Construction and Application of MALDI-TOF Mass Spectrometry for the Detection of Haemophilus parasuis.

To construct a protein fingerprint database of Haemophilus parasuis (H. parasuis), thus improving its clinical diagnosis efficiency. A total of 15 H. parasuis standard strains were collected to establish a protein fingerprint database of H. parasuis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the effects of different culture media and culture time on the quality and identification results of the protein fingerprint were investigated. The results showed that tryptone soy agar (TSA) and tryptone soy broth (TSB) media and different incubation times had no significant effect on the characteristic peaks of the protein profiles. In addition, 18 clinical isolates were used to compare the identification results of the self-built protein fingerprint database, PCR detection, and basic database. Only one strain was identified in the original VITEK-MS system database, while the self-made protein fingerprint database of H. parasuis was 100% accurate for the detection of 18 clinical isolate strains. The protein fingerprint database of H. parasuis built by our laboratory is suitable for rapid clinical diagnosis of H. parasuis, due to its high accuracy, efficiency, and strong specificity.

Comparative genome analysis of multidrug-resistant and susceptible Glaesserella parasuis strains isolated from diseased pigs in China.

Glaesserella parasuis (G. parasuis) is a respiratory pathogen of swine and the etiological agent of Glässer's disease. Although the emergence of multidrug-resistant (MDR) G. parasuis is a critical problem in the swine industry, there are few publications on the genetic basis of antimicrobial resistance of G. parasuis. In this study, comparative genome analyses were used to identify genomic differences between two phenotypically distinct isolates, an MDR isolate (HPS-1) and a susceptible isolate (HPS-2), from diseased swines in China. These isolates were both serovar 4, which is predominant in cases of Glässer's disease and is the most prevalent serovar in China. Based on clusters of orthologous group (COG) annotations, genes assigned to the extracellular structure category were only detected in HPS-1 and genes related to cell motility were more abundant in HPS-1 than in HPS-2. A comparative genomic analysis showed that these two isolates are closely related, although there was a large-scale genomic rearrangement. Eighteen percent of the genome consisted of strain-specific accessory genes of HPS-1. Notably, only the two genes aac(6')-Ie-aph(2'')-Ia and blaROB-1 on a plasmid were specific to HPS-1. We also detected 30,599 single nucleotide polymorphisms (SNPs), including nonsynonymous SNPs in the aminoglycoside resistance gene aph(3'')-Ib, the fusidic acid resistance gene fusA, and the two rifampicin resistance genes rpoC and rpoB in HPS-1. These findings improve our understanding of the differences between MDR and susceptible isolates and will aid the development of treatment strategies to decrease the prevalence and disease burden caused by G. parasuis.

Ct value-based real time PCR serotyping of Glaesserella parasuis.

Glaesserella parasuis is the causative agent of Glässer's disease in swine. Serotyping plays an essential role in prevalence investigations and in the development of vaccination strategies for the prevention of this disease. Molecular serotyping based on variation within the capsule loci of the 15 serovars is more accurate and efficient than traditional serological serotyping. To reduce the running time and facilitate ease of data interpretation, we developed a simple and rapid cycle threshold (Ct) value-based real time PCR (qPCR) method for the identification and serotyping of G. parasuis. The qPCR method distinguished between all 15 serovar reference strains of G. parasuis with efficiency values ranging between 85.5 % and 110.4 % and, R2 values > 0.98. The qPCR serotyping was evaluated using 83 clinical isolates with 43 of the isolates having been previously assigned to a serovar by the gel immuno-diffusion (GID) assay and 40 non-typeable isolates. The qPCR results of 41/43 (95.3 %) isolates were concordant with the GID assay except two isolates of serovar 12 were assigned to serovar 5. In addition, the qPCR serotyping assigned a serovar to each of the 40 non-typeable isolates. Of the 83 isolates tested to assign a serovar, a concordance rate of 98.8 % (82/83) was determined between the qPCR and the previously reported multiplex PCR of Howell et al. (2015) (including those that were either serovars 5 or 12). Despite the inability to differentiate between serovars 5 and 12, the Ct value-based qPCR serotyping represents an attractive alternative to current molecular serotyping method for G. parasuis and could be used for both epidemiological monitoring and the guidance of vaccination programs.

