Unidirectional mannitol synthesis of Acinetobacter baumannii MtlD is facilitated by the helix-loop-helix-mediated dimer formation.

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix­loop­helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.

K+ homeostasis is important for survival of Acinetobacter baumannii ATCC 19606 in the nosocomial environment.

Pathogenic bacteria have developed several mechanisms to thrive within the hostile environment of the human host, but it is often disregarded that their survival outside this niche is crucial for their successful transmission. Acinetobacter baumannii is very well adapted to both the human host and the hospital environment. The latter is facilitated by multifactorial mechanisms including its outstanding ability to survive on dry surfaces, its high metabolic diversity, and, of course, its remarkable osmotic resistance. As a first response to changing osmolarities, bacteria accumulate K+ in high amount to counterbalance the external ionic strength. Here, we addressed whether K+ uptake is involved in the challenges imposed by the harsh conditions outside its host and how K+ import influences the antibiotic resistance of A. baumannii. For this purpose, we used a strain lacking all major K+ importer ∆kup∆trk∆kdp. Survival of this mutant was strongly impaired under nutrient limitation in comparison to the wild type. Furthermore, we found that not only the resistance against copper but also against the disinfectant chlorhexidine was reduced in the triple mutant compared to the wild type. Finally, we revealed that the triple mutant is highly susceptible to a broad range of antibiotics and antimicrobial peptides. By studying mutants, in which the K+ transporter were deleted individually, we provide evidence that this effect is a consequence of the altered K+ uptake machinery. Conclusively, this study provides supporting information on the relevance of K+ homeostasis in the adaptation of A. baumannii to the nosocomial environment.

Detection of Novel Poxvirus from Gray Seal (Halichoerus grypus), Germany.

We detected a novel poxvirus from a gray seal (Halichoerus grypus) from the North Sea, Germany. The juvenile animal showed pox-like lesions and deteriorating overall health condition and was finally euthanized. Histology, electron microscopy, sequencing, and PCR confirmed a previously undescribed poxvirus of the Chordopoxvirinae subfamily, tentatively named Wadden Sea poxvirus.

Identification and characterization of a novel pathway for aldopentose degradation in Acinetobacter baumannii.

The nosocomial pathogen Acinetobacter baumannii is well known for its extraordinary metabolic diversity. Recently, we demonstrated growth on L-arabinose, but the pathway remained elusive. Transcriptome analyses revealed two upregulated gene clusters that code for isoenzymes catalysing oxidation of a pentonate to α-ketoglutarate. Molecular, genetic, and biochemical experiments revealed one branch to be specific for L-arabonate oxidation, and the other for D-xylonate and D-ribonate. Both clusters also encode an uptake system and a regulator that acts as activator (L-arabonate) or repressor (D-xylonate and D-ribonate). Genes encoding the initial oxidation of pentose to pentonate were not part of the clusters, but our data are consistent with the hypothesis of a promiscous, pyrroloquinoline quinone (PQQ)-dependent, periplasmic pentose dehydrogenase, followed by the uptake of the pentonates and their degradation by specific pathways. However, there is a cross-talk between the two different pathways since the isoenzymes can replace each other. Growth on pentoses was found only in pathogenic Acinetobacter species but not in non-pathogenic such as Acinetobacter baylyi. However, mutants impaired in growth on pentoses were not affected in traits important for infection, but growth on L-arabinose was beneficial for long-term survival and desiccation resistance in A. baumannii ATCC 19606.

