A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens

The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

Comparison of sampling sites and detection methods for Haemophilus parasuis

Objective To improve the isolation rate and identification procedures for Haemophilus parasuis from pig tissues. Design Thirteen sampling sites and up to three methods were used to confirm the presence of H. parasuis in pigs after experimental challenge. Procedure Colostrum-deprived, naturally farrowed pigs were challenged intratracheally with H parasuis serovar 12 or 4. Samples taken during necropsy were either inoculated onto culture plates, processed directly for PCR or enriched prior to being processed for PCR. The recovery of H parasuis from different sampling sites and using different sampling methods was compared for each serovar. Results H parasuis was recovered from several sample sites for all serovar 12 challenged pigs, while the trachea was the only positive site for all pigs following serovar 4 challenge. The method of solid medium culture of swabs, and confirmation of the identity of cultured bacteria by PCR, resulted in 38% and 14% more positive results on a site basis for serovars 12 and 4, retrospectively, than direct PCR on the swabs. This difference was significant in the serovar 12 challenge. Conclusion Conventional culture proved to be more effective in detecting H parasuis than direct PCR or PCR on enrichment broths. For subacute (serovar 4) infections, the most successful sites for culture or direct PCR were pleural fluid, peritoneal fibrin and fluid, lung and pericardial fluid. For acute (serovar 12) infections, the best sites were lung, heart blood, affected joints and brain. The methodologies and key sampling sites identified in this study will enable improved isolation of H parasuis and aid the diagnosis of Glässer's disease.

Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractive hybridization.

Haemophilus parasuis is the causative agent of Glässer's disease. Up to now 15 serovars of H. parasuis have been identified, with significant differences existing in virulence between serovars. In this study, suppression subtractive hybridization (SSH) was used to identify the genetic difference between Nagasaki (H. parasuis serovar 5 reference strain, highly virulent) and SW114 (H. parasuis serovar 3 reference strain, non-virulent). A total of 191 clones were obtained from the SSH library. Using dot hybridization and PCR, 15 clones were identified containing fragments that were present in the Nagasaki genome while absent in the SW114 genome. Among these 15 fragments, three fragments (ssh1, ssh13, ssh15) encode cell surface-associated components; three fragments (ssh2, ssh5, ssh9) are associated with metabolism and stress response; one fragment (ssh8) is involved in assembly of fimbria and one fragment (ssh6) is a phage phi-105 ORF25-like protein. The remaining seven fragments are hypothetical proteins or unknown. Based on PCR analysis of the 15 serovar reference strains, eight fragments (ssh1, ssh2, ssh3, ssh6, ssh8, ssh10, ssh11 and ssh12) were found in three to five of most virulent serovars (1, 5, 10, 12, 13 and 14), zero to two in three moderately virulent serovars (2, 4 and 15), but absent in the low virulent serovar (8) and non-virulent serovars (3, 6, 7, 9 and 11). In vivo transcription fragments ssh1, ssh2, ssh8 and ssh12 were identified in total RNA samples extracted from experimental infected pig lung by RT-PCR. This study has provided some evidence of genetic differences between H. parasuis strains of different virulence.

Haemophilus parasuis treatment

The aim of the project is to prove that live vaccination on the farm will protect the piglets from heterologous challenge with H. parasuis. The steps to achieve the aim of the project are to find a dose rate on the farm which guarantees colonisation of the vaccine strain and is safe On farm vaccinated and unvaccinated pigs are then shifted to CAAS at three weeks of age and challenged with a heterologous strains. The method is then applied on a large piggery for a period of nearly 12 months. We will also develop a freeze drying method and a technical manual of procedures to identify serovars prevailing on pig farms and which serovar to include into the vaccine.

Detection of a putative hemolysin operon, hhdBA, of Haemophilus parasuis from pigs with Glasser disease

The aim of the current study was to investigate whether polymerase chain reaction amplification of 16S ribosomal (r)RNA and a putative hemolysin gene operon, hhdBA, can be used to monitor live pigs for the presence of Haemophilus parasuis and predict the virulence of the strains present. Nasal cavity swabs were taken from 30 live, healthy, 1- to 8-week-old pigs on a weekly cycle from a commercial Thai nursery pig herd. A total of 27 of these pigs (90%) tested positive for H. parasuis as early as week 1 of age. None of the H. parasuis-positive samples from healthy pigs was positive for the hhdBA genes. At the same pig nursery, swab samples from nasal cavity, tonsil, trachea, and lung, and exudate samples from pleural/peritoneal cavity were taken from 30 dead pigs displaying typical pathological lesions consistent with Glasser disease. Twenty-two of 140 samples (15.7%) taken from 30 diseased pigs yielded a positive result for H. parasuis. Samples from the exudate (27%) yielded the most positive results, followed by lung, tracheal swab, tonsil, and nasal swab, respectively. Out of 22 positive samples, 12 samples (54.5%) harbored hhdA and/or hhdB genes. Detection rates of hhdA were higher than hhdB. None of the H. parasuis-positive samples taken from nasal cavity of diseased pigs tested positive for hhdBA genes. More work is required to determine if the detection of hhdBA genes is useful for identifying the virulence potential of H. parasuis field isolates.

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.

CsrA interacting small RNAs in Haemophilus spp genomes: a theoretical analysis

The csrA is a carbon storage regulator gene that encodes a protein with multiple RNA interaction sites. Bacterial non-coding small RNAs like csrB, csrC and their counterparts in diverse bacterial genus are identified to control the regulatory activities of CsrA and its orthologs. An attempt has been made in this study to identify 'novel' non-coding small RNAs that are involved in the regulatory activities of csrA gene. All CsrA-interacting small RNAs are computationally fingerprinted to have multiple occurrence of 7-nucleotide CsrA interacting repeats [CAGGA(U/A/C)G] along with a 18-nucleotide upstream binding site. However, in several of the genomes like Haemophilus spp, the upstream binding site is not identified. The current methodology overcomes this difficulty by identifying small RNA-specific orphan transcriptional units within the intergenic regions of the genome. The results could identify all known CsrA-interacting small RNAs in E. coli, Vibrio cholerae and Pseudomonas aeruginosa genomes and additionally has picked six new possible CsrA-interacting small RNA regions in E. coli. Our computational analysis indicates that known rygD and rprA sRNAs in E. coli could possibly interact with CsrA proteins. The study is extended to three of the Haemophilus genomes that could identify seven new possible CsrA interacting small RNAs.

Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

Haemophilus influenzae and Helicobacter pylori are major bacterial pathogens that face high levels of genotoxic stress within their host. UvrD, a ubiquitous bacterial helicase that plays important roles in multiple DNA metabolic pathways, is essential for genome stability and might, therefore, be crucial in bacterial physiology and pathogenesis. In this study, the functional characterization of UvrD helicase from Haemophilus influenzae and Helicobacter pylori is reported. UvrD from Haemophilus influenzae (HiUvrD) and Helicobacter pylori (HpUvrD) exhibit strong single-stranded DNA-specific ATPase and 3'5' helicase activities. Mutation of highly conserved arginine (R288) in HiUvrD and glutamate (E206) in HpUvrD abrogated their activities. Both the proteins were able to bind and unwind a variety of DNA structures including duplexes with strand discontinuities and branches, three- and four-way junctions that underpin their role in DNA replication, repair and recombination. HiUvrD required a minimum of 12 nucleotides, whereas HpUvrD preferred 20 or more nucleotides of 3'-single-stranded DNA tail for efficient unwinding of duplex DNA. Interestingly, HpUvrD was able to hydrolyze and utilize GTP for its helicase activity although not as effectively as ATP, which has not been reported to date for UvrD characterized from other organisms. HiUvrD and HpUvrD were found to exist predominantly as monomers in solution together with multimeric forms. Noticeably, deletion of distal C-terminal 48 amino acid residues disrupted the oligomerization of HiUvrD, whereas deletion of 63 amino acids from C-terminus of HpUvrD had no effect on its oligomerization. This study presents the characteristic features and comparative analysis of Haemophilus influenzae and Helicobacter pylori UvrD, and constitutes the basis for understanding the role of UvrD in the biology and virulence of these pathogens.

Haemophilus influenzae Genome Database (HIGDB): A single point web resource for Haemophilus influenzae

Background: Haemophilus influenzae (H. Influenzae) is the causative agent of pneumonia, bacteraemia and meningitis. The organism is responsible for large number of deaths in both developed and developing countries. Even-though the first bacterial genome to be sequenced was that of H. Influenzae, there is no exclusive database dedicated for H. Influenzae. This prompted us to develop the Haemophilus influenzae Genome Database (HIGDB). Methods: All data of HIGDB are stored and managed in MySQL database. The HIGDB is hosted on Solaris server and developed using PERL modules. Ajax and JavaScript are used for the interface development. Results: The HIGDB contains detailed information on 42,741 proteins, 18,077 genes including 10 whole genome sequences and also 284 three dimensional structures of proteins of H. influenzae. In addition, the database provides ``Motif search'' and ``GBrowse''. The HIGDB is freely accessible through the URL:http://bioserverl.physicslisc.ernetin/HIGDB/. Discussion: The HIGDB will be a single point access for bacteriological, clinical, genomic and proteomic information of H. influenzae. The database can also be used to identify DNA motifs within H. influenzae genomes and to compare gene or protein sequences of a particular strain with other strains of H. influenzae. (C) 2014 Elsevier Ltd. All rights reserved.

Validação dos processos envolvidos na obtenção do polissacarídeo capsular de Haemophilus influenzae tipo b conjugado com toxóide tetânico utilizado para produção de vacina

Haemophilus influenzae tipo b (Hib) é a principal causa de pneumonia bacteriana e meningite em crianças com menos de 5 anos de idade. A doença pode ser prevenida através da vacinação com uma vacina conjugada polissacarídeo-proteína, uma vez que a vacina de polissacarídeo não é eficaz. Desde 1999, o Ministério da Saúde incluiu a vacina conjugada Hib na rotina do calendário de imunização para crianças rotineiramente. Sendo este um processo de grande importância para as pessoas e demanda de produção significativa para o governo brasileiro, é necessário que os processos estejam de acordo com as normas nacionais e internacionais de regulamentação e devem ser validados. Uma operação validada assegura a produção de lotes uniformes que atendem as especificações exigidas, consequentemente, garantindo ao fabricante a capacidade de proporcionar um produto de qualidade. O trabalho propõe o desenvolvimento de uma metodologia que evidencie e garanta o processo de produção de um conjugado de polissacarídeo-proteína pelo método de conjugação química e que será produzido de forma consistente e de acordo com as suas especificações pré-definidas. Os atributos de qualidade garantem o não desabastecimento da vacina Haemophilus influenzae tipo b por desvios de processo, o que contribui com aumento do valor agregado ao produto em questão. Portanto, a metodologia de validação foi aplicada ao estudo de validação. O estudo baseou-se em três etapas. A primeira foi baseada em análise de parâmetros do processo de conjugação química para mapear e estabelecer intervalos críticos de controle quanto a estes parâmetros, a segunda fase foi realizada com a produção de três lotes consecutivos, em escala industrial, com monitorização regular dos parâmetros críticos mapeados anteriormente. A fase final consistiu em amostras recolhidas, em tempo pré-determinado, durante o processo de produção e de controle do produto final em tamanho 50% a mais que na rotina de produção. Estas amostras foram analisadas utilizando os métodos de análise que foram anteriormente validados, a fim de garantir a reprodutibilidade dos resultados. A robustez do processo foi assegurada uma vez que todos os parâmetros do processo avaliados apresentaram resultados dentro do padrão de conformidade com os resultados de controle determinados durante o processo, e no produto final para a população de amostragem dentro de especificações definidas pelas metodologias analíticas validadas garantindo os resultados. O trabalho proposto assegura status de processo validado para o conjugado polissacarídeo-proteína com excepcionais rendimentos obtidos, combinado com características químicas bem definidas

The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin.

The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases.

Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis

We have determined that gene HI#1181 of Haemophilus influenzae is a homolog of Escherichia coli gmhA (previously designated lpcA) (J. S. Brooke and M. A. Valvano, J. Biol. Chem. 271:3608-3614, 1996), which encodes a phosphoheptose isomerase catalyzing the first step of the biosynthesis of ADP-L-glycero-D-manno heptose. Mutations in this gene are associated with a heptoseless core lipopolysaccharide which determines an increased outer membrane permeability to hydrophobic compounds. The cloned H. influenzae gmhA restored the synthesis of a complete core in the gmhA-deleted E. coli strain chi711. Amino acid sequence comparisons of the GmhA proteins of E. coli and H. influenzae with other proteins in the databases revealed the existence of a novel family of phosphosugar a1do-keto isomerases.

Genotypic and phenotypic diversity of the noncapsulated Haemophilus influenzae:Adaptation and pathogenesis in the human airways

The human respiratory tract contains a highly adapted microbiota including commensal and opportunistic pathogens. Noncapsulated or nontypable Haemophilus influenzae (NTHi) is a human-restricted member of the normal airway microbiota in healthy carriers and an opportunistic pathogen in immunocompromised individuals. The duality of NTHi as a colonizer and as a symptomatic infectious agent is closely related to its adaptation to the host, which in turn greatly relies on the genetic plasticity of the bacterium and is facilitated by its condition as a natural competent. The variable genotype of NTHi accounts for its heterogeneous gene expression and variable phenotype, leading to differential host-pathogen interplay among isolates. Here we review our current knowledge of NTHi diversity in terms of genotype, gene expression, antigenic variation, and the phenotypes associated with colonization and pathogenesis. The potential benefits of NTHi diversity studies discussed herein include the unraveling of pathogenicity clues, the generation of tools to predict virulence from genomic data, and the exploitation of a unique natural system for the continuous monitoring of long-term bacterial evolution in human airways exposed to noxious agents. Finally, we highlight the challenge of monitoring both the pathogen and the host in longitudinal studies, and of applying comparative genomics to clarify the meaning of the vast NTHi genetic diversity and its translation to virulence phenotypes.

Host cell kinases, α5 and β1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase C?1 inhibition but was not affected by protein kinase inhibition. We also found that a5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.