Paragallinarum Strain Research Articles

Establishment of a Multilocus Sequence Typing Scheme for the Characterization of Avibacterium paragallinarum.

Infectious coryza is an acute respiratory infection caused by Avibacterium paragallinarum, which is widely distributed throughout the world. However, there is no effective molecular typing scheme to obtain basic knowledge about the Av. paragallinarum population structure. This study aimed to develop a multilocus sequence typing (MLST) scheme for Av. paragallinarum that allows for the worldwide comparison of sequence data. For this purpose, the genetic variability of 59 Av. paragallinarum strains from different geographical origins and serovars was analyzed to identify correlations. The MLST scheme was developed using seven conserved housekeeping genes, which identified eight STs that clustered all of the strains into three evolutionary branches. The analytical evaluation of the clone group relationship between the STs revealed two clone complexes (CC1 and CC2) and three singletons (ST2, ST5, and ST6). Most of the isolates from China belonged to ST1 and ST3 in CC1. ST8 from Peru and ST7 from North America together formed CC2. Our results showed that the Av. paragallinarum strains isolated from China had a distant genetic relationship with CC2, indicating strong regional specificity. The MLST scheme established in this study can monitor the dynamics and genetic differences of Av. paragallinarum transmission.

Complete genome sequences generated using hybrid Nanopore-Illumina assembly of two non-typical Avibacterium paragallinarum strains isolated from clinically normal chicken flocks.

We report the complete genome sequences of two non-typical Avibacterium paragallinarum (AP) strains isolated from chickens in the absence of clinical signs. The availability of these genomes can aid scientists in improving current diagnostics and increase our understanding of AP epidemiology and pathogenicity in chickens.

Regions Important for Hemagglutination Activity and Serotypes of Avibacterium paragallinarum HMTp210 Protein.

Avibacterium paragallinarum is an important respiratory pathogen of domestic chickens. Avibacterium paragallinarum has been subtyped into three serogroups and nine serovars according to the Page and revised Kume schemes. The major hemagglutinin antigen of A. paragallinarum is HMTp210, which is a large protein of about 2000 amino acids (aa), including a 70-aa signal peptide at its N-terminal end. However, the regions important for the hemagglutination (HA) activity and serotypes of HMTp210 remain unclear. In this study we constructed a series of A. paragallinarum strains expressing HMTp210 in-frame deletion mutants and determined their HA titers to identify the regions important for the HA activity and serotypes of HMTp210. Two distinct types of HA activities were found in HMTp210. The type 1 HA activity resided in the region spanning the full-length HA (aa 71-2084), whereas the type 2 resided in the region spanning aa 1003-2084. The putative ligand binding of the type 1 HA activity was located at aa 176-360, which had a structure similar to YadA of Yersinia enterocolitica. The putative ligand binding site of the type 2 HA activity was located at aa 1003-1125, which had a structure similar to UspA1 from Moraxella catarrhalis. The type 1 HA activity appeared to be Page serogroup specific, whereas type 2 appeared to be Kume serovar specific. Finally, sequence analyses of the regions spanning aa 1-400 and aa 1100-1600 of HMTp210 could be useful for the molecular serotyping (the Page and revised Kume schemes) of A. paragallinarum isolates.

Basic Characterization of Natural Transformation in Avibacterium paragallinarum.

