Serotype Reference Research Articles

AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes

AAV vectors are promising delivery tools for human gene therapy. However, broad tissue tropism and pre-existing immunity against natural serotypes limit their clinical use. We identified two AAV capsid variants, AAV2-THGTPAD and AAV2-NLPGSGD, by invivo AAV2 peptide display library screening in a murine model of pressure overload-induced cardiac hypertrophy. Both variants showed significantly improved efficacy in invivo cardiomyocyte transduction compared with the parental serotype AAV2 as indicated by a higher number of AAV vector episomes in the nucleus and significant improved transduction efficiency. Both variants also outcompeted the reference serotype AAV9 regarding cardiomyocyte tropism, reaching comparable cardiac transduction efficiencies accompanied with liver de-targeting and decreased transduction efficiency of non-cardiac cells. Capsid modification influenced immunogenicity as sera of mice treated with AAV2-THGTPAD and AAV2-NLPGSGD demonstrated a poor neutralization capacity for the parental serotype and the novel variants. In a therapeutic setting, using the long non-coding RNA H19 in low vector dose conditions, novel AAV variants mediated superior anti-hypertrophic effects and revealed a further improved target-to-noise ratio, i.e., cardiomyocyte tropism. In conclusion, AAV2-THGTPAD and AAV2-NLPGSGD are promising novel tools for cardiac-directed gene therapy outperforming AAV9 regarding the specificity and therapeutic efficiency of invivo cardiomyocyte transduction.

Vaccinomics-driven proteome-wide screening of Haemophilus influenzae for the prediction of common putative vaccine candidates.

Haemophilus influenzae colonizes the respiratory tract and is associated with life-threatening invasive infections. The recent rise in its global prevalence, even in the presence of multiple vaccines, indicates an urgent need to develop effective cross-strain vaccine strategies. Our work focused on identifying the universally conserved antigenic regions of H. influenzae that can be used to develop new vaccines. A variety of bioinformatics tools were applied for the comprehensive geno-proteomic analysis of H. influenzae type a strain, as reference serotype, through which subcellular localization, essentiality, virulence, and non-host homology were determined. B and T cell epitope mapping of the 3D protein structures were performed. Thereafter, molecular docking with HLA_DRB1*0101 and comparative genome analysis established the candidature of the identified regions. Based on the established vaccinomics criteria, five target proteins were predicted as novel vaccine candidates. Among these, nine epitopic regions that could regulate lymphocyte activity through strong protein-protein interactions were identified. Comparative genomic analysis revealed that the identified regions were highly conserved among the different strains of H. influenzae. Based on multiple immunogenic factors, five prioritized proteins and their predicted epitopes were identified as ideal common putative vaccine candidates against typeable strains.

Antibiotic susceptibility and phylogenetic analyses for the origins and serotypes of Listeriamonocytogenes strains isolated from ice cream and cream cakes

Listeria monocytogenes is a zoonotic bacterium which also infects humans. The aim of this study was to isolate this organism from cream cakes and ice cream and obtain 16S rRNA and hlyA gene sequences from isolates in order to perform phylogenetic analyses. Serotypes and antibiotic susceptibility were also determined. The cream cake and ice cream samples were examined for L. monocytogenes according to ISO 11290-1 and using the mini Vidas LMO 2 kit procedure. Antibiotic susceptibilities were investigated using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) standards. The Sanger DNA sequencing method for phylogenetic analysis was used for L. monocytogenes isolates. A total of 16 (n =128, 12.50%) L. monocytogenes strains, 9 (12.16%) from cream cake samples and 7 (12.96%) from ice cream samples, were isolated. This was the first investigation involving sequencing of L. monocytogenes isolated from cream cakes and ice creams in Turkey. All isolates were susceptible to sulfamethoxazole/trimethoprim, tetracycline, streptomycin, gentamicin, meropenem, and erythromycin. The numbers of isolates resistant to sulbactam/ampicillin, penicillin G, chloramphenicol, and ampicillin were 16, 2, 1, and 1, respectively. Moreover, 3 isolates showed intermediate resistance to amikacin and 2 to ciprofloxacin. The hlyA gene sequences of 11 of the L. monocytogenesisolates isolated from milk were closely related to the hlyA gene sequences of the GenBank reference strains. The comparison of the 16s rRNA gene sequences of the L. monocytogenes strains with the GenBank reference serotypes identified 1 isolate as serotype 1/2c, 1 as serotype 1/2a, and 1 as serotype 4. Nucleic acid sequencing is useful for identification of L. monocytogenes. The 16S rRNAand hlyA sequences can be used to determine the origin and relationship between L. monocytogenesisolates, as well as the serotype.

