Incidence of tick-borne spotted fever group Rickettsia species in rodents in two regions in Kazakhstan.

Records on the distribution of Rickettsia spp. in their natural hosts in Central Asia are incomplete. Rodents and small mammals are potential natural reservoirs for Rickettsiae in their natural lifecycle. Studies about the maintenance of Rickettsia in wild animals are available for Western nations, but-to our knowledge-no studies and data are available in the Republic of Kazakhstan so far. The first case description of Rickettsioses in Kazakhstan was made in the 1950ies in the Almaty region and now Kyzylorda, East Kazakhstan, Pavlodar and North Kazakhstan are endemic areas. The existence of murine and endemic typhus was proven in arthropod vectors in the regions Kyzylorda and Almaty. Here we show for the first time investigations on tick-borne Rickettsia species detected by a pan-rickettsial citrate synthase gene (gltA) real-time PCR in ear lobes of small mammals (n = 624) in Kazakhstan. From all analysed small mammals 2.72% were positive for Rickettsia raoultii, R. slovaca or R. conorii. Sequencing of the rickettsial gene OmpAIV and the 23S-5S interspacer region revealed a similar heritage of identified Rickettsia species that was observed in ticks in previous studies from the region. In summary, this study proves that rodents in Kazakhstan serve as a natural reservoir of Rickettsia spp.

Molecular and genomic characterization of a novel equine molluscum contagiosum-like virus.

Cases of pox-like lesions in horses and donkeys have been associated with poxviruses belonging to different genera of the family Poxviridae. These include the orthopoxviruses vaccinia virus (VACV), horsepoxvirus (HPXV) and cowpoxvirus (CPXV), as well as a potentially novel parapoxvirus and molluscum contagiosum virus (MOCV). However, with the exception of VACV, HPXV and CPXV, the genomic characterization of the causative agents remains largely elusive with only single short genome fragments available. Here we present the first full-length genome sequence of an equine molluscum contagiosum-like virus (EMCLV) directly determined from skin biopsies of a horse with generalized papular dermatitis. Histopathological analysis of the lesions revealed severe epidermal hyperplasia with numerous eosinophilic inclusion bodies within keratinocytes. Virions were detected in the lesions in embedded tissue by transmission electron microscopy. The genome sequence determined by next- and third-generation sequencing comprises 166 843 nt with inverted terminal repeats (ITRs) of 3473 nt. Overall, 20 of the predicted 159 ORFs have no equivalents in other poxviruses. Intriguingly, two of these ORFs were identified to encode homologues of mammalian proteins involved in immune signalling pathways, namely secreted and transmembrane protein 1 (SECTM1) and insulin growth factor-like family receptor 1 (IGFLR1), that were not described in any virus family so far. Phylogenetic analysis with all relevant representatives of the Poxviridae suggests that EMCLV should be nominated as a new species within the genus Molluscipoxvirus.

High prevalence of genetically diverse Borrelia bavariensis-like strains in Ixodes persulcatus from Selenge Aimag, Mongolia.

In Mongolia, Lyme borreliosis was first reported in 2003. To determine which Borrelia species may contribute to the occurrence of Lyme borreliosis in Mongolia, real-time PCR was conducted on 372 adult Ixodes persulcatus ticks collected in Selenge Aimag, the province with the highest incidence of human Lyme borreliosis. 24.5% of ticks were identified to be positive for Borrelia burgdorferi sensu lato DNA. Species differentiation using an SNP-based real-time PCR and multi-locus sequence analysis revealed that strains phylogenetically closely related to B. bavariensis (previously known as B. garinii OspA serotype 4) is the most prevalent species, showing an unexpectedly high genetic diversity.

A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.

Relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central Europe.

Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8×10(-4) substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus in zdár Kaplice proved for the first time that TBEV is indeed subject to local evolution.

Non-human primates in outdoor enclosures: risk for infection with rodent-borne hantaviruses.

Different species of non-human primates have been exploited as animal disease models for human hantavirus infections. To study the potential risk of natural hantavirus infection of non-human primates, we investigated serum samples from non-human primates of three species living in outdoor enclosures of the German Primate Center (GPC), Göttingen, located in a hantavirus endemic region of central Germany. For that purpose we used serological assays based on recombinant antigens of the bank vole (Myodes glareolus) transmitted Puumala virus (PUUV) and the common and field vole (Microtus arvalis, Microtus agrestis) associated Tula virus (TULV) which are both broadly geographically distributed in Germany. In 24 out of 251 (9.6%) monkey sera collected in 2006 PUUV- and/or TULV-reactive immunoglobulin G (IgG) antibodies were detected. Investigation of follow-up sera from 13 animals confirmed for two animals a seroconversion due to hantavirus exposure at the GPC. To prove the origin of the infection, wild rodents from the surrounding regions were analyzed by hantavirus-specific reverse transcriptase-PCR analysis. In 6 of the 73 investigated bank voles and 3 of the 19 investigated Microtus spp. PUUV- and TULV-specific nucleic acid sequences, respectively, were detected. In conclusion, our investigations demonstrate for the first time natural infections of non-human primates in outdoor enclosures in Germany. These findings highlight the importance of hantavirus surveillance in those primate housings and corresponding preventive measures against wild rodents, particularly in hantavirus endemic regions.

