In Vitro Antibacterial Activity of Microbial Natural Products against Bacterial Pathogens of Veterinary and Zoonotic Relevance.

Antimicrobial resistance (AMR) is considered one of the greatest threats to both human and animal health. Efforts to address AMR include implementing antimicrobial stewardship programs and introducing alternative treatment options. Nevertheless, effective treatment of infectious diseases caused by bacteria will still require the identification and development of new antimicrobial agents. Eight different natural products were tested for antimicrobial activity against seven pathogenic bacterial species (Brachyspira sp., Chlamydia sp., Clostridioides sp., Mannheimia sp., Mycobacterium sp., Mycoplasma sp., Pasteurella sp.). In a first pre-screening, most compounds (five out of eight) inhibited bacterial growth only at high concentrations, but three natural products (celastramycin A [CA], closthioamide [CT], maduranic acid [MA]) displayed activity at concentrations <2 µg/mL against Pasteurella sp. and two of them (CA and CT) also against Mannheimia sp. Those results were confirmed by testing a larger collection of isolates encompassing 64 Pasteurella and 56 Mannheimia field isolates originating from pigs or cattle, which yielded MIC90 values of 0.5, 0.5, and 2 µg/mL against Pasteurella and 0.5, 4, and >16 µg/mL against Mannheimia for CA, CT, and MA, respectively. CA, CT, and MA exhibited higher MIC50 and MIC90 values against Pasteurella isolates with a known AMR phenotype against commonly used therapeutic antimicrobial agents than against isolates with unknown AMR profiles. This study demonstrates the importance of whole-cell antibacterial screening of natural products to identify promising scaffolds with broad- or narrow-spectrum antimicrobial activity against important Gram-negative veterinary pathogens with zoonotic potential.

Description, occurrence and significance of Mycoplasma seminis sp. nov. isolated from semen of a gyrfalcon (Falco rusticolus).

The Mycoplasma strain ARNO was isolated from the semen of a clinically healthy gyrfalcon (Falco rusticolus). Colonies of strain ARNO grew in fried-egg shape on Mycoplasma agar plates (SP4). The organism did not ferment glucose or hydrolyze arginine or urea; hence, organic acids are assumed as energy source. Growth was sterol-dependent and optimal growth temperature 42 °C, with a temperature range from 20 to 44 °C. Strain ARNO was not identified as a representative of any of the currently described Mycoplasma species by alignment of the 16S rRNA gene sequence and 16 S-23 S intergenic transcribed spacer region, or immunobinding assay. Hence, strain ARNO represents a novel Mycoplasma species for which the name Mycoplasma seminis sp. nov. is proposed (DSM 27653, NCTC 13927). After developing a species-specific PCR, the prevalence of M. seminis sp. nov. was determined in adult and juvenile falcons in a commercial breeding center for falcons. Semen samples (n = 171) were obtained from 113 male adults, due to repeated sampling of 39 birds. Female adults (n = 26) were sampled once, while 105 of the 152 juvenile birds were sampled twice via choanal swabs. Mycoplasma seminis sp. nov. was found in the semen of clinically healthy adult males (3.5 %) as well as in the respiratory tract of female (34.6 %) and juvenile birds (59.2 %). After comparison of semen samples with (2.9 %) and without M. seminis sp. nov. identification, no indications for a potential influence on the semen quality were demonstrated. Hence, M. seminis sp. nov. seems likely to be of commensal character in falcons.

Differential expression of hemolysin genes in weakly and strongly hemolytic Brachyspira hyodysenteriae strains.

