreductases (easA, easD, easE, easG, and easH). These oxidoreductases are most likely involved in the early steps within the pathway, but none of them has been functionally analyzed so far (15).We initiated a functional analysis of the putative oxidoreductase-encoding gene ccsA (formerly easE) (Fig. ​(Fig.2).2). The coding region of ccsA ({"type":"entrez-nucleotide","attrs":{"text":"AJ011965","term_id":"4499842","term_text":"AJ011965"}}AJ011965; 1,503 bp) is composed of two exons interrupted by an intron of 52 bp, yielding a coding capacity of 483 amino acids (aa). The gene product shows highest similarity to putative oxidoreductases of other ergot alkaloid-producing fungi: EasE of C. fusiformis (e⁻¹⁶⁰; {"type":"entrez-protein","attrs":{"text":"ABV57823","term_id":"157488556","term_text":"ABV57823"}}ABV57823), EasE of Neotyphodium lolii (e⁻¹¹⁸; {"type":"entrez-protein","attrs":{"text":"ABM91450","term_id":"124110194","term_text":"ABM91450"}}ABM91450) and CpoX1 of Aspergillus fumigatus (e⁻⁹⁶; {"type":"entrez-nucleotide","attrs":{"text":"XM_751049","term_id":"71002921","term_text":"XM_751049"}}XM_751049). Analyses of the protein sequence using the program PROSITE revealed a flavin adenine dinucleotide (FAD)-binding domain (pfam01565) spanning the region from amino acids 14 to 161 and a berberine bridge enzyme domain (BBE domain; pfam08031) from amino acids 412 to 457. The role of CcsA in the alkaloid biosynthesis pathway was investigated by knockout of the corresponding gene, followed by functional and biochemical analyses of the deletion mutants.     

Factors influencing the production of stilbenes by the knotweed, <Emphasis Type="Italic">Reynoutria</Emphasis> × <Emphasis Type="Italic">bohemica</Emphasis>

Background  

Japanese knotweed, Reynoutria japonica, is known for its high growth rate, even on adverse substrates, and for containing organic substances that are beneficial to human health. Its hybrid, Reynoutria × bohemica, was described in the Czech Republic in 1983 and has been widespread ever since. We examined whether Reynoutria × bohemica as a medicinal plant providing stilbenes and emodin, can be cultivated in spoil bank substrates and hence in the coalmine spoil banks changed into arable fields. We designed a pot experiment and a field experiment to assess the effects of various factors on the growth efficiency of Reynoutria × bohemica on clayish substrates and on the production of stilbenes and emodin in this plant.     

Molecular detection of Babesia canis in Dermacentor reticulatus ticks collected in Slovakia

Dermacentor reticulatus ticks are recognized as the most important vectors of Babesia canis, the aetiological agent of canine babesiosis occurring throughout Europe. Vector competence of D. reticulatus for B. canis is well described and experimentally determined; however, by using molecular analysis it was proven so by one recent study in Russia. Herein, the additional molecular evidence of B. canis infection in D. reticulatus ticks collected in Slovakia is provided. Using PCR followed by sequencing of distinctive amplicons we determined the presence of Babesia canis canis in one of 100 tested adult ticks. Two zoonotic pathogens, Francisella tularensis and Coxiella burnetii, were previously isolated from D. reticulatus ticks in Slovakia. In our samples, we detected only the presence of F. tularensis.     

Evidence for a fifth (G) region in theH-2 complex of the mouse

Antisera (B10.129×A)F₁ anti-P and (B10×A)F₁ anti-B10.P contain antibodies that define, in the PVP hemagglutination test, an antigen originally described as G or H-2.7. Of the independentH-2 haplotypes, the H-2.7 antigen is present inf, j, k, p, ands. In addition, the antisera also contain a weak cytotoxic antibody, distinct from anti-H-2.7. The cytotoxic antibody reacts with antigens controlled by theK orI regions. The hemagglutinating H-2.7 antibody does not have cytotoxic activity. The genetic determinant coding for antigen H-2.7 can be mapped into the chromosomal segment between theS andD regions. The H-2.7 antigen thus serves as a marker for a new region of theH-2 complex. The locus coding for antigen H-2.7 is designatedH-2 G and the correspondingH-2 regionG. The H-2.7 antigen has a tissue distribution distinct from that of the H-2 antigens controlled by theK orD regions. So far it could be detected primarily on erythrocytes.     

Large‐scale disturbance legacies and the climate sensitivity of primary Picea abies forests

Determining the drivers of shifting forest disturbance rates remains a pressing global change issue. Large‐scale forest dynamics are commonly assumed to be climate driven, but appropriately scaled disturbance histories are rarely available to assess how disturbance legacies alter subsequent disturbance rates and the climate sensitivity of disturbance. We compiled multiple tree ring‐based disturbance histories from primary Picea abies forest fragments distributed throughout five European landscapes spanning the Bohemian Forest and the Carpathian Mountains. The regional chronology includes 11,595 tree cores, with ring dates spanning the years 1750–2000, collected from 560 inventory plots in 37 stands distributed across a 1,000 km geographic gradient, amounting to the largest disturbance chronology yet constructed in Europe. Decadal disturbance rates varied significantly through time and declined after 1920, resulting in widespread increases in canopy tree age. Approximately 75% of current canopy area recruited prior to 1900. Long‐term disturbance patterns were compared to an historical drought reconstruction, and further linked to spatial variation in stand structure and contemporary disturbance patterns derived from LANDSAT imagery. Historically, decadal Palmer drought severity index minima corresponded to higher rates of canopy removal. The severity of contemporary disturbances increased with each stand's estimated time since last major disturbance, increased with mean diameter, and declined with increasing within‐stand structural variability. Reconstructed spatial patterns suggest that high small‐scale structural variability has historically acted to reduce large‐scale susceptibility and climate sensitivity of disturbance. Reduced disturbance rates since 1920, a potential legacy of high 19th century disturbance rates, have contributed to a recent region‐wide increase in disturbance susceptibility. Increasingly common high‐severity disturbances throughout primary Picea forests of Central Europe should be reinterpreted in light of both legacy effects (resulting in increased susceptibility) and climate change (resulting in increased exposure to extreme events).     

Global diversity of dipteran families (Insecta Diptera) in freshwater (excluding Simulidae, Culicidae, Chironomidae, Tipulidae and Tabanidae)

Today’s knowledge of worldwide species diversity of 19 families of aquatic Diptera in Continental Waters is presented. Nevertheless, we have to face for certain in most groups a restricted knowledge about distribution, ecology and systematic, particularly in the tropical environments. At the same time we realize a dramatically decline or even lack of specialists being able, having the time or the opportunity to extend or even secure the present information. The respective families with approximate numbers of aquatic species are: Blephariceridae (308), Deuterophlebiidae (14), Nyphomyiidae (7), Psychodidae (∼2.000), Scatopsidae (∼5), Tanyderidae (41), Ptychopteridae (69), Dixidae (173), Corethrellidae (97), Chaoboridae (∼50), Thaumaleidae (∼170), Ceratopogonidae (∼6.000), Stratiomyidae (∼43), Empididae (∼660), Lonchopteridae (2), Syrphidae (∼1.080), Sciomyzidae (∼190), Ephydridae (∼1.500), Muscidae (∼870). Numbers of aquatic species will surely increase with increased ecological and taxonomical efforts. Guest editors: E. V. Balian, C. Lévêque, H. Segers & K. Martens Freshwater Animal Diversity Assessment