Avibacterium paragallinarum is the pathogen involved in infectious coryza (IC), an acute infectious upper respiratory disease in chickens. The prevalence of IC has increased in China in recent years. There is a lack of reliable and effective procedures for gene manipulation, which has limited the research on the bacterial genetics and pathogenesis of A. paragallinarum. Natural transformation has been developed as a method of gene manipulation in Pasteurellaceae by the introduction of foreign genes or DNA fragments into bacterial cells, but there has been no report on natural transformation in A. paragallinarum. In this study, we analyzed the existence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum and established a method for transformation in it. Through bioinformatics analysis, we identified 16 homologs of Haemophilus influenzae competence proteins in A. paragallinarum. We found that the uptake signal sequence (USS) was overrepresented in the genome of A. paragallinarum (1,537 to 1,641 copies of the core sequence ACCGCACTT). We then constructed a plasmid, pEA-KU, that carries the USS and a plasmid, pEA-K, without the USS. These plasmids can be transferred via natural transformation into naturally competent strains of A. paragallinarum. Significantly, the plasmid that carries USS showed a higher transformation efficiency. In summary, our results demonstrate that A. paragallinarum has the ability to undergo natural transformation. These findings should prove to be a valuable tool for gene manipulation in A. paragallinarum. IMPORTANCE Natural transformation is an important mechanism for bacteria to acquire exogenous DNA molecules during the process of evolution. Additionally, it can also be used as a method to introduce foreign genes into bacteria under laboratory conditions. Natural transformation does not require equipment such as an electroporation apparatus. It is easy to perform and is similar to gene transfer under natural conditions. However, there have been no reports on natural transformation in Avibacterium paragallinarum. In this study, we analyzed the presence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum. Our results indicate that natural competence could be induced in A. paragallinarum serovars A, B, and C. Furthermore, the method that we established to transform plasmids into naturally competent A. paragallinarum strains was stable and efficient.

Pathogenicity of Avibacterium paragallinarum Strains from Peru and the Selection of Candidate Strains for an Inactivated Vaccine.

Worldwide, Avibacterium paragallinarum is the aetiological agent of infectious coryza in poultry. Vaccines are the best means of control, helping reduce clinical signs and colonization of this bacterium. Most vaccines are based on international reference strains, or, lately, regional strains, but, generally, without any information regarding their virulence. The characterization of the pathogenicity of 24 Av. paragallinarum strains of the three Page serogroups, including four variant strains of serogroup B, all isolated from infectious coryza outbreaks in Peru, was performed. After experimental inoculation into the infraorbital sinuses, information regarding their capacity to induce infectious coryza typical clinical signs, spreading, and colonization was recorded. Furthermore, after intraperitoneal inoculation, septicaemia and death were registered. Differences among strains in these parameters were observed, even within strains from the same serogroup. Finally, the four most pathogenic strains, one from each serogroup, were chosen to formulate an experimental vaccine that was tested successfully against homologous challenges in reducing clinical signs and colonization in vaccinated birds compared to unvaccinated ones. This is the first time that Av. paragallinarum strains from Peru were studied thoroughly for their virulence in a search for improving vaccine formulation.

ВЫЯВЛЕНИЕ УСТОЙЧИВОСТИ К АНТИБИОТИКАМ У ВОЗБУДИТЕЛЯ ГИСТОФИЛЕЗА КРУПНОГО РОГАТОГО СКОТА Histophilus somni

ВЫЯВЛЕНИЕ УСТОЙЧИВОСТИ К АНТИБИОТИКАМ У ВОЗБУДИТЕЛЯ ГИСТОФИЛЕЗА КРУПНОГО РОГАТОГО СКОТА Histophilus somni

PROPERTIES OF EXPERIMENTAL SAMPLES OF VACCINE AGAINST AVIAN INFECTIOUS CORYZA

PROPERTIES OF EXPERIMENTAL SAMPLES OF VACCINE AGAINST AVIAN INFECTIOUS CORYZA

Characterization of emergent Avibacterium paragallinarum strains and the protection conferred by infectious coryza vaccines against them in China

Infectious coryza (IC), an acute respiratory disease of chickens, is caused by Avibacterium paragallinarum. Here, the current epidemiological status of IC was investigated in China over 5 yr (2013 to 2018). A total of 28 Av. paragallinarum field isolates were identified by PCR tests and by sequence analysis of the hemagglutinin gene. The pathogenicities of 4 field isolates, the efficacy of 2 commercial inactivated oil-emulsion IC vaccines and vaccines containing different Av. paragallinarum isolates were also evaluated. The PCRs revealed a high rate (51.5%) of sample positivity for Av. paragallinarum during 2013 to 2018. Phylogenetic analysis showed that most field strains fell into the same cluster and had a farther genetic relationship with the early isolates from China. Pathogenicity testing revealed that the Chinese Av. paragallinarum isolates were able to induce the typical clinical signs of IC; hence, they were clearly pathogenic to chickens. Vaccine efficacy tests revealed that the 2 commercial inactivated oil-emulsion IC vaccines we tested had low protection rates against 2 selected Av. paragallinarum isolates after a single immunization, whereas the inactivated vaccine containing the Av. paragallinarum BJ26 isolate generated a relatively high protection rate against the field isolates compared with other three tested vaccines. The results indicate that IC is currently prevalent in China, and that commercial vaccines have not counteracted its presence in this country.