BACKGROUND: Swine dysentery (SD) is a diarrheal disease in fattening pigs that is caused by the strongly hemolytic species Brachyspira (B.) hyodysenteriae, B. hampsonii and B. suanatina. As weakly hemolytic Brachyspira spp. are considered less virulent or even non-pathogenic, the hemolysin is regarded as an important factor in the pathogenesis of SD. Four hemolysin genes (tlyA, tlyB, tlyC, and hlyA) and four putative hemolysin genes (hemolysin, hemolysin activation protein, hemolysin III, and hemolysin channel protein) have been reported, but their role in strong hemolysis is not entirely clear. Our study aimed to assess the transcriptional activity of eight (putative) hemolysin genes in a strongly hemolytic (B204) and a weakly hemolytic (G423) B. hyodysenteriae strain during non-hemolytic and hemolytic growth stages. RESULTS: Strongly and weakly hemolytic B. hyodysenteriae strains caused hemolysis on blood agar at different growth stages, namely during log phase (B204) and stationary/death phase (G423). During the lag, early log, late log (stationary phase in G423) and death phase (time points 1-4) strains differed in their hemolysin gene transcription patterns. At time point 1, transcription of the putative hemolysin gene was higher in B204 than in G423. At time point 2, tlyA and tlyC were upregulated in B204 during hemolysis. TlyB and hlyA were upregulated in both strains at all time points, but higher transcription rates were observed in the weakly hemolytic strain G423. The transcription activity of the hemolysin channel protein gene was quite similar in both strains, whereas the hemolysin activation protein gene was upregulated in the non-hemolytic stage of B204 at time point 4. Sequence analysis revealed deletions, insertions and single nucleotide polymorphisms in the G423 hlyA promoter, although without altering the transcription activity of this gene. CONCLUSION: Our data indicate a combined activity of TlyA and TlyC as the most probable underlying mechanism of strong hemolysis in B. hyodysenteriae. Further studies should verify if the expression of tlyA is upregulated by the putative hemolysin gene. Depending on their immunogenic potential TlyA and TlyC may serve as possible vaccine candidates, especially since vaccines for an effective control of swine dysentery are currently not available.

Canine Brucellosis: Insights Into the Epidemiologic Situation in Europe.

Brucella canis is one of many responsible pathogens of discospondylitis in dogs and infections require specific management. Little is known about the epidemiologic situation in Europe. The purpose of the study was to get insights into the occurrence of brucellosis in dogs in Europe. The database of a European veterinary laboratory was screened for Brucella positive samples. Additionally, medical records of a veterinary hospital in Germany were screened for diagnosis of discospondylitis and brucellosis. The laboratory received samples from 20 European countries for Brucella testing in dogs: 3.7% of submitted samples were Brucella spp. PCR-positive (61/1,657), and Brucella canis antibodies were identified in 5.4% of submitted samples (150/2,764). Brucella spp. PCR-positive samples originated from Spain (11.1% of submitted samples), Poland (6.7% of submitted samples) and rarely from Italy and France. Samples with Brucella canis antibodies originated from 13 European countries (Sweden, Belgium, Austria, Switzerland, Italy, Finland, Germany, Denmark, Hungary, Norway, Poland, France, Netherlands). Young dogs (0-24 months) had a 5.4-fold increased risk of PCR positive samples. The supplementary medical records search identified four young female dogs (7-30 months) with Brucella canis discospondylitis in Germany. The four dogs had been imported to Germany from Eastern European countries (Moldavia, Romania, Macedonia). In conclusion, infection with Brucella canis needs to be considered in dogs in Europe and diagnostics for Brucella canis infection appear indicated in young dogs with discospondylitis.

Phylogenetic diversity, antimicrobial susceptibility and virulence gene profiles of Brachyspira hyodysenteriae isolates from pigs in Germany.