Importance of strain selection in the generation of heterologous immunity to Glaesserella (Haemophilus) parasuis.

Glaesserella (Haemophilus) parasuis is a part of the microbiota of healthy pigs and also causes the systemic condition called Glässer's disease. G. parasuis is categorized by it capsular polysaccharide into 15 serovars. Because of the serovar and strain specific immunity generated by whole cell vaccines and the rapid onset of disease, G. parasuis has been difficult to control in the swine industry. This report investigated the protection afforded by the use of two serovar 5 isolates (Nagasaki and HS069) as whole cell, killed bacterins against homologous challenge and heterologous challenge with the serovar 1 strain 12939 to better understand bacterin generated immunity. Both bacterins induced a high antibody titer to the vaccine strain and the heterologous challenge strain. Protection was seen with both bacterins against homologous challenge; however, after heterologous challenge, the HS069 bacterin provided complete protection and all Nagasaki bacterin vaccinated animals succumbed to disease. The difference in protection appears to be due to differences in antibody specificity and the capacity of induced antibody to fix complement and opsonize G. parasuis, as shown by Western blotting and functional assays. This report shows the importance of strain selection when developing bacterin vaccines, as some strains are better able to generate heterologous protection. The difference in protection seen here can also be utilized to detect proteins of interest for subunit vaccine development.

Phylogenomic analysis of Haemophilus parasuis and proposed reclassification to Glaesserella parasuis, gen. nov., comb. nov.

The Gram-negative bacterium Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs, and causes significant economic losses to the swine industry. This bacterium has been classified as a member of the family Pasteurellaceae in the genus Haemophilus, but phylogenetic relatedness has not been adequately examined to support this genus classification. Phenotypically, all 38 strains of H. parasuis tested were positive for catalase activity, oxidase activity, V-factor requirement, and acid formation from maltose and d-galactose without gas. All strains were negative for X-factor requirement, formation of indole from tryptophan, urease, l-arabinose, and α-glucosidase activity. To determine whether H. parasuis belongs to one of the current Pasteurellaceae genera 40 H. parasuis genomes, plus those of representative Pasteurellaceae, were subjected to phylogenetic analysis of concatenated, multi-protein alignments. Sequence variation at 16S rRNA and rpoB loci allowed the 15 reference serovars of H. parasuis to be integrated into the whole-genome tree. The phylogenetic analysis showed H. parasuis to be a distinct and tight clade whose sister taxon is the genus Bibersteinia. Within H. parasuis two clades were identified with individual serovars distributed between the two. As a result, H. parasuis was confirmed as a member of the family Pasteurellaceae, but was distinct from other genera in this family. Therefore, we propose the name Glaesserella parasuis, gen. nov., comb. nov. for bacterial strains currently classified as H. parasuis. The reference strain of this species is ATCC 19417 (1374)T, NCTC 4557T, DSM 21448T, CCUG 3712T.

Virulence-associated gene profiling, DNA fingerprinting and multilocus sequence typing of Haemophilus parasuis isolates in Australia.

OBJECTIVE: Determine if there is a link between virulence-associated genes of Haemophilus parasuis and the genotype and serovar of isolates. METHODS: Isolates of H. parasuis from 38 farms across six Australian states, representing all serovars present in Australia, were assessed for the presence of virulence-associated genes (vtaA, hhdBA, fhuA, lsgB and capD). Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) and multilocus sequence typing (MLST), together with existing knowledge of the serovar of the isolates and the health status of the source pig, were used to examine 75 Australian isolates of H. parasuis. RESULTS: An analysis of the ERIC-PRC patterns revealed six main clusters. One cluster of 25 isolates lacked virulence-associated genes and on the basis of serovar and field data, appeared to be mostly non-pathogenic. Another cluster of five isolates containing most of the virulence-associated genes appeared to be pathogenic based on the field and serovar data. The remaining four clusters were a mix of apparently pathogenic and apparently non-pathogenic isolates. The MLST results revealed a high degree of variation, with 54 sequence types of which 41 had not been previously recognised. CONCLUSION: Not all virulence-associated genes are present in potentially pathogenic strains of H. parasuis. Australian isolates of H. parasuis are both genetically diverse and markedly different from isolates in other countries. These key findings suggest that vaccine development will be challenging.