Differences in the replicative capacities of clinical isolates of dengue virus in C6/36 cells and in urban populations of Aedes aegypti from Colombia, South America

Dengue, the most prevalent arboviral disease worldwide, is caused by any of the four dengue virus (DENV) serotypes that co-circulate constantly in hyperendemic areas such as Medellin (Colombia), and these serotypes are transmitted by mosquitoes of the genus Aedes. In this study, we evaluated the replicative capacity of strains isolated in Medellin between 2003 and 2007 in C6/36 cells and in colonies of Aedes aegypti collected during 2010–2011 from high or low-incidence areas within the same city. The phylogenetic analysis grouped isolates according to the predominant genotypes found in the Americas, and the in vitro characterization showed differences in the morphological changes induced by the isolates of each of the isolated serotypes compared to the reference serotypes. In vitro replicative capacity studies demonstrated that genomic copy number increased at four days post-infection and that cell viability decreased significantly compared to the control for all serotypes. The largest number of genomic copies in C6/36 was produced by DENV-2, followed by DENV-1 and DENV-4; DENV-3 produced the smallest number of genomic copies and had the smallest negative effect on cell viability. Finally, differences in the in vivo replication of intercolonial serotypes between the Rockefeller colony and the field colonies and among the intracolonial serotypes were found. The replication of DENV-2 at 7 and 14 days in both high- and low-incidence colonies was higher than that of the other serotypes, and replication of DENV-3 in the mosquito colonies was the most stable on the days evaluated. Our results support the notion that replication and, possibly, DENV transmission and severity depend on many factors, including serotype and vector characteristics.

The relative invasive disease potential of Streptococcus pneumoniae among children after PCV introduction: A systematic review and meta-analysis

Burden of pneumococcal disease depends on the prevalence and invasive disease potential of serotypes. We aimed to estimate the invasive disease potential of serotypes in children under 5 years of age by combining data from different settings with routine immunisation with pneumococcal conjugate vaccines (PCV). We conducted a systematic review, supplemented by unpublished data, to identify data on the frequency of pneumococcal serotypes in carriage and invasive pneumococcal disease (IPD). We estimated the invasive disease potential of serotypes as the ratio of IPD in relation to carriage (odds ratio and 95%CI) compared with 19A (reference serotype) by meta-analysis. We report results based on a random effects model for children aged 0-23, 24-29, and 0-59 months and by invasive clinical syndromes. In comparison with 19A, serotypes 1, 7F, and 12F had a significantly higher invasive disease potential in children aged 0-23 and 0-59 months for all IPD and clinical syndromes (OR > 5). Several non-vaccine types (NVTs) (6C, 15A, 15BC, 16F, 23B, in these two age groups) had a lower invasive disease potential than 19A (OR 0.1-0.3). NVTs 8, 12F, 24F, and 33F were at the upper end of the invasiveness spectrum. There is substantial variation among pneumococcal serotypes in their potential to cause IPD and disease presentation, which is influenced by age and time after PCV introduction. Surveillance of IPD and carriage is critical to understand the expected effectiveness of current PCVs (in the longer term) and guide the development of future vaccines.