K+ and its role in virulence of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions worldwide. The success of A. baumannii is based on the rise of multiple antibiotic resistances and its outstanding potential to persist in the human host and under conditions of low water activity in hospital environments. Combating low water activities involves osmoprotective measures such as uptake of compatible solutes and K+. To address the role of K+ uptake in the physiology of A. baumannii we have identified K+ transporter encoding genes in the genome of A. baumannii ATCC 19606. The corresponding genes (kup, trk, kdp) were deleted and the phenotype of the mutants was studied. The triple mutant was defective in K+ uptake which resulted in a pronounced growth defect at high osmolarities (300 mM NaCl). Additionally, mannitol and glutamate synthesis were strongly reduced in the mutant. To mimic host conditions and to study its role as an uropathogen, we performed growth studies with the K+ transporter deletion mutants in human urine. Both, the double (ΔkupΔtrk) and the triple mutant were significantly impaired in growth. This could be explained by the inability of ΔkupΔtrkΔkdp to metabolize various amino acids properly. Moreover, the reactive oxygen species resistance of the triple mutant was significantly reduced in comparison to the wild type, making it susceptible to one essential part of the innate immune response. Finally, the triple and the double mutant were strongly impaired in Galleria mellonella killing giving first insights in the importance of K+ uptake in virulence.

A novel alphaherpesvirus associated with fatal diseases in banded Penguins.

A novel avian alphaherpesvirus, preliminarily designated sphenicid alphaherpesvirus 1 (SpAHV-1), has been independently isolated from juvenile Humboldt and African penguins (Spheniscus humboldti and Spheniscus demersus) kept in German zoos suffering from diphtheroid oropharyngitis/laryngotracheitis and necrotizing enteritis (collectively designated as penguin-diphtheria-like disease). High-throughput sequencing was used to determine the complete genome sequences of the first two SpAHV-1 isolates. SpAHV-1 comprises a class D genome with a length of about 164 kbp, a G+C content of 45.6 mol% and encodes 86 predicted ORFs. Taxonomic association of SpAHV-1 to the genus Mardivirus was supported by gene content clustering and phylogenetic analysis of herpesvirus core genes. The presented results imply that SpAHV-1 could be the primary causative agent of penguin-diphtheria-like fatal diseases in banded penguins. These results may serve as a basis for the development of diagnostic tools in order to investigate similar cases of penguin diphtheria in wild and captive penguins.

A decade of research into classical swine fever marker vaccine CP7_E2alf (Suvaxyn® CSF Marker): a review of vaccine properties.

Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. However, until recently, the available live vaccines did not allow a serological marker concept that is essentially important to circumvent long-term trade restrictions. In 2014, a new live attenuated marker vaccine, Suvaxyn® CSF Marker (Zoetis), was licensed by the European Medicines Agency. This vaccine is based on pestivirus chimera "CP7_E2alf" that carries the main immunogen of CSF virus "Alfort/187", glycoprotein E2, in a bovine viral diarrhea virus type 1 backbone ("CP7"). This review summarizes the available data on design, safety, efficacy, marker diagnostics, and its possible integration into control strategies.

Deletion mutants of Schmallenberg virus are avirulent and protect from virus challenge.

UNLABELLED: Since its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were tested in vitro and in vivo in type I interferon receptor knockout mice (IFNAR(-/-)) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growth in vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR(-/-) mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses. IMPORTANCE: SBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genus Orthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire family Bunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

[Indurative mastitis in a herd of Dorper sheep caused by an infection with Maedi Visna virus]. / Indurative Mastitis in einer Herde Dorperschafe als Folge einer Infektion mit dem Maedi-Visna-Virus.

This case report describes indurative mastitis in a herd of sheep caused by Maedi Visna virus (MVV) infection. Reduced udder formation after delivery, small, indurated udders and increased losses of lambs were observed in a herd of Dorper sheep. Examination of the mammary gland and milk did not reveal findings characteristic of chronic bacterial mastitis. The protein supply was insufficient which may have contributed to reduced milk yield, but was considered unlikely as cause for the induration of the mammary gland. Nineteen of the 21 mothers were positive for MVV by serology. Mammary gland and supramammary lymph nodes were collected in a sheep with indurated udder at the time of slaughter. Meat inspection did not reveal lesions in any other organs. One part of the mammary gland showed a mild to moderate multifocal lymphohistiocytic mastitis, the other exhibited a severe diffuse lymphohistiocytic mastitis with atrophy of the glandular acini, vasculopathy, fibrosis and calcification. MVV antigen was visualized by immunohistochemistry in macrophages, dendritic cells, epithelial cells and endothelial cells in the mammary gland, and macrophages and dendritic cells in the supramammary lymph nodes. A large amount of MVV provirus was detected in the supramammary lymph nodes and the severely indurated part of the mammary gland by PCR. In conclusion, indurative mastitis as a result of a systemic infection may occur independently of the commonly known manifestations of Maedi Visna in the lung and central nervous system. MVV should be considered as differential diagnosis in mastitis of sheep. The MVV status of the herd can be tested by serological detection of specific antibodies. Additionally, characteristic histological lesions are present in the mammary gland. MVV antigen can also be detected by immunohistochemistry and MVV provirus by PCR in the altered mammary gland and regional lymph nodes.