Network "Rodent-borne pathogens" in Germany: longitudinal studies on the geographical distribution and prevalence of hantavirus infections.

Hantavirus infections are known in Germany since the 1980s. While the overall antibody prevalence against hantaviruses in the general human population was estimated to be about 1-2%, an average of 100-200 clinical cases are recorded annually. In the years 2005 and 2007 in particular, a large increase of the number of human hantavirus infections in Germany was observed. The most affected regions were located in the federal states of Baden-Wuerttemberg, Bavaria, North Rhine Westphalia, and Lower Saxony. In contrast to the well-documented situation in humans, the knowledge of the geographical distribution and frequency of hantavirus infections in their rodent reservoirs as well as any changes thereof was very limited. Hence, the network "Rodent-borne pathogens" was established in Germany allowing synergistic investigations of the rodent population dynamics, the prevalence and evolution of hantaviruses and other rodent-associated pathogens as well as their underlying mechanisms in order to understand their impact on the frequency of human infections. A monitoring of hantaviruses in rodents from endemic regions (Baden-Wuerttemberg, Bavaria, North Rhine Westphalia, Lower Saxony) and regions with a low number of human cases (Mecklenburg Western-Pomerania, Brandenburg, Saxony, Saxony-Anhalt) was initiated. Within outbreak regions, a high prevalence of Puumala virus (PUUV) was detected in bank voles. Initial longitudinal studies in North Rhine Westphalia (city of Cologne), Bavaria (Lower Bavaria), and Lower Saxony (rural region close to Osnabrück) demonstrated a continuing presence of PUUV in the bank vole populations. These longitudinal studies will allow conclusions about the evolution of hantaviruses and other rodent-borne pathogens and changes in their distribution, which can be used for a risk assessment of human infections. This may become very important in order to evaluate changes in the epidemiology of rodent-borne pathogens in the light of expected global climate changes in the future.

Substitution of vaccinia virus Elstree by modified vaccinia virus Ankara to test the virucidal efficacy of chemical disinfectants.

After the eradication of variola in 1980, the smallpox vaccination was considered to be no longer required and was subsequently abandoned mainly because of possible adverse effects of vaccinia virus especially in first-time vaccinees. Despite a growing number of humans without immunity against vaccinia virus, vaccinia virus Lister Elstree (VACV) is still prescribed for testing virucidal efficacy of chemical disinfectants in the guidelines of the German Veterinary Medical Society [Deutsche Veterinärmedizinische Gesellschaft (DVG)], the German Association for the Control of Virus Diseases [Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV)] and the Robert Koch Institute (RKI). To evaluate a possible substitution of VACV, with the attenuated modified vaccinia virus Ankara (MVA) the virucidal efficacy of four different DVG-listed commercially available chemical disinfectants representing different groups of chemicals was tested against these two viruses. Quantitative suspension tests and qualitative carrier tests with poplar wood and gauze were performed. Distinction of VACV and MVA was confirmed by cytopathogenic effects, such as differences in plaque morphology. No significant difference in disinfection efficacy between VACV and MVA was observed for any of the disinfectants tested. Implying that vaccinia virus poses a risk after inadvertent inoculation, our results show that MVA, which does not replicate in humans, should replace VACV in the chemical disinfectant testing guidelines.

Long-lasting stability of vaccinia virus (orthopoxvirus) in food and environmental samples.

Poxviruses are known to remain infectious in the scabs of patients for months to years. The aim of this study was to investigate viral stability in storm water, food or gauze spiked with vaccinia virus strain Munich 1 (VACV M1). Storm water, storm water supplemented with either fetal calf serum (FCS) or potting soil was stored at two different temperatures (refrigerator, room temperature; 4 degrees C/25 degrees C). In addition, we analysed the viability of VACV M1 on the surface of bread, salad, sausages and gauze bandages stored at 4 degrees C. Samples were titrated in MA 104 cells and the presence of viral DNA was demonstrated by orthopoxvirus-specific PCRs. After 2 weeks, reisolation of VACV M1 from all kinds of food, bandage and water samples except for storm water supplemented with potting soil was possible. Viral DNA was detected in almost all samples by PCR. Prolonged experiments with VACV M1-spiked storm water and storm water supplemented with FCS revealed that samples kept at 4.5 degrees C are infectious for up to 166 days. Our data demonstrate that VACV M1 has a longlasting stability in water and food. The results obtained during this study should be taken into account for risk assessment calculations for poxvirus transmission. Implying that variola virus and vaccinia virus behave in a similar way, our data call for sophisticated countermeasures in cases of a variola release in biological warfare.