Swine dysentery (SD) is an economically important diarrheal disease in pigs caused by different strongly hemolytic Brachyspira (B.) species, such as B. hyodysenteriae, B. suanatina and B. hampsonii. Possible associations of epidemiologic data, such as multilocus sequence types (STs) to virulence gene profiles and antimicrobial susceptibility are rather scarce, particularly for B. hyodysenteriae isolates from Germany. In this study, B. hyodysenteriae (n = 116) isolated from diarrheic pigs between 1990 and 2016 in Germany were investigated for their STs, susceptibility to the major drugs used for treatment of SD (tiamulin and valnemulin) and genes that were previously linked with virulence and encode for hemolysins (tlyA, tlyB, tlyC, hlyA, BHWA1_RS02885, BHWA1_RS09085, BHWA1_RS04705, and BHWA1_RS02195), outer membrane proteins (OMPs) (bhlp16, bhlp17.6, bhlp29.7, bhmp39f, and bhmp39h) as well as iron acquisition factors (ftnA and bitC). Multilocus sequence typing (MLST) revealed that 79.4% of the isolates belonged to only three STs, namely ST52 (41.4%), ST8 (12.1%), and ST112 (25.9%) which have been observed in other European countries before. Another 24 isolates belonged to twelve new STs (ST113-118, ST120-123, ST131, and ST193). The temporal distribution of STs revealed the presence of new STs as well as the regular presence of ST52 over three decades (1990s-2000s). The proportion of strains that showed resistance to both tiamulin und valnemulin (39.1%) varied considerably among the most frequent STs ranging from 0% (0/14 isolates resistant) in ST8 isolates to 46.7% (14/30), 52.1% (25/48), and 85.7% (6/7) in isolates belonging to ST112, ST52, and ST114, respectively. All hemolysin genes as well as the iron-related gene ftnA and the OMP gene bhlp29.7 were regularly present in the isolates, while the OMP genes bhlp17.6 and bhmp39h could not be detected. Sequence analysis of hemolysin genes of selected isolates revealed co-evolution of tlyB, BHWA1_RS02885, BHWA1_RS09085, and BHWA1_RS02195 with the core genome and suggested independent evolution of tlyA, tlyC, and hlyA. Our data indicate that in Germany, swine dysentery might be caused by a limited number of B. hyodysenteriae clonal groups. Major STs (ST8, ST52, and ST112) are shared with other countries in Europe suggesting a possible role of the European intra-Community trade of pigs in the dissemination of certain clones. The identification of several novel STs, some of which are single or double locus variants of ST52, may on the other hand hint towards an ongoing diversification of the pathogen in the studied area. The linkage of pleuromutilin susceptibility and sequence type of an isolate might reflect a clonal expansion of the underlying resistance mechanism, namely mutations in the ribosomal RNA genes. A linkage between single virulence-associated genes (VAGs) or even VAG patterns and the phylogenetic background of the isolates could not be established, since almost all VAGs were regularly present in the isolates.

Isolation and antimicrobial susceptibility of Brachy -spira species from feces of layer chickens in Germany.

OBJECTIVE: Anaerobic spirochetes of the genus Brachyspira are important pathogens causing swine dysentery (Brachyspira [B.] hyodysenteriae) and porcine intestinal spirochetosis (B. pilosicoli, PIS). In addition, avian intestinal spirochetosis (AIS) is caused by B. pilosicoli, B. intermedia and B. alvinipulli. Despite the economic impact of AIS, the disease has not received appropriate attention in Germany. This study was aimed at identifying Brachyspira spp. in Germany and determining their antimicrobial susceptibility. MATERIAL AND METHODS: From 2009 to 2013, a total of 71 fecal swabs were obtained from clinically healthy layer hens from eight different commercial flocks. Brachyspira spp. culture was performed in trypticase soybean agar added with 5% sheep blood. Species determination was conducted by PCRs targeting the NADH-gen and the 16S rDNA or by nox-gene sequencing. Antimicrobial susceptibility to macrolides, lincosamides and pleuromutilins was tested by a microdilution assay. RESULTS: Brachyspira spp. were isolated from 40 (56.3%) swabs distributed over all eight flocks. In 26 cases, the following species were determined by PCR: B. pilosicoli (n = 16), B. intermedia (2), B. innocens (3), B. murdochii (1), mixtures of B. pilosicoli/B. intermedia (2), B. innocens/B. intermedia (1), B. innocens/B. murdochii (1). Remaining isolates were characterized by noxgene sequencing as B. "pulli" (n = 9), B. alvinipulli (3), B. intermedia (1) and as not identifiable (1). Antimicrobial susceptibility testing of 37 isolates revealed minimal inhibitory concentrations 90 (MIC90) of > 128 mg/l (tylosin), 64 mg/l (lincomycin), 8 mg/l (tiamulin) and 4 mg/l (valnemulin), respectively. Comparing to breakpoints applied to pigs, these values lie within the range of resistance. CONCLUSION: The demonstration of different Brachyspira spp., particularly B. pilosicoli, intermedia and alvinipulli in commercial layers, indicates the need of further research to assess their potential role in causing AIS in German poultry flocks. The increased antimicrobial resistance of Brachyspira spp. isolates to tylosin and pleuromutilins is likely associated with extensive use of these drugs in poultry medicine.