Development of Serotype-Specific PCR Assays for Typing of Haemophilus parasuis Isolates Circulating in Southern China.

The bacterium Haemophilus parasuis is the specific pathogenic cause of Glässer's disease in swine. Fifteen serotypes of H. parasuis have been reported. A method to serotype H. parasuis isolates accurately would help to prevent and control Glässer's disease outbreaks through appropriate vaccination and to understand the epidemiology in specific geographic areas. However, according to traditional serotyping, the rate of nontypeable (NT) strains is 10 to 40%, which gives low accuracy. In the present study, we developed a set of PCR assays that are able to identify all the currently known H. parasuis serotypes, with a detection limit of 5 CFU. This PCR method is particularly useful to distinguish serotype 5 from serotype 12. We then surveyed the serotype prevalence of H. parasuis isolates from southern China using both the traditional indirect hemagglutination (IHA) and current PCR methods. Of the 298 isolates tested, 228 (76.51%) and 281 (94.30%) were serotyped by the IHA and PCR tests, respectively, with a concordance rate of 80.87% (241/298). The most prevalent serotypes obtained by PCR were 4, 5, 12, 13, NT, and 2, and the most prevalent obtained by IHA were NT, 5, 4, 12, 13, and 2. In conclusion, the PCR assays developed in this study provide a rapid and specific method for the molecular serotyping of H. parasuis.

Analysis of efficacy obtained with a trivalent inactivated Haemophilus parasuis serovars 4, 5, and 12 vaccine and commercial vaccines against Glässer's disease in piglets.

The objective of this study was to assess the efficacy of a trivalent inactivated Haemophilus parasuis serovars 4, 5, and 12 vaccine with polymeric adjuvant gel (GEL) and commercial vaccines against Glässer's disease in piglets. Commercial vaccines containing inactivated H. parasuis serovars 4 and 5 (China), inactivated H. parasuis serovars 1 and 6 (Spain), and inactivated H. parasuis serovar 5 (USA) were also evaluated. Our results demonstrated that the trivalent inactivated H. parasuis serovars 4, 5, and 12 vaccine with GEL adjuvant can provide better protection against the 3 most common pathogenic serovars circulating in China than other commercial vaccines tested. Our findings also indicated that inactivated H. parasuis serovars 1 and 6 vaccine cross-protects piglets against H. parasuis serovars 4 and 5; inactivated H. parasuis serovar 5 vaccine cross-protects piglets against H. parasuis serovar 4 challenge; but none of the commercial vaccines tested in this study protected piglets against H. parasuis serovar 12. Our results provide a basis for further identification of common protective antigens that can induce cross-protection against heterogeneous serovars.

L'objectif de la présente étude était d'évaluer l'efficacité d'un vaccin inactivé trivalent contre Haemophilus parasuis contenant les sérovars 4, 5 et 12 avec un adjuvant en gel polymérique (GEL) et des vaccins commerciaux contre la maladie de Glässer chez des porcelets. Des vaccins commerciaux contenant du H. parasuis inactivé sérovars 4 et 5 (Chine), du H. parasuis inactivé sérovars 1 et 6 (Espagne), et du H. parasuis inactivé sérovar 5 (États-Unis) ont également été évalués. Nos résultats démontrent que le vaccin inactivé trivalent contenant H. parasuis sérovars 4, 5 et 12 avec l'adjuvant GEL peut fournir une meilleure protection contre les trois sérovars les plus fréquents circulant en Chine que les autres vaccins commerciaux testés. Nos trouvailles indiquent également que le vaccin contenant H. parasuis inactivé sérovars 1 et 6 offre une protection croisée contre H. parasuis sérovars 4 et 5; le vaccin inactivé H. parasuis sérovar 5 offre une protection croisée contre un challenge avec H. parasuis sérovar 4; mais qu'aucun des vaccins commerciaux testés dans l'étude ne protège les porcelets contre H. parasuis sérovar 12. Nos résultats fournissent une base pour l'identification future d'antigènes protecteurs communs qui peuvent induire une protection contre des sérovars hétérologues.(Traduit par Docteur Serge Messier).

