Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures.

BACKGROUND: We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation "short-read" and third-generation "long-read" sequencing methods. RESULTS: We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a "long-read first" approach. CONCLUSIONS: Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Influenza A(H5N8) Virus Similar to Strain in Korea Causing Highly Pathogenic Avian Influenza in Germany.

Highly pathogenic avian influenza (H5N8) virus, like the recently described H5N8 strain from Korea, was detected in November 2014 in farmed turkeys and in a healthy common teal (Anas crecca) in northeastern Germany. Infected wild birds possibly introduced this virus.

BVD-2 outbreak leads to high losses in cattle farms in Western Germany.

In November 2012, a dairy farmer in the district Kleve first observed a reduction in milk yield, respiratory symptoms, nasal discharge, fever, sporadic diarrhoea and sudden deaths in dairy cows and calves. In the following months, further farms were found infected with cattle showing similar clinical signs. An epidemiological investigation was carried out to identify the source of infection, the date of introduction, potential transmission pathways and to analyse the extent of the epidemic. Furthermore, laboratory analyses were conducted to characterise the causative agent. BVDV had been diagnosed in the index herd in December 2012, but due to the atypical clinical picture, the virus was not immediately recognised as the causative agent. Further laboratory analysis showed that this outbreak and subsequent infections in the area were caused by a BVD type 2c virus with a characteristic genome insertion, which seems to be associated with the occurrence of severe clinical symptoms in infected cattle. Epidemiological investigations showed that the probable date of introduction was in mid-October 2012. The high risk period was estimated as three months. A total of 21 affected farms with 5325 cattle were identified in two German Federal States. The virus was mainly transmitted by person contacts, but also by cattle trade and vehicles. The case-fatality rate was up to 60% and mortality in outbreak farms varied between 2.3 and 29.5%. The competent veterinary authorities imposed trade restrictions on affected farms. All persons who had been in contact with affected animals were advised to increase biosecurity measures (e.g. using farm-owned or disposable protective clothing). In some farms, affected animals were vaccinated against BVD to reduce clinical signs as an "emergency measure". These measures stopped the further spread of the disease.

Prevalence of antibodies against feline panleukopenia virus in client-owned cats in Southern Germany.

Feline panleukopenia is a frequent and commonly fatal disease of cats. Recent published studies have raised suspicions that some cats fail to develop antibodies after vaccination. The purpose of this study was to assess the prevalence of antibodies against feline panleukopenia virus (FPV) in cats in Southern Germany, and to identify factors that are associated with a lack of antibodies. In total, 350 cats presented to the Clinic of Small Animal Medicine, Ludwig-Maximilians-Universitaet were randomly included in the study. Information regarding signalment, origin, environment, lifestyle, housing conditions, health status, chronic diseases, glucocorticoid therapy, and vaccination status were collected. Antibodies were detected by haemagglutination inhibition test. Asymptomatic chi-squared tests and univariable logistic regression were used to investigate associations between a lack of antibodies and the different variables. Associations determined to be statistically significant at P<0.1 were verified by a multivariable logistic regression analysis. Of the 350 cats, 103 (29.4%) had no antibodies against FPV. Chronic kidney disease, neoplasia, glucocorticoid therapy, and vaccination status were significantly associated with a lack of antibodies. The cats with no antibodies were likely to have inadequate immunity against panleukopenia and those with chronic diseases or receiving glucocorticoids were less likely to be protected.

Population dynamics and in vitro antibody pressure of porcine parvovirus indicate a decrease in variability.

To estimate the impact of porcine parvovirus (PPV) vaccines on the emergence of new phenotypes, the population dynamic history of the virus was calculated using the Bayesian Markov chain Monte Carlo method with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed with consecutive passages of the 'Challenge' strain (a virulent field strain) and NADL2 strain (a vaccine strain) in a PK-15 cell line supplemented with polyclonal antibodies raised against the vaccine strain. A decrease in genetic diversity was observed in the presence of antibodies in vitro or after vaccination (as estimated by the in silico model). We hypothesized that the antibodies induced a selective pressure that may reduce the incidence of neutral selection, which should play a major role in the emergence of new mutations. In this scenario, vaccine failures and non-vaccinated populations (e.g. wild boars) may have an important impact in the emergence of new phenotypes.

An updated TaqMan real-time PCR for canine and feline parvoviruses.