Protein profiles of bacteriophages of the family Myoviridae-like induced on M. haemolytica

The aim of study was to isolate, characterize and analyse the protein profiles of Myoviridae-like bacteriophages obtained from M. haemolytica using MALDI TOF mass spectrometry. The material consisted of the M. haemolytica reference strain ATCC® BAA410, reference serotypes A1, A2, A5, A6, A7, A9, and A11, and wild-type isolates of serotype A1. Bacteriophage morphology was examined with a transmission electron microscope. The proteins were separated in SDS-PAGE and two-dimensional electrophoresis and characterized by MALDI-TOF. Among the phages obtained, seven were specific for strains A1, A2, A5, A6, A7 and 25, and PHL-1 was specific for the BAA410 strain. The protein profiles for the phages were very similar to one another, but differed from the reference phage in that they lacked protein fractions with molecular weights of 22.9, 56.3 and 73.1 kDa. 2D electrophoresis revealed significant differences in the size of proteins and their localization in the pH gradient. The most similar profiles were observed in phages specific for strains BAA-410 and A6. In all profiles two main spots were observed in the molecular weight range from 44 to 70 kDa at pH < 4. The results indicate that 2D electrophoresis is a very useful tool for characterization of phage protein profiles. An important objective was to determine the molecular differences between morphologically similar phages belonging to one family and to find similarities to phages specific for other pathogens. The study also assessed the suitability of the methods used to characterize phages.

Genotyping and phylogenetic analysis of infectious bronchitis virus isolated from broiler chickens in Kashmir.

Infectious bronchitis virus (IBV) is responsible for significant economic losses to the poultry industry across the world. The enormous genetic diversity of IBV poses difficulty in diagnosing and controlling the virus. To understand the nature of IBV prevalent in the Kashmir Himalayas, we characterized two field strains, isolated from non-immunized broiler chickens, by sequence and phylogenetic analysis of S1 subunit of the spike glycoprotein. The analysis revealed both the isolates are identical to each other, with nucleotide and amino acid sequence identities of 99.4% and 98.4%, respectively. They exhibit variable sequence divergence in the S1 gene to that of the reference serotypes. Both are of “Massachusetts type” belonging to GI-1 lineage of the IBV génotype. The phylogenetic analysis revealed both of the isolates clustered into the same branch as that of many IBV strains recently reported from China and Iran. Likely, these regionally-related isolates represent revertant vaccine strains which may have been disseminated across the region by wild migratory birds. This study provides the first report of molecular evidence and phylogenetic characterization of the IBV from the Kashmir Himalayas and implicate the possible role of wild migratory birds in the spread of IBV in the region.Electronic supplementary materialThe online version of this article (10.1007/s13337-017-0416-2) contains supplementary material, which is available to authorized users.

Pro-inflammatory cytokine responses in human gingival epithelial cells after stimulation with cell wall extract of Aggregatibacter actinomycetemcomitans subtypes

Varying cytokine responses of human gingival epithelial cells (HGECs) by Aggregatibacter actinomycetemcomitans subtypes have been found. Most studies have used reference strains, whereas a few has evaluated the cytokine expression in response to clinical subtypes of this bacterial species. This study aimed to examine whether there was any difference in cytokine responses of HGECs stimulated with cell wall extract (CWE) from A. actinomycetemcomitans subtypes included clinical strains from Thai adult periodontitis, various serotypes and non-serotypeable strains, strains from deep or shallow pockets, and reference serotype strains. Totally 50 clinical strains and 7 reference strains of A. actinomycetemcomitans were analyzed for the expression of IL-1β, IL-6, IL-8, and TNF-α mRNAs in HGECs by real time-PCR, and the IL-8 concentrations in cell-free supernatant measured using ELISA. An in vitro effect of released IL-8 on neutrophil migration was examined using transwell chambers. Result showed that among four cytokines studied, IL-8 mRNA was highly up-regulated by both clinical and reference strains. Serotype f revealed the highest expression compared to other serotypes. The JP2-like leukotoxin promoter gene and non-serotypeable (NS1 and NS2) demonstrated lower IL-8 responses compared to serotypeable strains, and IL-8 responses upon stimulation with clinical strains from deep pockets were also significantly lower than those isolated from shallow pockets (P < 0.01). Our findings suggest that the clinical isolates of A. actinomycetemcomitans associating with deep pockets, JP2-like leukotoxin promoter gene, NS1, and NS2 may interfere neutrophil function via minimal and immunosuppressing IL-8 responses, which may enhance their survival and virulence.