The VBNC state: a fundamental survival strategy of Acinetobacter baumannii.

IMPORTANCE: Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.

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

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.

Bungowannah Pestivirus Chimeras as Novel Double Marker Vaccine Strategy against Bovine Viral Diarrhea Virus.

Marker or DIVA (differentiation of infected from vaccinated animals) vaccines are beneficial tools for the eradication of animal diseases in regions with a high prevalence of the designated disease. Bovine viral diarrhea virus (BVDV)-1 (syn. Pestivirus A) is a flavivirus that infects predominantly cattle resulting in major economic losses. An increasing number of countries have implemented BVDV eradication programs that focus on the detection and removal of persistently infected cattle. No efficient marker or DIVA vaccine is yet commercially available to drive the eradication success, to prevent fetal infection and to allow serological monitoring of the BVDV status in vaccinated farms. Bungowannah virus (BuPV, species Pestivirus F), a related member of the genus Pestivirus with a restricted prevalence to a single pig farm complex in Australia, was chosen as the genetic backbone for a marker vaccine candidate. The glycoproteins E1 and E2 of BuPV were substituted by the heterologous E1 and E2, which are major immunogens, of the BVDV-1 strain CP7. In addition, the candidate vaccine was further attenuated by the introduction of a deletion within the Npro protein coding sequence, a major type I interferon inhibitor. Immunization of cattle with the chimeric vaccine virus BuPV_ΔNpro_E1E2 CP7 (modified live or inactivated) followed by a subsequent experimental challenge infection confirmed the safety of the prototype strain and provided a high level of clinical protection against BVDV-1. The serological discrimination of vaccinated cattle could be enabled by the combined detection of BVDV-1 E2- in the absence of both BVDV NS3- and BVDV Erns-specific antibodies. The study demonstrates for the first time the generation and application of an efficient BVDV-1 modified double marker vaccine candidate that is based on the genetic background of BuPV accompanied by commercially available serological marker ELISA systems.

Development of a Modular Vaccine Platform for Multimeric Antigen Display Using an Orthobunyavirus Model.

Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.

A first response to osmostress in Acinetobacter baumannii: transient accumulation of K+ and its replacement by compatible solutes.

The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+ , in osmostress response. Growth of A. baumannii was K+ -dependent and the K+ -dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+ . After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.

Full-genome sequencing of German rabbit haemorrhagic disease virus uncovers recombination between RHDV (GI.2) and EBHSV (GII.1).

Rabbit haemorrhagic disease virus (RHDV; genotypes GI.1 and GI.2) and European brown hare syndrome virus (EBHSV; genotype GII.1) are caliciviruses belonging to the genus Lagovirus. These viruses pose a serious threat to wild and domestic rabbit and hare populations around the world. In recent years, an expanding genetic diversity has been described within the genus, with recombination events occurring between the different genotypes. Here, we generated and analysed 56 full-genome sequences of RHDV and EBHSV from rabbit and hare livers, collected in Germany between the years 2013 and 2020. We could show that genotype Gl.2 (RHDV-2) almost entirely replaced Gl.1 (classical RHDV) in the German rabbit population. However, GI.1 is still present in Germany and has to be included into disease control and vaccination strategies. Three recombinant strains were identified from rabbit samples that contain the structural genes of genotype Gl.2 and the non-structural genes of genotype Gl.1b. Of special interest is the finding that sequences from two hare samples showed recombination events between structural genes of RHDV Gl.2 and non-structural genes of EBHSV GII.1, a recombination between different genogroups that has not been described before. These findings lead to the assumption that also a recombination of the non-structural genes of RHDV Gl.2 with the structural genes of EBHSV Gll.1 might be possible and therefore increase the potential genetic variability of lagoviruses immensely. Our findings underline the importance of whole genome analysis with next-generation sequencing technology as one of new tools now available for in-depth studies that allow in depth molecular epidemiology with continuous monitoring of the genetic variability of viruses that would otherwise likely stay undetected if only routine diagnostic assays are used.