Characterization of Chinese Haemophilus parasuis Isolates by Traditional Serotyping and Molecular Serotyping Methods.

Haemophilus parasuis is classified mainly through serotyping, but traditional serotyping always yields non-typable (NT) strains and unreliable results via cross-reactions. Here, we surveyed the serotype prevalence of Chinese H. parasuis isolates using traditional serotyping (gel immuno-diffusion test, GID) and molecular serotyping (multiplex PCR, mPCR). We also investigated why discrepant results between these methods were obtained, and investigated mPCR failure through whole-genome sequencing. Of the 100 isolate tested, 73 (73%) and 93 (93%) were serotyped by the GID test and mPCR, respectively, with a concordance rate of 66% (66/100). Additionally, mPCR reduced the number of NT isolates from 27 (27%) for the GID testing, to seven (7%). Eleven isolates were sequenced, including nine serotype-discrepant isolates from mPCR and GID typing (excluding strains that were NT by GID only) and two NT isolates from both methods, and their in silico serotypes were obtained from genome sequencing based on their capsule loci. The mPCR results were supported by the in silico serotyping of the seven serotype-discrepant isolates. The discrepant results and NT isolates determined by mPCR were attributed to deletions and unknown sequences in the serotype-specific region of each capsule locus. Compared with previous investigations, this study found a similar predominant serotype profile, but a different prevalence frequency for H. parasuis, and the five most prevalent serotypes or strain groups were serotypes 5, 4, NT, 7 and 13 for mPCR, and serotypes 5, NT, 4, 7 and 13/10/14 for GID. Additionally, serotype 7 was recognized as a principal serotype in this work.

The BALB/c mouse infection model for improving the Haemophilus parasuis serotyping scheme.

The use of BALB/c mouse as an alternative model to study Haemophilus parasuis (HPS) infections was evaluated, supplying the serotyping scheme by comparing the pathogenicity of different serovar HPS in pigs and mice challenge using statistical analysis. Results showed that the pathogenicity of different serovar HPS in mouse was consistent with in pigs, proving that this model is a viable alternative to pigs. It provides a convenient methodology for determining the virulence of HPS strains.

Multilocus sequence typing and virulence analysis of Haemophilus parasuis strains isolated in five provinces of China.

Haemophilus parasuis is the etiological agent of Glässers disease, which causes high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars with the Kielstein-Rapp-Gabrielson serotyping scheme, a large number of isolates cannot be classified and have been designated 'nontypeable' strains. In this study, multilocus sequence typing (MLST) of H. parasuis was used to analyze 48 H. parasuis field strains isolated in China and two strains from Australia. Twenty-six new alleles and 29 new sequence types (STs) were detected, enriching the H. parasuis MLST databases. A BURST analysis indicated that H. parasuis lacks stable population structure and is highly heterogeneous, and that there is no association between STs and geographic area. When an UPGMA dendrogram was constructed, two major clades, clade A and clade B, were defined. Animal experiments, in which guinea pigs were challenged intraperitoneally with the bacterial isolates, supported the hypothesis that the H. parasuis STs in clade A are generally avirulent or weakly virulent, whereas the STs in clade B tend to be virulent.

Development and antigenic characterization of three recombinant proteins with potential for Glässer's disease prevention.