Genomic Islands in the Full-Genome Sequence of an NAD-Hemin-Independent Avibacterium paragallinarum Strain Isolated from Peru.

Here, we report the full-genome sequence of an NAD-hemin-independent Avibacterium paragallinarum serovar C-2 strain, FARPER-174, isolated from layer hens in Peru. This genome contained 12 potential genomic islands that include ribosomal protein-coding genes, a nadR gene, hemocin-coding genes, sequences of fagos, an rtx operon, and drug resistance genes.

Identification and Characterization of an RTX Toxin–Like Gene and Its Operon from Avibacterium paragallinarum

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Whole-genome sequencing analysis showed that A. paragallinarum strain H18 contains an RTX toxin-like operon with strong similarity to the RTX operons of other members of the Pasteurellaceae. Four genes, designated avxIC, avxIA, avxIB, and avxID, were found in this operon. The avxIA gene encodes the structural RTX toxin-like protein, which has a predicted molecular mass of about 250 kDa. The AvxIA protein contains a peptidase S8 domain and a proprotein convertase P-domain, neither of which has been found in other RTX toxins. Recombinant AvxIA proteins expressed in Escherichia coli showed neither hemolytic nor cytotoxic activity. Polymerase chain reaction and sequencing analysis revealed that the avxIA gene was present in all strains and field isolates of A. paragallinarum examined in this study. Sera collected from chickens exposed to A. paragallinarum exhibited strong reactivity to the AvxIA protein, which suggests that AvxIA is immunogenic. This is the first report of the identification of an RTX toxin-like operon from A. paragallinarum. The gene products of this operon may be related to disease pathogenesis and potentially represent a useful vaccine target of A. paragallinarum.

Vaccination of One-Day-Old Broiler Chicks Against Infectious Coryza

In order to evaluate the protection conferred by an experimental inactivated vaccine against infectious coryza, three challenge trials were undertaken using 112 1-day-old broilers. The vaccine "Hepa Inmuno NC" included bacterial antigens of Avibacterium paragallinarum (serogroups A, B, variant B, and C) as well as antigens of Newcastle virus and hepatitis virus. Fifty-six broiler chicks were vaccinated at the first day of life at the hatchery while another 56 chicks were left unvaccinated. Three infection trials were conducted simultaneously using each of the three serogroups A, B, or C of Av. paragallinarum. In each trial, 17 vaccinated and 17 unvaccinated broilers were used. Challenge was performed at day 31 of life by injection, into the left infraorbital sinus, of approximately 1 x 10(5) colony forming units of the corresponding Av. paragallinarum strain. Clinical signs were recorded on day 2 postchallenge. All broilers were euthanatized and both infraorbital sinuses were bacteriologically examined for the presence of Av. paragallinarum on day 5 postchallenge. In comparison with the unvaccinated broilers, the vaccine significantly reduced the number of broilers with clinical signs after challenge with serogroup B, and significantly fewer vaccinated broilers were positive for the presence of Av. paragallinarum after challenge with serogroup C. On the other hand, no significant protection was observed when broilers were challenged with Av. paragallinarum from serogroup A. Despite the high infection rates in vaccinated chicks after an experimental infection with Av. paragallinarum, it was possible to reduce colonization of Av. paragallinarum (serogroup B) and clinical signs (serogroup C) in broiler chicks by vaccination at the first day of life. Further cross-protection trials should be done, including other Av. paragallinarum strains in the vaccine, especially those from serogroup A.