An update of Brachyspira hyodysenteriae serotyping.

Brachyspira (B.) hyodysenteriae the causative agent of swine dysentery (SD) has been divided into 9 serotypes on basis of its lipooligosaccharide (LOS). Knowledge on circulating serotypes in Europe, however, is rare. Regarding that immunity to SD is serotype specific an update of B. hyodysenteriae serotyping was undertaken. A LOS band of 10 to 25kDa was identified being appropriate for this purpose. Isolates from Germany, Spain, Denmark, USA and Japan were characterized in the immunoblot by sera raised to serotypes 1 through 7, serogroups H and I (reference strains) and to eight German strains. In total, 57 (51%) isolates responded to at least one of the antisera. Regarding German isolates (n=75) only 35 (46.7%) were identified but mainly by antisera to German strains. Positive Spanish isolates (12 of 17) yielded similar results. In contrast, positively reacting Danish isolates (9 of 12) were mainly identified by antisera to the reference strains as it was the case for recent U.S. (1 of 8) and Japanese isolates (3 of 5). Results indicate that B. hyodysenteriae has a high degree of serological heterogeneity that has probably differently developed in diverse geographical areas over time. This situation represents a challenge for vaccine development.

Detection of virus particles resembling circovirus and porcine circovirus 2a (PCV2a) sequences in feces of dogs.

During routine electron microscopy of fecal samples from diarrheic dogs dated from 2000 virus particles resembling circovirus in shape and size were detected in two samples (V2177/00; V3374/00). Polymerase chain reaction (PCR) using primers specific for porcine circovirus type 2 (PCV2) amplified DNA recovered from both samples. Sequencing of PCR amplificates (V2177/00) obtained with PCV2-specific primer pairs revealed a genome size of 1768bp. The nucleotide sequence was highly similar (98% nucleotide identity) to the PCV2a reference sequence.

Influenza A (H10N7) Virus Causes Respiratory Tract Disease in Harbor Seals and Ferrets.

Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7 (Seal/H10N7) in harbor seals (Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.

Description and prevalence of Mycoplasma ciconiae sp. nov. isolated from white stork nestlings (Ciconia ciconia).

The mycoplasma strain ST 57T was isolated from the trachea of a clinically healthy, free-ranging white stork nestling in Nielitz, Mecklenburg-Western Pomerania, Germany. Strain ST 57T grew in fried-egg-shaped colonies on mycoplasma (SP4) agar plates and was dependent on sterol for growth. The organism fermented glucose and did not hydrolyse arginine or urea. The optimal growth temperature was 37 °C, with a temperature range from 23 to 44 °C. Strain ST 57Tcould not be identified as a representative of any of the currently described mycoplasma species by alignment of the 16S rRNA gene sequence or 16S-23S intergenic transcribed spacer region, or by immunobinding assays. Thus, this organism appears to be a representative of a novel species, for which the name Mycoplasma ciconiae sp. nov. is proposed. The type strain is ST 57T (=ATCC BAA-2401T=DSM 25251T). Four further strains of this species are included in this description (ST 24=DSM 29908, ST 56 Clone 1=DSM 29054, ST 99=DSM 29909, ST 102=DSM 29010). The prevalence of this mycoplasma species in clinically healthy, white stork nestlings in northern Germany was determined. Our species-specific PCR detected 57.8 % (48/83) of the samples positive for M. ciconiae sp. nov. As this species appears to be widespread in the healthy free-ranging white stork population, we conclude that this species is either apathogenic or an opportunistic pathogen in white storks.

Isolation and propagation of the animal rotaviruses in MA-104 cells--30 years of practical experience.