Re-emergence of Francisella tularensis in Germany: fatal tularaemia in a colony of semi-free-living marmosets (Callithrix jacchus).

Francisella tularensis was identified as the cause of a die-off which occurred among a colony of semi-free-living common marmosets (Callithrix jacchus). During the outbreak 5 out of 62 animals died of tularaemia in a research facility located in the district of Goettingen, Germany. All animals had been born at the facility suggesting an endemic infection. A total of five culture isolates were recovered and characterized as F. tularensis holarctica, biovar I. These cultures represent the first isolates obtained in the Federal Republic of Germany for more than 45 years. The outbreak area shows several geographical and ecological characteristics known to favour long-term presence of F. tularensis. Persistence of the pathogen in the remote region along the former German-German border, continuous re-introduction from eastern European countries after destruction of the 'Iron curtain' or introduction through migrating birds are testable hypotheses which could explain the emergence of tularaemia in this particular region.

Rickettsia spp. in Ixodes ricinus ticks in Bavaria, Germany.

This study aims to provide information on the occurrence of spotted fever rickettsiae in Ixodes ricinus ticks in southern Germany. A total of 2,141 I. ricinus ticks was collected in Bavaria. Pools of 5-10 ticks were studied by a PCR targeting the rickettsial citrate synthase gene gltA. The average prevalence rate was 12% (257 of 2,141). Sequencing data exclusively identified Rickettsia helvetica DNA. Results and other data demonstrate the possible role of R. helvetica in I. ricinus as a source of human infections in southern Germany.

A new Puumala hantavirus subtype in rodents associated with an outbreak of Nephropathia epidemica in South-East Germany in 2004.

A micro-epidemic of hantavirus infections occurred in Lower Bavaria, South-East Germany, starting in April 2004. While only three cases were registered from 2001 to 2003, a dramatically increased number of clinically apparent human hantavirus infections (n=38) was observed in 2004, plus seven additional cases by June 2005. To determine the reservoir responsible for the infections, a total of 43 rodents were trapped in Lower Bavaria. Serological and genetic investigations revealed that Puumala virus (PUUV) is dominant in the local population of bank voles. Partial PUUV S segment nucleotide sequences originating from bank voles at four different trapping sites in Lower Bavaria showed a low divergence (up to 3.1%). This is contrasted by a nucleotide sequence divergence of 14-16% to PUUV strains detected in Belgium, France, Slovakia or North-Western Germany. PUUV sequences from bank voles in Lower Bavaria represent a new PUUV subtype which seems to be responsible for the observed increase of human hantavirus infections in 2004-2005.

[Zoonotic poxviruses]. / Zoonotische Pockenviren.

In the last few years, the mass media and also scientists have been showing an increasing interest in poxviruses. What kind of infections can be found in animals and men today? The following review gives an overview on current taxonomy, properties, epidemiology, and diagnosis of zoonotic poxviruses.

Unusual presentations of cowpox infection in cats.

Cowpox virus infections are reported typically to cause focal ulcerated, crusted skin lesions, sometimes with mild systemic illness and concurrent oral lesions. Severe systemic illness usually only occurs in young or immunosuppressed individuals. This report describes four cases of cowpox infection in cats which illustrate variations to the usual presentation of the virus. The poxvirus infections were confirmed histopathologically, serologically and by PCR analysis.

Avipoxvirus infection in peregrine falcons (Falco peregrinus) from a reintroduction programme in Germany.

Poxvirus infections are common in domestic birds in Germany, but they are rare in birds of prey. Only species of falconidae imported from Arabian or Asian countries have so far tested positive for poxvirus, and, among these, only raptors kept for falconry. As part of a reintroduction programme in the northern county of Mecklenburg-Western Pomerania, which is adjacent to the Baltic Sea, 21 young peregrine falcons were released into the wild; six of them died and one was examined postmortem, its tissues being examined by light and electron microscopy. In addition, an ELISA for fowlpox, pigeonpox and canarypox was applied. No virus could be isolated and propagation in culture failed, but virus particles were detected by electron microscopy in lesions from its skin and tongue.

Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA.