Canine parvovirus type 2 (CPV-2) emerged in late 1970s from the feline panleukopenia virus (FPLV) and developed, since then, into novel genetic and antigenic variants (CPV-2a, -2b and -2c). Canine and feline parvoviruses cause an acute enteric disease in their hosts, with high level of viral shedding. In this study, a quantitative TaqMan PCR for detection and quantitation of canine and feline parvoviruses in serum and fecal samples was developed. The primers were designed based upon the entire GenBank content for CPV and FPLV. A standard curve was generated, and validation tests were performed using 10-fold serial dilutions of CPV-2 virus in CPV/FPLV-negative feces and CPV/FPLV-negative serum samples. As a result, the 100% detection limit of the PCR was 18 copies of the viral genome per µl of serum and fecal sample. All canine parvovirus types as well as FPLV were detected. In conclusion, the real-time PCR represents an upgraded and useful tool to identify and quantify canine and feline parvoviruses in different sample matrices.

Analysis of porcine parvoviruses in tonsils and hearts from healthy pigs reveals high prevalence and genetic diversity in Germany.

In the present study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for porcine parvoviruses (PPV, PPV2, PPV3 and PPV4). PPV was observed in 60 of 100 hearts and 61 of 100 tonsils, and PPV2 was observed in 55 of 100 hearts and 78 of 100 tonsils. PPV3 and PPV4 were found in 20 and 7, respectively, of the 100 tonsils tested, but not in the heart samples. Positive samples of PPV, PPV2 and PPV3 were analyzed by nucleotide sequencing, and phylogenetic analysis revealed at least two distinct lineages for each virus in the German samples. The high detection rate of PPV, PPV2 and PPV3 in healthy animals and their genetic diversity highlights the importance of continuous monitoring of these viruses and their zoonotic potential.

High rate of viral evolution in the capsid protein of porcine parvovirus.

In recent years, it has been shown that some parvoviruses exhibit high substitution rates, close to those of RNA viruses. In order to monitor and determine new mutations in porcine parvovirus (PPV), recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analysed. These samples, together with sequences retrieved from GenBank, were included in three datasets, consisting of the complete NS1 and VP1 genes and a partial VP1 gene. For each dataset, the nucleotide substitution rate and the molecular clock were determined. Analysis of the PPV field isolates revealed that a recently described amino acid substitution, S436T, appeared to be common in the VP2 protein in the Austrian, Brazilian and German virus populations. Furthermore, new amino acid substitutions were identified, located mainly in the viral capsid loops. By inferring the evolutionary dynamics of the PPV sequences, nucleotide substitution rates of approximately 10(-5) substitutions per site per year for the non-structural protein gene and 10(-4) substitutions per site per year for the capsid protein gene (for both viral protein datasets) were found. The latter rate is similar to those commonly found in RNA viruses. An association of the phylogenetic tree with the molecular clock analysis revealed that the mutations on which the divergence for both capsid proteins was based occurred in the past 30 years. Based on these findings, it was concluded that PPV variants are continuously evolving and that vaccines, which are based mainly on strains isolated about 30 years ago, should perhaps be updated.

The GimA locus of extraintestinal pathogenic E. coli: does reductive evolution correlate with habitat and pathotype?

IbeA (invasion of brain endothelium), which is located on a genomic island termed GimA, is involved in the pathogenesis of several extraintestinal pathogenic E. coli (ExPEC) pathotypes, including newborn meningitic E. coli (NMEC) and avian pathogenic E. coli (APEC). To unravel the phylogeny of GimA and to investigate its island character, the putative insertion locus of GimA was determined via Long Range PCR and DNA-DNA hybridization in 410 E. coli isolates, including APEC, NMEC, uropathogenic (UPEC), septicemia-associated E. coli (SEPEC), and human and animal fecal isolates as well as in 72 strains of the E. coli reference (ECOR) collection. In addition to a complete GimA (approximately 20.3 kb) and a locus lacking GimA we found a third pattern containing a 342 bp remnant of GimA in this strain collection. The presence of GimA was almost exclusively detected in strains belonging to phylogenetic group B2. In addition, the complete GimA was significantly more frequent in APEC and NMEC strains while the GimA remnant showed a higher association with UPEC strains. A detailed analysis of the ibeA sequences revealed the phylogeny of this gene to be consistent with that obtained by Multi Locus Sequence Typing of the strains. Although common criteria for genomic islands are partially fulfilled, GimA rather seems to be an ancestral part of phylogenetic group B2, and it would therefore be more appropriate to term this genomic region GimA locus instead of genomic island. The existence of two other patterns reflects a genomic rearrangement in a reductive evolution-like manner.

Signature-tagged mutagenesis in a chicken infection model leads to the identification of a novel avian pathogenic Escherichia coli fimbrial adhesin.

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.

The chicken as a natural model for extraintestinal infections caused by avian pathogenic Escherichia coli (APEC).

E. coli infections in avian species have become an economic threat to the poultry industry worldwide. Several factors have been associated with the virulence of E. coli in avian hosts, but no specific virulence gene has been identified as being entirely responsible for the pathogenicity of avian pathogenic E. coli (APEC). Needless to say, the chicken would serve as the best model organism for unravelling the pathogenic mechanisms of APEC, an extraintestinal pathogen. Five-week-old white leghorn SPF chickens were infected intra-tracheally with a well characterized APEC field strain IMT5155 (O2:K1:H5) using different doses corresponding to the respective models of infection established, that is, the lung colonization model allowing re-isolation of bacteria only from the lung but not from other internal organs, and the systemic infection model. These two models represent the crucial steps in the pathogenesis of APEC infections, including the colonization of the lung epithelium and the spread of bacteria throughout the bloodstream. The read-out system includes a clinical score, pathomorphological changes and bacterial load determination. The lung colonization model has been established and described for the first time in this study, in addition to a comprehensive account of a systemic infection model which enables the study of severe extraintestinal pathogenic E. coli (ExPEC) infections. These in vivo models enable the application of various molecular approaches to study host-pathogen interactions more closely. The most important application of such genetic manipulation techniques is the identification of genes required for extraintestinal virulence, as well as host genes involved in immunity in vivo. The knowledge obtained from these studies serves the dual purpose of shedding light on the nature of virulence itself, as well as providing a route for rational attenuation of the pathogen for vaccine construction, a measure by which extraintestinal infections, including those caused by APEC, could eventually be controlled and prevented in the field.

Avian pathogenic, uropathogenic, and newborn meningitis-causing Escherichia coli: how closely related are they?

Avian pathogenic Escherichia coli (APEC), uropathogenic E. coli (UPEC), and newborn meningitis-causing E. coli (NMEC) establish infections in extraintestinal habitats (extraintestinal pathogenic E. coli; ExPEC) of different hosts. As diversity, epidemiological sources, and evolutionary origins of ExPEC are so far only partially defined, we screened a collection of 526 strains of medical and veterinary origin of various O-types for assignment to E. coli reference collection (ECOR) group and virulence gene patterns. Results of ECOR typing confirmed that human ExPEC strains mostly belong to groups B2, followed by group D. Although a considerable portion of APEC strains did also fell into ECOR group B2 (35.1%), a higher amount (46.1%) belonged to group A, which has previously been described to also harbour strains with a high pathogenic potential for humans. The number of virulence-associated genes of single strains ranged from 5 to 26 among 33 genes tested and high numbers were rather related to K1-positive and ECOR B2 strains than to a certain pathotype. With a few exceptions (iha, afa/draB, sfa/foc, and hlyA), which were rarely present in APEC strains, most chromosomally located genes were widely distributed among all ExPEC strains irrespective of host and pathotype. However, prevalence of invasion genes (ibeA and gimB) and K1 capsule-encoding gene neuC indicated a closer relationship between APEC and NMEC strains. Genes associated with ColV plasmids (tsh, iss, and the episomal sit locus) were in general more prevalent in APEC than in UPEC and NMEC strains, indicating that APEC could be a source of ColV-located genes or complete plasmids for other ExPEC strains. Our data support the hypothesis that (a) poultry may be a vehicle or even a reservoir for human ExPEC strains, (b) APEC potentially serve as a reservoir of virulence-associated genes for UPEC and NMEC, (c) some ExPEC strains, although of different pathotypes, may share common ancestors, and (d) as a conclusion certain APEC subgroups have to be considered potential zoonotic agents. The finding of different evolutionary clusters within these three pathotypes implicates an independently and parallel evolution, which should be resolved in the future by thorough phylogenetic typing.