A total of 136 rotavirus positive samples from diarrhoeic animals of different species were submitted for isolation and cultural propagation of rotavirus on MA-104 cells. The samples were collected from animals with diarrhoea, between 1980 and 2010, originating from herds or farms located in several parts of Germany. Rotaviruses of species A were isolated from 102 faecal samples in cultures of MA-104 cells under the following conditions: pre-treatment of virus with trypsin, incorporation of trypsin into culture medium, use of roller cultures, and centrifugation of the samples on the cells. The cell culture adapted viruses produced a cytopathic effect, accompanied by the release of cells from the glass surface of the cultivation vessels. After 10 passages the virus isolates yielded titres between 10(5.5) and 10(7.5)ml(-1) TCID50. Isolation and serial propagation of the virus in MA-104 cells was confirmed by immunofluorescence assay, transmission electron microscopy, and polyacrylamide-gel electrophoresis of viral dsRNA. Eight (5.9%) of the electrophoretic profiles were characteristic of species B or D rotaviruses, which were not replicated in MA-104 cells.

Analysis of Multiple Brachyspira hyodysenteriae Genomes Confirms That the Species Is Relatively Conserved but Has Potentially Important Strain Variation.

The intestinal spirochete Brachyspira hyodysenteriae is an important pathogen in swine, causing mucohemorrhagic colitis in a disease known as swine dysentery. Based on the detection of significant linkage disequilibrium in multilocus sequence data, the species is considered to be clonal. An analysis of the genome sequence of Western Australian B. hyodysenteriae strain WA1 has been published, and in the current study 19 further strains from countries around the world were sequenced with Illumina technology. The genomes were assembled and aligned to over 97.5% of the reference WA1 genome at a percentage sequence identity better than 80%. Strain regions not aligned to the reference ranged between 0.2 and 2.5%. Clustering of the strain genes found on average 2,354 (88%) core genes, 255 (8.6%) ancillary genes and 77 (2.9%) unique genes per strain. Depending on the strain the proportion of genes with 100% sequence identity to WA1 ranged from 85% to 20%. The result is a global comparative genomic analysis of B. hyodysenteriae genomes revealing potential differential phenotypic markers for numerous strains. Despite the differences found, the genomes were less varied than those of the related pathogenic species Brachyspira pilosicoli, and the analysis supports the clonal nature of the species. From this study, a public genome resource has been created that will serve as a repository for further genetic and phenotypic studies of these important porcine bacteria. This is the first intra-species B. hyodysenteriae comparative genomic analysis.

Mutations in the 50S ribosomal subunit of Brachyspira hyodysenteriae associated with altered minimum inhibitory concentrations of pleuromutilins.

Brachyspira hyodysenteriae, the causative agent of swine dysentery, is responsible for severe mucohaemorrhagic colitis with considerable financial loss to worldwide swine production. Antimicrobial resistance against macrolides and lincosamides is widespread and the mechanisms are well known. Currently, the most common treatment for swine dysentery is the use of pleuromutilins and resistance to these drugs also is increasingly being reported. Although resistance mechanisms against pleuromutilins are less clear than for other drugs, they seem to involve alterations of the peptidyl transferase centre (PTC), including ribosomal RNA and the ribosomal protein L3. The present study was conducted to examine molecular mechanisms of resistance on a representative set of B. hyodysenteriae field strains with different resistance patterns. In total, we identified 24 single nucleotide polymorphisms (SNPs) in the 23S rRNA gene and genes of the ribosomal proteins L3, L4, L2 and L22. The SNP in the ribosomal protein gene L3 at position 443 led to an amino acid substitution of asparagine (Asn) by serine (Ser) at position 148, significantly associated with MICs for pleuromutilins. Based on this SNP a correct assignment of 71% of the strains with respect to a threshold of >0.625 µg tiamulin/ml was reached. Unexpectedly low MICs in some of the Asn-strains were explained by a second SNP at position 2535 of the 23S rRNA. Our results clearly show the associations between MICs for pleuromutilins and mutations in their binding site. A complete list of SNPs that influence MICs of B. hyodysenteriae strains is needed to enable the interpretation of future molecular susceptibility testing.

Adherence of Brachyspira hyodysenteriae to porcine intestinal epithelial cells is inhibited by antibodies against outer membrane proteins.