Interdisciplinary Comparison of Endoscopic Laser-Assisted Diverticulotomy vs. Transcervical Myotomy as a Treatment for Zenker's Diverticulum.

BACKGROUND: Currently, there are different competing techniques for the treatment of Zenker's diverticulum (ZD). To improve patient selection, we compared endoscopic laser-assisted diverticulotomy (ELAD) with transcervical myotomy (TCM) with regard to possible risk factors for treatment failure. METHODS: Data of ZD patients (n = 104) treated between 2004 and 2016 with either TCM (38%) or ELAD (62%) were analyzed retrospectively. Univariate and multivariate analyses were performed. RESULTS: TCM is associated with a higher morbidity (27.8% vs. 10.2%; p = 0.095) but lower recurrence rate (7.3% vs. 19.3%; p = 0.095). Preoperative reflux disease (OR 8.755; p = 0.021) was identified as an independent risk factor for complications. CONCLUSIONS: Although short-term outcome and symptom relief are similar, TCM tends to have a higher complication rate but better long-term results. Preoperative reflux disease is an independent risk factor for postoperative complications.

A Synthetic Modified Live Chimeric Marker Vaccine against BVDV-1 and BVDV-2.

Bovine viral diarrhea virus (BVDV), a pestivirus which exists in the two distinct species BVDV-1 (syn. Pestivirus A) and BVDV-2 (syn. Pestivirus B), is the causative agent of one of the most widespread and economically important virus infections in cattle. For economic as well as for animal health reasons, an increasing number of national BVDV control programs were recently implemented. The main focus lies on the detection and removal of persistently infected cattle. The application of efficient marker or DIVA (differentiation of infected from vaccinated animals) vaccines would be beneficial for the eradication success in regions with a high BVDV prevalence to prevent fetal infection and it would allow serological monitoring of the BVDV status also in vaccinated farms. Therefore, a marker vaccine based on the cytopathic (cp) BVDV-1b strain CP7 was constructed as a synthetic backbone (BVDV-1b_synCP7). For serological discrimination of vaccinated from infected animals, the viral protein Erns was substituted by the heterologous Erns of Bungowannah virus (BuPV, species Pestivirus F). In addition, the vaccines were attenuated by a deletion within the type I interferon inhibitor Npro protein encoding sequence. The BVDV-2 vaccine candidate is based on the genetic sequence of the glycoproteins E1 and E2 of BVDV-2 strain CS8644 (CS), which were introduced into the backbone of BVDV-1b_synCP7_ΔNpro_Erns Bungo in substitution of the homologous glycoproteins. Vaccine virus recovery resulted in infectious cytopathic virus chimera that grew to titers of up to 106 TCID50/mL. Both synthetic chimera BVDV-1b_synCP7_ΔNpro_Erns Bungo and BVDV-1b_synCP7_ΔNpro_Erns Bungo_E1E2 BVDV-2 CS were avirulent in cattle, provided a high level of protection in immunization and challenge experiments against both BVDV species and allowed differentiation of infected from vaccinated cattle. Our study presents the first report on an efficient BVDV-1 and -2 modified live marker vaccine candidate and the accompanying commercially available serological marker ELISA system.

Serological Cross-Reactivity Between Bovine alphaherpesvirus 2 and Bovine alphaherpesvirus 1 in a gB-ELISA: A Case Report in Italy.