Preparation and use of autogenous vaccine from Avibacterium paragallinarum (strain Tan 1-05) in layer chickens

The study was conducted to prepare and evaluate the use of autogenous vaccine from Avibacterium paragallinarum (strain Tan 1-05) in layer chickens. The results showed that all chickens vaccinated with autogenous vaccine with 10(8)CFU/mL in aluminum phosphate gel developed MAT antibodies (GMT of 2.8 log2 to 5.3 log2) against A. paragallinarum infection. Moreover, the results indicated that all chickens (n=6) selected from vaccinated chickens were protected against A. paragallinarum infection after challenge. No A. paragallinarum was isolated from these chickens. Nevertheless, all unvaccinated chickens did not develop antibodies, and all selected unvaccinated chickens (n=6) showed clinical signs consistent with infectious coryza (IC) where two of them died from the disease after challenge. The findings from the present and previous studies showed that the development of an inactivated IC vaccine from local strains if optimized and adopted may be the best possible way of controlling this economically important poultry disease.

<i>Haemophilus paragallinarum</i> haemagglutinin : role in adhesion, serotyping and pathogenicity

It is suggested that Haemophilus paragallinarum requires at least three haemagglutinins for adhesion during infection. This paper reports the partial purification and characterization of the HA-L haemagglutinin from H. paragallinarum strain 46-C3, a heat sensitive, trypsin sensitive haemagglutinin that has been shown to be the serovar specific haemagglutinin in this organism. Using the pl and molecular mass obtained, it was shown that this protein shares similarities with other types of adhesins found in Gram-negative bacteria. The haemagglutination assay conditions were optimized at pH 7.5 at 37 degrees C. It was also shown that activity is enhanced by the addition of Ca2+ and Mn2+ ions.

Amplified 16S Ribosomal DNA Restriction Analysis for Identification of Avibacterium paragallinarum

A molecular technique based on the restriction fragment length polymorphism of the 16S ribosomal genes amplified by a polymerase chain reaction (PCR), referred to as amplified 16S ribosomal DNA restriction analysis (ARDRA), was designed to identify 19 Avibacterium paragallinarum strains isolated from infraorbital sinus and nasal turbinate bone samples of broiler chickens, breeders, and laying hens from different regions of Peru. The 16S rDNA was amplified by PCR using a pair of bacterial universal primers and restriction analysis of 16S rDNA sequences was done to select endonucleases with the highest number of cutting points inside the 16S rDNA. The DNA patterns with DdeI and RsaI endonucleases were identical for the 19 A. paragallinarum strains, but differed from those obtained for Ornithobacterium rhinotracheale, a bacterium with a high genetic and phenotypic resemblance to A. paragallinarum, as well as from Escherichia coli, a bacterium associated with infectious coryza. The ARDRA method could prove to be valuable for molecular identification of A. paragallinarum, a microorganism implicated in respiratory diseases in commercial birds.

Antimicrobial susceptibility, plasmid profiles and haemocin activities of Avibacterium paragallinarum strains

In this study, 18 Avibacterium paragallinarum isolates collected in Taiwan from 1990 to 2003 were serotyped and tested for resistance to antimicrobial agents. Serotyping revealed that 13 isolates were Page serovar A and 5 isolates were Page serovar C. More than 75% of the isolates were resistant to neomycin, streptomycin and erythromycin. The most common resistance pattern (15 isolates, 83.3%) was neomycin–streptomycin. Furthermore, 88.9% of the isolates were resistant to two or more antibiotics. About 72% of isolates contained plasmids (pYMH5 and/or pA14). Plasmid pYMH5 encoded functional streptomycin, sulfonamide, kanamycin and neomycin resistance genes and revealed significant homology to a broad host-range plasmid, pLS88. Plasmid pA14 encoded a putative MglA protein and RNase II, both of which might be associated with virulence. Furthermore, seven isolates showed haemocin activity. Plasmid pYMH5 is the first multidrug-resistance plasmid reported in A. paragallinarum and it may facilitate the spread of antibiotic-resistance genes between bacteria. The putative virulence plasmid pA14 and haemocin-like activity in A. paragallinarum indicate two possible mechanisms which might be responsible for the pathogenesis.