Viruses were isolated in cell culture from tissue homogenates of flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Neutralization and immunofluorescence tests with aquabirnavirus (West Buxton strain)-specific polyclonal antisera indicated that both viruses were aquabirnaviruses belonging to Serogroup A, the most common aquabirnavirus serogroup in the United States. This was confirmed by RT-PCR, with primers targeting the VP3 and VP2 gene of aquabirnaviruses. The VP2-specific RT-PCR cDNA amplification product was sequenced and deduced amino-acid sequences were compared with known sequences of the type strains of the 9 serotypes of aquabirnavirus Serogroup A. This demonstrated that the viruses from both flounder and mummichog belong to aquabirnavirus Genogroup 1. The flounder isolate exhibited deduced amino acid sequence similarities of 98.1% with the Jasper strain of serotype A9, and 97.7% with the West Buxton strain of serotype A1. The isolate from mummichog exhibited deduced amino acid sequence similarities of 99.1% with the West Buxton strain of Serotype A1 and 94.8% with the Jasper isolate of Serotype A9. Similarities of deduced amino acid sequences ranged from 79.9 to 86.9%, with representatives of the other 7 serotypes. This is the first report of an aquabirnavirus from mummichog F. heteroclitus and only the fifth report of an aquabirnavirus from a flounder species.

[Human cowpox/catpox infection. A potentially unrecognized disease]. / Kuh-/Katzenpocken-Infektion beim Menschen. Ein potenziell verkanntes Krankheitsbild.

HISTORY AND ADMISSION FINDINGS: A 36-year-old woman initially noticed a red spot, about pea-sized, with a central pimple over the right eyebrow and a swollen submandibular lymph node. A pressure-sensitive, 4 cm large, node developed out of this small spot, with a central, black, tightly-adhering crust bearing several varioliform vesicles around its edge. In addition to swelling of the right half of the face, the patient had a fever up to 39.5 degrees C, general malaise, nausea and vomiting. Various antibiotics were ineffective. The woman was hospitalized with a diagnosis of facial erysipelas. She owned a cat which had developed a purulent nodule on a forepaw a few days before onset of the patient's disease. LABORATORY TEST: ESR and CRP were moderately elevated, no leukocytosis and blood cultures were sterile. Wound smears showed colonization with Klebsiella pneumoniae and Enterobacter cloacae. DIAGNOSIS, TREATMENT AND COURSE: The patient's general condition improved under initially calculated antibiotic dosages, which was later adapted to the measured resistance. The black-crusted nodes became larger, however, and incision was performed on the 8 th day after hospitalization, under the suspicion of fluctuation. However, no pus was removed, but there was massive inflammatory infiltration of the soft tissue. Examination of samples of skin and part of the crust revealed orthopox virus (cowpox virus). Spontaneous healing followed within 3 weeks, leaving only a small scar. CONCLUSIONS: This was a cowpox virus in the sense of a zoonosis transmitted by the cat. In Germany, now that smallpox has been eradicated, the clinical presentation of infections with the orthopox virus, which are closely related to variola virus, are too little recognized. Atopic and immunocompromised patients are at risk of a cutaneous dissemination with a more severe course of the infectious illness; even a lethal outcome has been reported in Germany.

The epizootic haematopoietic necrosis virus (Iridoviridae) induces apoptosis in vitro.

The epizootic haematopoietic necrosis virus (EHNV) is an iridovirus causing severe disease in different fish species. We investigated the induction of apoptosis during EHNV infection of the epithelioma carp papulosum (EPC) cell line. Apoptosis reveals several characteristic morphological changes, such as chromatin condensation, nuclear fragmentation, cytoplasm membrane disorientation, or mitochondrial changes. During EHNV infection of EPC cells the occurrence of apoptosis was analysed using a fluorescein-isothiocyanate (FITC) conjugate of annexin-V to detect phosphatidylserines that have changed cytoplasm membrane localization. Annexin-V labelling was obvious 12 h after infection. At 54 h after EHNV infection 39% of the investigated EPC cells exhibited fluorescence. Furthermore, EHNV-infected cells were stained with 4'-6'-diamidino-2-phenylindole (DAPI) to detect pycnotic nuclei. Appearance of DAPI-positive nuclei was found beginning at 18 h after infection. At 54 h after EHNV infection approximately 56% of the EPC cells showed fragmented nuclei. Assays to inhibit a protein kinase-dependent (e.g. double-stranded RNA-dependent protein kinase) apoptosis pathway with 2-aminopurine revealed a reduction of EHNV titres, e.g. titres were reduced 1000-fold in the presence of 100 and 200 mM 2-aminopurine. Apoptosis takes place during iridovirus infection in vitro and it seems to involve the activation of protein kinases.