Haemophilus parasuis is the causative agent of Glässer's disease, which causes high morbidity and mortality in piglets, leading to severe economic losses. The lack of a commercial vaccine against a broad spectrum of strains has limited the disease control. H. parasuis outer membrane proteins (OMPs) are potentially essential components for vaccine formulation. In this study, seven putative OMPs were selected from the annotated H. parasuis serovar 5 genome; they were predicted by bioinformatics and annotated as potential virulence-related factors. These proteins were cloned, expressed, and purified as His-tagged proteins. Antigenicity of the candidate proteins was assessed using Western blotting with convalescent sera against H. parasuis serovar 5. The immunogenicity of the seven OMPs was assessed in a guinea pig model. Except VacJ, all the other six recombinant proteins elicited a detectable antibody response. Antisera against four of the selected proteins effectively killed the bacteria in vitro. Three proteins (Omp26, VacJ, and HAPS_0742) were found to confer significant protection against challenge with a lethal dose of H. parasuis in a guinea pig model. The results suggest that these three proteins have a strong potential to be vaccine candidates against Glässer's disease.

First comparison of adjuvant for trivalent inactivated Haemophilus parasuis serovars 4, 5 and 12 vaccines against Glässer's disease.

Haemophilus parasuis has had a huge impact in the swine industry throughout the world. Inactivated bacterium for H. parasuis is a traditional vaccine that can elicit efficient protection against homologous challenges. The objective of this study was to screen for the adjuvant-enhanced immune effect of trivalent inactivated H. parasuis serovars 4, 5 and 12 (prevalent serovars in China) vaccines against Glässer's disease. The adjuvants of mineral oil, aluminum hydroxide, Montanide GEL 01 PR, Montanide IMS 1313N VG and Montanide ISA 760 VG were used to make emulsified inactivated H. parasuis serovars 4, 5 and 12, respectively. Safety, antibody titer and protective efficacy of these vaccines were examined separately in piglets, and the feasibility of microagglutination test for detecting antibody titer of H. parasuis was confirmed for the first time. Due to easy of injection, high safety, rapidly immune responses, high concentrations of antibody, and 100% of protective efficacy for piglets, Montanide GEL 01 PR adjuvant can provide more homologous serovar protection than other domestically developed inactivated vaccines and should be used as a candidate adjuvant.

Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis.

Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/µl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.

Fifteen novel immunoreactive proteins of Chinese virulent Haemophilus parasuis serotype 5 verified by an immunoproteomic assay.

Haemophilus parasuis (H. parasuis) is associated with meningitis, polyserositis, polyarthritis and bacterial pneumonia. At present, its prevention and control is difficult because of the lack of suitable subunit vaccines. Nowadays, high-throughput methods, immunoproteomics, are available to screen for more vaccine candidates. A protein extraction method for H. parasuis and two-dimensional electrophoresis (2-DE) were optimized to provide high-resolution profiles covering pH 3 to 10. Twenty immunoreactive spots were excised from gels after strict comparison between 2-DE Western blot membranes and the relevant gels. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and MALDI-TOF-TOF-MS successfully identified 16 different proteins. Fifteen of them were reported as immunoreactive proteins in H. parasuis for the first time. In addition, recombinant HP5-7 (ABC transporter, periplasmic-binding protein) showed immunoreactivity both with hyperimmune rabbit serum and convalescent swine serum. Four recombinants of the 14 successfully expressed genes showed immunoreactivity with hyperimmune rabbit serum.

The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis.