Attachment of Brachyspira hyodysenteriae to intestinal epithelial cell lines and its possible mediation by outer membrane proteins (OMPs) of the spirochete were examined. Different B. hyodysenteriae serotypes were shown to adhere to rat and swine intestinal epithelial cells (IEC-18 and IPEC-J2) in vitro but not to the human rectal tumor cell line (HRT-18). Adherence of strain B204 to IPEC-J2 cells was reduced by rOMP-specific antisera in amounts of 29 % (anti-rBhlp29.7), 59 % (anti-rBhlp16), 70 % (anti-rBhmp39h), and 74 % (anti-rBhmp39h), respectively. By use of pooled antisera against Bhlp16 and Bhmp39f inhibition rates of the other serotypes ranged from 53 to 91 %. In a western blot assay OMPs of all serotypes but one were detected by the respective rOMP antisera. Altogether the results indicated that OMPs of B. hyodysenteriae displayed a serotype overlapping antigenicity and mediated adherence of the spirochetes to animal cell cultures.

Demonstration of genes encoding virulence and virulence life-style factors in Brachyspira spp. isolates from pigs.

The distribution of many genes encoding virulence and virulence life-style (VL-S) factors in Brachyspira (B.) hyodysenteriae and other Brachyspira species are largely unknown. Their knowledge is essential e.g. for the improvement of diagnostic methods targeting the detection and differentiation of the species. Thus 121 German Brachyspira field isolates from diarrhoeic pigs were characterized down to the species level by restriction fragment length polymorphism analysis of the nox gene and subsequently subjected to polymerase chain reaction detecting VL-S genes for inner (clpX) and outer membrane proteins (OMPs: bhlp16, bhlp17.6, bhlp29.7, bhmp39f, bhmp39h), hemolysins (hlyA/ACP, tlyA), iron metabolism (ftnA, bitC), and aerotolerance (nox). For comparison, B. hyodysenteriae reference strains from the USA (n=7) and Australia (2) were used. Of all genes tested only nox was detected in all isolates. The simultaneous presence of both the tlyA and hlyA/ACP was restricted to the species B. hyodysenteriae. The hlyA infrequently occurred also in weakly hemolytic Brachyspira. Similarly to tlyA and hlyA all B. hyodysenteriae strains contained the ferritin gene ftnA which was also found in two Brachyspira intermedia isolates. OMP encoding genes were present in B. hyodysenteriae field isolates in rates of 0% (bhlp17.6, bhmp39h), 58.1% (bhlp29.7), and 97.3% (bhmp39f). Since the study revealed a high genetic heterogeneity among German B. hyodysenteriae field isolates differentiating them from USA as well as Australian strains, targets for diagnostic PCR were limited to the nox gene (genus specific PCR) as well as to the species specific nox(hyo) gene and the combination of hlyA and tlyA which allow to specifically detect B. hyodysenteriae.

Tenacity of mammalian viruses in the gut of leeches fed with porcine blood.

Leech therapy is currently considered to be of high therapeutic value in medicine. However, feeding leeches with fresh animal blood during the maintenance and reproduction phase bears the risk of transmission of zoonotic viruses to the patient. We hypothesize that this would be abolished by subjecting leeches to quarantine measures prior to use. The required duration of quarantine would depend on the maximum survival time of pathogens in contaminated leeches. In order to be able to estimate this survival time reliably, experiments were conducted with enveloped and non-enveloped mammalian viruses possessing either RNA or DNA. Leeches were fed porcine blood contaminated with bovine parvovirus (BPV), feline calicivirus (FCV), equine arteritis virus (EAV) and equine herpesvirus type 1 (EHV-1) and kept in aquaria at 10 °C. From week 6 after feeding onwards, some leeches were held at 30 °C. Before feeding and at different time points thereafter, blood samples were taken from the leeches to determine residual virus infectivity. Prototype mammalian viruses were able to survive in inoculated leeches for considerable periods of time. When leeches were kept at 10 °C throughout, reisolation of infectious virus from the leeches' abdominal cavity blood was no longer possible at 23 (FCV), 23 (EAV), 27 (EHV-1) and 29 (BPV) weeks after inoculation. Shifting the temperature to 30 °C in week 6 slightly reduced the duration of detection of infectious viruses to 15 (EAV and EHV-1), 21 (FCV) and 27 (BPV) weeks. These data indicate that the ability of mammalian viruses to survive in leeches theoretically poses a possible risk for patients unless adequate precautionary measures are adopted. Application of a quarantine period, e.g. 31 weeks (i.e. including an additional safety period) at 10 °C, may be a suitable measure to significantly decrease this risk.