Molecular study of an outer fragment of Haemophilus parasuis neuraminidase and utility with diagnostic and immunogen purposes

Haemophilus parasuis is a swine pathogenic organism, being the causative agent of Glässer's disease. It has got some virulence factors, some of which act as potential candidates for the vaccine developing. Among them there is the neuraminidase enzyme, which is located inside the outer membrane and contains a β-barrel domain with seven external loops. By using the polymerase chain reaction technique, the β-barrel fragment was amplified, sequenced and analysed for the 15 H. parasuis reference serotypes. The results showed a small diversity for them, except for serotype 2, which has a deletion that covers the loops with potential to be used as vaccine antigen. However, some of the other serotypes showed the same nucleotidic sequence between them, such those 6 and 7 or those 12 and 13. This fact was also confirmed by means of phylogenetic analysis. For these reasons, the tested fragment might result in a putative candidate for the development of subunit vaccines against all the serotypes causing Glässer's disease outbreaks, with the exception of serotype 2, alone or in combination with other proven immunogenicity molecules. Anyway, further studies should be carried out in pigs in order to confirm this hypothesis. Finally, this outer fragment of H. parasuis neuraminidase could be used as a suitable diagnostic tool at a species level, for instance, by PCR.

Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

Aim of StudyThe objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves.Material and MethodsThe material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR).ResultsEight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101.ConclusionsThe results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

Direct amplification, sequencing and profiling of Chlamydia trachomatis strains in single and mixed infection clinical samples.

Sequencing bacterial genomes from DNA isolated directly from clinical samples offers the promise of rapid and precise acquisition of informative genetic information. In the case of Chlamydia trachomatis, direct sequencing is particularly desirable because it obviates the requirement for culture in mammalian cells, saving time, cost and the possibility of missing low abundance strains. In this proof of concept study, we developed methodology that would allow genome-scale direct sequencing, using a multiplexed microdroplet PCR enrichment technology to amplify a 100 kb region of the C. trachomatis genome with 500 1.1–1.3 kb overlapping amplicons (5-fold amplicon redundancy). We integrated comparative genomic data into a pipeline to preferentially select conserved sites for amplicon design. The 100 kb target region could be amplified from clinical samples, including remnants from diagnostics tests, originating from the cervix, urethra and urine, For rapid analysis of these data, we developed a framework for whole-genome based genotyping called binstrain. We used binstrain to estimate the proportion of SNPs originating from 14 C. trachomatis reference serotype genomes in each sample. Direct DNA sequencing methods such as the one described here may have an important role in understanding the biology of C. trachomatis mixed infections and the natural genetic variation of the species within clinically relevant ecological niches.

Intraneural convection enhanced delivery of AAVrh20 for targeting primary sensory neurons.

Gene therapy using adeno-associated virus (AAV) is an attractive strategy to treat disorders of the peripheral nervous system (PNS), such as chronic pain or peripheral neuropathies. Although intrathecal (IT) administration of AAV has been the standard in the field for targeting the PNS, it lacks anatomical specificity and results in wide rostro-caudal distribution of the vector. An alternative approach is to deliver AAV directly to the peripheral nerve axon. The present study employed convection-enhanced delivery (CED) of a novel AAV serotype, AAVrh20, expressing enhanced green fluorescent protein (EGFP) into rat sciatic nerve investigating its efficacy, anatomical selectivity, and safety, compared to the IT route. Intraneural CED resulted in transduction confined to the ipsilateral L4 and L5 DRG while IT administration led to promiscuous DRG transduction encompassing the entire lumbar region bilaterally. The transduction rate for intraneural AAV administration was similar to IT delivery (24% for L4 and 31.5% for L5 DRG versus 50% for L4 and 19.5% for L5 DRG). The use of hyperosmotic diluent did not further improve the transduction efficiency. AAVrh20 was superior to reference serotypes previously described to be most active for each route. Intraneural CED of AAV was associated with transient allodynia that resolved spontaneously. These findings establish intraneural CED as an alternative to IT administration for AAV mediated gene transfer to the PNS and, based on a reference rodent model, suggest AAVrh20 as a superior serotype for targeting the PNS.