In this study, we demonstrated for the first time in Italy, the serological cross-reactivity between Bovine alphaherpesvirus 2 (BoHV-2) and Bovine alphaherpesvirus 1 (BoHV-1). Five months after arriving at a performance test station in Central Italy, a 6-month-old calf, which was part of a group of 57 animals, tested positive for BoHV-1 in a commercial gB-ELISA test. It was immediately transferred to the quarantine unit and subjected to clinical observation and serological and virological investigations. During this period, the calf showed no clinical signs. The results from laboratory investigations demonstrated the presence of antibodies via competitive glycoprotein B (gB) ELISAs, indirect BoHV-1 ELISAs, and indirect BoHV-2 ELISAs. Furthermore, the plaque reduction assay provided evidence for the presence of antibodies only for BoHV-2, whereas the virus neutralization test showed negative results for both BoHV-1 and BoHV-5. These findings strongly suggest the occurrence of a serological cross-reactivity between BoHV-2 and BoHV-1. Interference of BoHV-2 antibodies in serological BoHV-1 diagnostics should be considered during routine IBR tests, especially when animals are kept in a performance test station.

Fetal infection with Schmallenberg virus - An experimental pathogenesis study in pregnant cows.

Since its first appearance in 2011, Schmallenberg virus (SBV) has been repeatedly detected in aborted ruminant foetuses or severely malformed newborns whose mothers were naturally infected during pregnancy. However, especially the knowledge about dynamics of foetal infection in cattle is still scarce. Therefore, a total of 36 pregnant heifers were experimentally infected during two animal trials with SBV between days 60 and 150 of gestation. The foetuses were collected between 10 and 35 days after infection and virologically and pathologically investigated. Overall, 33 heifers yielded normally developed, macroscopically inconspicuous foetuses, but abundant virus replication was evident at the maternal/foetal interface and viral genome was detectable in at least one organ system of 18 out of 35 foetuses. One heifer was found to be not pregnant at autopsy. One of the animals aborted at day 4 after infection, viral RNA was detectable in the lymphatic tissue of the dam, in the maternal and foetal placenta, and in organs and lymphatic tissue of the foetus. In another foetus, SBV typical malformations like torticollis and arthrogryposis were observed. The corresponding dam was infected at day 90 of pregnancy and viral genome was detectable in the cerebellum of the unborn. Interestingly, no common patterns of infected foetal organs or maternal/foetal placentas could be identified, and both, sites of virus replication and genome loads, varied to a high degree in the individual foetuses. It is therefore concluded, that SBV infects in many cases also the bovine foetus of naïve pregnant cattle, however, the experimentally observed low abortion/malformation rate is in concordance to the reported low rates in the field during the first outbreak wave following the introduction of SBV. This observation speaks for a natural resistance of most bovine foetuses even during the vulnerable phase of early pregnancy, which has to be further studied in the future.

Salt induction and activation of MtlD, the key enzyme in the synthesis of the compatible solute mannitol in Acinetobacter baumannii.

Mannitol is the major compatible solute, next to glutamate, synthesized by the opportunistic human pathogen Acinetobacter baumannii under low water activities. The key enzyme for mannitol biosynthesis, MtlD, was identified. MtlD is highly similar to the bifunctional mannitol-1-phosphate dehydrogenase/phosphatase from Acinetobacter baylyi. After deletion of the mtlD gene from A. baumannii ATCC 19606T cells no longer accumulated mannitol and growth was completely impaired at high salt. Addition of glycine betaine restored growth, demonstrating that mannitol is an important compatible solute in the human pathogen. MtlD was heterologously produced and purified. Enzyme activity was strictly salt dependent. Highest stimulation was reached at 600 mmol/L NaCl. Addition of different sodium as well as potassium salts restored activity, with highest stimulations up to 41 U/mg protein by sodium glutamate. In contrast, an increase in osmolarity by addition of sugars did not restore activity. Regulation of mannitol synthesis was also assayed at the transcriptional level. Reporter gene assays revealed that expression of mtlD is strongly dependent on high osmolarity, not discriminating between different salts or sugars. The presence of glycine betaine or its precursor choline repressed promoter activation. These data indicate a dual regulation of mannitol production in A. baumannii, at the transcriptional and the enzymatic level, depending on high osmolarity.