Deficient incorporation of spike protein into virions contributes to the lack of infectivity following establishment of a persistent, non-productive infection in oligodendroglial cell culture by murine coronavirus.

Infection of mouse oligodendrocytes with a recombinant mouse hepatitis virus (MHV) expressing a green fluorescence protein facilitated specific selection of virus-infected cells and subsequent establishment of persistence. Interestingly, while viral genomic RNAs persisted in infected cells over 14 subsequent passages with concomitant synthesis of viral subgenomic mRNAs and structural proteins, no infectious virus was isolated beyond passage 2. Further biochemical and electron microscopic analyses revealed that virions, while assembled, contained little spike in the envelope, indicating that lack of infectivity during persistence was likely due to deficiency in spike incorporation. This type of non-lytic, non-productive persistence in oligodendrocytes is unique among animal viruses and resembles MHV persistence previously observed in the mouse central nervous system. Thus, establishment of such a culture system that can recapitulate the in vivo phenomenon will provide a powerful approach for elucidating the mechanisms of coronavirus persistence in glial cells at the cellular and molecular levels.

[Examination of biocides for their effectiveness against animal viruses according to European Union Standards with emphasis on the selection of a suitable test virus]. / Prüfung von Bioziden auf Wirksamkeit gegen animale Viren nach EU-Norm im Hinblick auf die Auswahl eines geeigneten Testvirus.

Because of the changes to be expected in the methods for testing disinfectants deemed to be used in the veterinary field, candidate viral species were evaluated for their suitability as test virus. Considered viral species included different non-enveloped viruses [bovine enterovirus type 1 (ECBO (Enteric Cytopathogenic Bovine Orphan) virus), mammalian reovirus type 1, feline calici virus (FCV), and bovine parvovirus (BPV)], as well as enveloped viruses, as equine arteritisvirus (EAV), bovine herpesvirus type 1 (BHV1), Newcastle disease virus (NDV) and vaccinia virus. Viruses were tested for their tenacity against different biocidal agents (formaldehyde, formic acid, peracetic acid, and sodium hypochlorite) in the suspension test at a temperature of 20 degrees C which is given as an optional test temperature according to prEN 14675 "Quantitative suspension test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in veterinary field--Test method and requirements"elaborated by the "Comite Européen de Normalisation"(CEN) (Anonym, 2004). Of the animal viruses tested for their tenacity highest tenacity against the disinfectants. FCV and the enveloped viruses were of lower resistance. In addition to the tenacity of viruses, other parameters, such as the ability of the virus to replicate in permanent cells, the magnitude of the virus titre that can be obtained from such cultures, as well as the threat a virus poses to humans and animals are to be considered when selecting a suitable test virus. Based on these criteria and despite its tenacity being inferior to that of BPV, the ECBO virus was chosen as the most suitable test virus. The result of the efficacy of disinfectants is not based on the most resistant virus in this case. This circumstance is to be considered when giving recommendations for the practical use of disinfectants.

Isolation and molecular characterisation of equine rotaviruses from Germany.

A total of 26 rotavirus positive faecal samples of diarrhoeal foals, and 8 equine rotavirus isolates were examined. Viral RNA patterns were generated, G typing was performed by PCR, and a P[12]-specific DNA probe was developed for P typing. Furthermore, five equine rotavirus isolates were sequenced in the genomic regions coding for VP7 and part of VP4. Rotaviruses of genotype G3 P[12] were found in 22 faecal samples and G14 P[12] type could be found in 4 faecal samples. These findings confirm that in Germany G3 P[12] is the predominating type of equine rotaviruses.