Characterization of the omlA gene from different serotypes of Actinobacillus pleuropneumoniae: a new insight into an old approach

The OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein.

Streptococcus suis serotype 9 bacterin immunogenicity and protective efficacy

Streptococcus suis diseases in pigs, most importantly meningitis, are worldwide responsible for major economic losses in the pig industry. About one fourth of invasive S. suis diseases are caused by S. suis serotype 9 strains in Europe. However, little is known about serotype 9 since most studies were performed with serotype 2. The objective of this study was to determine the immunogenicity and protective efficacy of a serotype 9 bacterin in piglets. Challenge was conducted with a reference serotype 9 strain, belonging to the same clonal complex but to a different sequence type as the bacterin strain. The bacterin induced protection against mortality but not morbidity. Eleven days post infection, 3 of 7 vaccinated survivors were not fully convalescent and had not eliminated the challenge strain from inner organs completely. In accordance with the clinical findings, the majority of piglets showed fibrinous–suppurative lesions in at least one inner organ or tissue. In contrast to the placebo group such lesions were not detected in one third of bacterin-vaccinated piglets. Determination of specific serum IgG titers revealed that the bacterin elicited seroconversion against muramidase-released protein and basic membrane lipoprotein. Furthermore, vaccination was associated with induction of opsonizing antibodies against the serotype 9 challenge strain. However, titers of opsonizing antibodies were rather low in comparison to those found in our previous serotype 2 vaccination trial. Piglets developed substantially higher titers of opsonizing antibodies after challenge. Opsonizing antibodies were absorbable with the serotype 9 challenge strain but not with an unencapsulated isogenic mutant of a serotype 2 strain indicating their specificity. The results indicate that a serotype 9 bacterin is less protective than a serotype 2 bacterin, most likely due to inducing only low titers of opsonizing antibodies. This might contribute to emergence of serotype 9 strains, in particular strains of this clonal complex, in Europe.

Fetal RHD genotype detection from circulating cell‐free fetal DNA in maternal plasma in non‐sensitized RhD negative women

To examine the performance of the SensiGene Fetal RHD Genotyping Laboratory Developed Test (RHD Genotyping LDT) using circulating cell-free fetal DNA (ccff DNA) extracted from maternal plasma. ccff DNA was extracted from maternal blood from non-sensitized women with singleton pregnancies in two cohorts, one with a serotype reference (11-13 weeks' gestation) and one with the reference source (6-30 weeks' gestation). The presence of three RHD exon sequences (exons 4, 5, 7), the psi-pseudogene, three Y-chromosome sequences (SRY, DBY and TTTY2), and the X/Y-chromosome TGIF gene control were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-the RHD Genotyping LDT. The cohort with a serotype RhD reference showed correct classification in 201 of 207 patients, a test accuracy of 97.1%, with a sensitivity and specificity for prediction of RhD serotype of 97.2 and 96.8%, respectively. The cohort with a genotype RHD reference showed correct classification in 198 of 199 patients, indicating a test accuracy of 99.5% with a sensitivity and specificity for prediction of RHD genotype of 100.0 and 98.3%, respectively. Fetal RHD genotyping can accurately be determined using ccff DNA in the first and second trimesters of pregnancy.