BACKGROUND: Haemophilus parasuis is the etiologic agent of Glässer's disease in pigs and causes devastating losses to the farming industry. Whilst some hyper-virulent isolates have been described, the relationship between genetics and disease outcome has been only partially established. In particular, there is weak correlation between serovar and disease phenotype. We sequenced the genomes of 212 isolates of H. parasuis and have used this to describe the pan-genome and to correlate this with clinical and carrier status, as well as with serotype. RESULTS: Recombination and population structure analyses identified five groups with very high rates of recombination, separated into two clades of H. parasuis with no signs of recombination between them. We used genome-wide association methods including discriminant analysis of principal components (DAPC) and generalised linear modelling (glm) to look for genetic determinants of this population partition, serovar and pathogenicity. We were able to identify genes from the accessory genome that were significantly associated with phenotypes such as potential serovar specific genes including capsule genes, and 48 putative virulence factors that were significantly different between the clinical and non-clinical isolates. We also show that the presence of many previously suggested virulence factors is not an appropriate marker of virulence. CONCLUSIONS: These genes will inform the generation of new molecular diagnostics and vaccines, and refinement of existing typing schemes and show the importance of the accessory genome of a diverse species when investigating the relationship between genotypes and phenotypes.

Identification of putative virulence-associated genes among Haemophilus parasuis strains and the virulence difference of different serovars.

This study was aimed at determining virulence-associated genes among Haemophilus parasuis (H. parasuis) strains, and supplying for the Kielstein-Rapp-Gabrielson serotyping scheme. The subtractive fragments, obtained through suppression subtractive hybridization and reverse Southern blot hybridization, were found to encode genes representative of 7 different functions. PCR was used to investigate the distribution of these fragments in H. parasuis strains isolated from different infection sites in pigs. Mice challenge was then used to analyze the correlationship between subtractive fragments, infection sites and bacterial virulence. Eight weeks old female BALB/c mice (10 mice/group) were inoculated intraperitoneally with 3.0 × 10(9) CFU suspension (0.5 ml/mouse) of H. parasuis strains in PBS. Results indicated that H. parasuis possessed varied virulence even among the same serovar strains. Transcription units hsdR, hsdS, gpT and ompP2, identified from the subtractive fragments, were uniformly expressed in highly virulent strains, while absent in weakly virulent strains, and demonstrated variable degrees of expression in moderately virulent strains. Moreover, H. parasuis strains, isolated from pericardium and heart blood, were all highly virulent strains, while from nasal cavity and joint were moderately or weakly virulent strains. This study indicated that fragments hsdR, hsdS, gpT and ompP2 were associated with the virulence of H. parasuis. The virulence of H. parasuis strains isolated from different infection sites was different. The current research provides a new reference for determining bacterial virulence in different H. parasuis strains.

Genome comparison of three serovar 5 pathogenic strains of Haemophilus parasuis: insights into an evolving swine pathogen.

Haemophilus parasuis is the causative agent of Glässer's disease, a systemic disorder characterized by polyarthritis, polyserositis and meningitis in pigs. Although it is well known that H. parasuis serovar 5 is the most prevalent serovar associated with the disease, the genetic differences among strains are only now being discovered. Genomes from two serovar 5 strains, SH0165 and 29755, are already available. Here, we present the draft genome of a third H. parasuis serovar 5 strain, the formal serovar 5 reference strain Nagasaki. An in silico genome subtractive analysis with full-length predicted genes of the three H. parasuis serovar 5 strains detected 95, 127 and 95 strain-specific genes (SSGs) for Nagasaki, SH0165 and 29755, respectively. We found that the genomic diversity within these three strains was high, in part because of a high number of mobile elements. Furthermore, a detailed analysis of large sequence polymorphisms (LSPs), encompassing regions ranging from 2 to 16 kb, revealed LSPs in virulence-related elements, such as a Toll-IL receptor, the AcrA multidrug efflux protein, an ATP-binding cassette (ABC) transporter, lipopolysaccharide-synthetizing enzymes and a tripartite ATP-independent periplasmic (TRAP) transporter. The whole-genome codon adaptation index (CAI) was also calculated and revealed values similar to other well-known bacterial pathogens. In addition, whole-genome SNP analysis indicated that nucleotide changes tended to be increased in membrane-related genes. This analysis provides further evidence that the genome of H. parasuis has been subjected to multiple lateral gene transfers (LGTs) and to fine-tuning of virulence factors, and has the potential for accelerated genome evolution.