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov.

This paper describes a phenotypic and genotypic investigation of the taxonomy of [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium, a major subcluster within the avian 16S rRNA cluster 18 of the family Pasteurellaceae. An extended phenotypic characterization was performed of the type strain of [Haemophilus] paragallinarum, which is NAD-dependent, and eight NAD-independent strains of [Haemophilus] paragallinarum. Complete 16S rRNA gene sequences were obtained for one NAD-independent and four NAD-dependent [Haemophilus] paragallinarum strains. These five sequences along with existing 16S rRNA gene sequences for 11 other taxa within avian 16S rRNA cluster 18 as well as seven other taxa from the Pasteurellaceae were subjected to phylogenetic analysis. The analysis demonstrated that [Haemophilus] paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium formed a monophyletic group with a minimum of 96.8 % sequence similarity. This group can also be separated by phenotypic testing from all other recognized and named taxa within the Pasteurellaceae. As both genotypic and phenotypic testing support the separate and distinct nature of this subcluster, the transfer is proposed of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium to a new genus Avibacterium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov. The type strains are NCTC 1118T (Avibacterium gallinarum), NCTC 11296T (Avibacterium paragallinarum), NCTC 11297T (Avibacterium avium) and NCTC 3438T (Avibacterium volantium). Key characteristics that separate these four species are catalase activity (absent only in Avibacterium paragallinarum) and production of acid from galactose (negative only in Avibacterium paragallinarum), maltose (negative only in Avibacterium avium) and mannitol (negative in Avibacterium gallinarum and Avibacterium avium).

Virulence of the Nine Serovar Reference Strains of Haemophilus paragallinarum

The virulence of the reference strains of the nine currently recognized Kume serovars of Haemophilus paragallinarum was investigated. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. All nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). Our results indicate that virulence differences exist among the serovars of H. paragallinarum.

Typing of Haemophilus paragallinarum Strains by Using Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction

The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used for fingerprinting of reference strains and Mexican isolates of Haemophilus paragallinarum. A total of nine ERIC patterns were given by the nine serovar reference strains of this bacteria. Two Modesto (C-2) reference strains from different sources showed the same ERIC pattern. Seventeen ERIC patterns were obtained among 29 Mexican isolates included in the study, belonging to serovars prevalent in Mexico (A-1, A-2, B-1, and C-2). Obtained results indicate that the ERIC-PCR technique could be used as a molecular laboratory tool for subtyping of H. paragallinarum.

Genetic analysis of a plasmid encoding haemocin production in Haemophilus paragallinarum.

The full sequence of plasmid p250, isolated from Haemophilus paragallinarum strain HP250, has been obtained. The plasmid contains seven ORFs: a putative integrase, a putative replication protein (repB) and five ORFs similar to those from the haemocin (bacteriocin) hmcDCBAI operon from Haemophilus influenzae. Of 19 other non-plasmid-containing H. paragallinarum strains screened (11 serovar reference strains and 8 field isolates), 17 strains produced haemocin and were resistant to killing by strain HP250. These strains, unlike strain HP250, have a chromosomally encoded haemocin operon. A number of other members of the family Pasteurellaceae were tested for haemocin sensitivity. Pasteurella avium, Pasteurella volantium and Pasteurella species A, all non-pathogenic bacteria found in the respiratory tract of chickens suffering from respiratory diseases, were sensitive to H. paragallinarum haemocin. However, amongst the pathogenic Pasteurellaceae, 50 % of P. multocida isolates and all five isolates of Pasteurella haemolytica tested were sensitive to the haemocin. Given the prevalence of haemocin production in H. paragallinarum strains, it may play a role in aiding colonization by inhibiting other Gram-negative bacteria that are associated with the respiratory tract in chickens. The origin of replication from plasmid p250 has been used to generate an Escherichia coli-H. paragallinarum shuttle vector which may be useful in genetically manipulating H. paragallinarum.