Streptococcus pneumoniae Clonal Complex 199: Genetic Diversity and Tissue-Specific Virulence

Streptococcus pneumoniae is an important cause of otitis media and invasive disease. Since introduction of the heptavalent pneumococcal conjugate vaccine, there has been an increase in replacement disease due to serotype 19A clonal complex (CC)199 isolates. The goals of this study were to 1) describe genetic diversity among nineteen CC199 isolates from carriage, middle ear, blood, and cerebrospinal fluid, 2) compare CC199 19A (n = 3) and 15B/C (n = 2) isolates in the chinchilla model for pneumococcal disease, and 3) identify accessory genes associated with tissue-specific disease among a larger collection of S. pneumoniae isolates. CC199 isolates were analyzed by comparative genome hybridization. One hundred and twenty-seven genes were variably present. The CC199 phylogeny split into two main clades, one comprised predominantly of carriage isolates and another of disease isolates. Ability to colonize and cause disease did not differ by serotype in the chinchilla model. However, isolates from the disease clade were associated with faster time to bacteremia compared to carriage clade isolates. One 19A isolate exhibited hypervirulence. Twelve tissue-specific genes/regions were identified by correspondence analysis. After screening a diverse collection of 326 isolates, spr0282 was associated with carriage. Four genes/regions, SP0163, SP0463, SPN05002 and RD8a were associated with middle ear isolates. SPN05002 also associated with blood and CSF, while RD8a associated with blood isolates. The hypervirulent isolate's genome was sequenced using the Solexa paired-end sequencing platform and compared to that of a reference serotype 19A isolate, revealing the presence of a novel 20 kb region with sequence similarity to bacteriophage genes. Genetic factors other than serotype may modulate virulence potential in CC199. These studies have implications for the long-term effectiveness of conjugate vaccines. Ideally, future vaccines would target common proteins to effectively reduce carriage and disease in the vaccinated population.

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.

A pyrosequencing-based method for genotyping pathogenic serotypes of S. suis

Streptococcus suis (S. suis for short) can cause a variety of infections in pigs, and the infections have brought about great losses in the swine industry and some cases of deaths in human beings. In order to rapidly diagnose and control the infections of S. suis, we designed a pyrosequencing-based assay to identify the serotypes of S. suis. In the assay, pyrosequencing is used to genotype most of the pathogenic serotypes of S. suis by detecting five informative regions on the Chaperonin 60 (cpn60) gene and one species-specific region on the 16S rRNA gene, and further a few undistinguished serotypes by pyrosequencing were finely discriminated by multiplex PCR of serotype-specific fragments on the cpsgene as well as species-specific fragments on the 16S rRNA gene. Through carefully designing the dispensing order of dNTP for each pyrosequencing reaction, the serotypes of S. suis could be discriminated by four pyrosequencing reactions within three hours. Five reference serotypes and three clinical strains were successfully detected and genotyped by our assay. The results indicated that our assay is a reliable, information-rich diagnostic method for the accurate detection ofS. suis serotypes.

Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real‐time PCR assay

The aim of this study was to develop a multiplex real-time PCR assay for the identification and discrimination of Erysipelothrix rhusiopathiae, tonsillarum and Erysipelothrix sp. strain 2 for direct detection of Erysipelothrix spp. from animal specimens. A primer set and three species-specific probes with different end labelling were designed from the noncoding region downstream of the 5S rRNA coding region. The sensitivity, specificity and repeatability of the assay were validated by analysing 27 Erysipelothrix spp. reference serotype strains and ten septicemia-associated non-Erysipelothrix spp. bacterial isolates. Cross-reactivity with Erysipelothrix sp. strain 1 was not observed with any of the primer probe combinations. The detection limit was determined to be <10 colony forming units and as low as one genome equivalent per PCR . Further evaluation of the Erysipelothrix spp. multiplex PCR was performed by comparing an enrichment isolation culture method and a conventional differential PCR on 15 samples from pigs experimentally inoculated with Erysipelothrix spp. and 22 samples from pigs with suspected natural infection. The multiplex real-time PCR assay was found to be simple, rapid, reliable, specific and highly sensitive. The developed real-time multiplex PCR assay does not require cumbersome and lengthy cultivation steps prior to DNA extraction, obtained comparable results to enrichment isolation, and will be useful in diagnostic laboratories for rapid detection of Erysipelothrix spp.

Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens.