Genotyping, pathogenicity, and immunogenicity of Avibacterium paragallinarum serovar B-1 isolates from the Americas.

The bacterium Avibacterium paragallinarum is the etiologic agent of infectious coryza of chickens. Among the nine Kume serovars currently recognized in this bacterium, serovar B-1 is a common serovar in the Americas. In the current study, serovar B-1 isolates from Ecuador (seven isolates), Mexico (seven isolates) and Panama (two isolates) were genotyped. In addition one Panamanian, one Ecuadorian, and two Mexican isolates were used in a vaccination-challenge trial in which the vaccine was based on the 2671 serovar B-1 reference strain. Genotyping by enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR) resulted in ten distinguishable ERIC patterns for the 16 isolates and the two reference strains of Av. paragallinarum included in the study. No ERIC patterns were shared among isolates of the three different countries. In the vaccination-challenge trial, one isolate from Panama showed a significantly lower virulence than did the three other isolates. In terms of cross-protection, chickens vaccinated with reference strain 2671 and challenged with an Ecuadorian strain showed 40% protection, a significantly lower protection than the homologous protection level. The other three field isolates gave a similar protection level to the homologous challenge.

Transcriptional profiling of chicken immunity-related genes during infection with Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of Infectious Coryza (IC), which is an upper respiratory tract disease in chickens. The occurrence of outbreaks has emphasized the significance of the disease globally in the chicken industry. Studies have demonstrated that early immune responses are critical in defining the severity and physiological outcome of an infection. This prompted the need to investigate the regulation of immune functions by the number of genes that are expressed during the chickens' response to A. paragallinarum serovar C3 insult. This study consisted of 15 male leghorn birds that were scored into groups (score 1, 2, 3) according to severity of symptoms after they were challenged. Expression patterns of immunity-related genes were followed as symptoms progressed from a disease score of 1 to 3. The data proposed that initial pathogen recognition was either through Toll-like receptors 2 or 4. Unique expression patterns were observed such as the up-regulation of TLR7 which recognizes viral-like particles. This substantiated the presence of prophages reported in the genome of A. paragallinarum. Significant down-regulation of metabolic pathways was observed, which led us to hypothesize that the host may rely on an oxidative stress response as initial immune response. The data sheds light onto the mechanisms that govern the immune system towards infection and/or towards the initial response to infections with highly virulent A. paragallinarum.

Serovar identification, antimicrobial sensitivity, and virulence of Avibacterium paragallinarum isolated from chickens in Thailand.

Avibacterium paragallinarum causes infectious coryza in chickens, an acute respiratory disease that has worldwide economic significance. The objectives of this study were to determine the serovars, antimicrobial resistance, and pathogenicity of A. paragallinarum isolated from chickens in Thailand. Eighteen field isolates of A. paragallinarum were confirmed by PCR. When examined by serotyping in a hemagglutination inhibition test, 10 isolates were serovar A, five isolates were serovar B, and three isolates were serovar C. The susceptibility of the isolates to 16 antimicrobial agents was tested by a disk diffusion method. All isolates were susceptible to amoxicillin-clavulanic acid. There was a high level of resistance to lincomycin and erythromycin. All isolates were resistant to cloxacillin and neomycin. A study of bacterial entry into, and survival within, chicken macrophages showed variation between isolates but no clear connection to serovar. A virulence test was performed by challenging 4-wk-old layers via the nasal route with 400 dl of bacteria (10(8) colony-forming units/ml). Clinical signs were observed daily for 7 days, and the birds were subjected to a postmortem necropsy at 7 days postchallenge. All 18 field isolates caused the typical clinical signs of infectious coryza and could be re-isolated at 7 days after challenge. There was no significant difference in the clinical scores of the isolates except that two isolates (112179 and 102984, serovars A and B, respectively) gave a significantly higher score than did isolate CMU1009 (a serovar A isolate). No correlation between serovar and severity of clinical signs was found.

Development of a multiplex PCR and PCR-RFLP method for serotyping of Avibacterium paragallinarum.

Avibacterium (Haemophilus) paragallinarum (A. paragallinarum) is a causative agent of infectious coryza in chickens and is classified into three serovars by agglutination tests. In an effort to identify the serovars easily, PCR and PCR-RFLP were employed. As the target gene for PCR, the hypervariable region of HMTp210, which encodes the HA antigen, was used. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively. Alternatively, the 1.6 kbp fragments were amplified with another primer pair encompassing the hypervariable region and was subjected to digestion with Bgl II, which resulted in the detection of serovar-specific digestion patterns. These results indicate that PCR and PCR-RFLP using the hypervariable region of HMTp210 are alternative methods to identify the serovar of A. paragallinarum.

Hemagglutinin serotyping of Avibacterium paragallinarum isolates from Ecuador.

Avibacterium paragallinarum is the causative agent of infectious coryza, an acute respiratory disease of chickens. In this study, a total of 28 isolates of A. paragallinarum from Ecuador were serotyped by the hemagglutinin scheme which recognizes nine serovars. Out of 28 isolates, 17 isolates belonged to serovar A-3, and five isolates to each serovars B-1 and C-1, whereas one isolate was non-typeable. This is the first report of A. paragallinarum serovar A-3 outside Brazil and serovar C-1 outside Japan.

Identification of Avibacterium paragallinarum serovar B-1 from severe infectious coryza outbreaks in Panama.

The isolation and identification of Avibacterium paragallinarum serovar B-1 from severe infectious coryza outbreaks in broiler breeders in Panama is reported for the first time. Infectious coryza was absent for over a decade in the breeder farms area. Disease outbreaks were characterized by an up to 45% drop in egg production and increased mortality. Use of a commercial trivalent bacterin and a strengthened biosecurity program prevented outbreaks in susceptible flocks in the farm.

Analysis of the biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum.

The aim of this study was to investigate biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum. The sequence of a 10-kb region containing the capsule biosynthetic locus of Av. paragallinarum was determined. Two reference strains, i.e., 221 (serovar A) and H18 (serovar C), together with four Taiwanese field strains (all serovar C) were sequenced. The results showed that there are two genotypes (I and II) of the capsule biosynthetic locus in Av. paragallinarum, and the capsule genotype is independent of the serovar. The capsule biosynthetic loci of genotypes I and II consisted of six and five genes, respectively. The genotype I genes encoded proteins that are most similar to proteins from Pasteurella multocida capsule types A and F while the genotype II genes encoded proteins most similar to proteins from P. multocida capsule type D and Escherichia coli K5. The results suggested that genotype I strains contain hyaluronan or chondroitin in the capsule wall while genotype II contain heparosan. Enzymatic digestion of the capsule materials extracted from Av. paragallinarum showed that genotype I strains contained chondroitin while genotype II strains contained heparosan in the capsule. This is the first report on the existence of different genotypes of capsule biosynthesis genes in Av. paragallinarum and the presence of chondroitin and heparosan as chemical components of the capsule of Av. paragallinarum.

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.

Effects of differences in virulence of different serovars of Haemophilus paragallinarum on perceived vaccine efficacy.

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum and two field isolates of NAD-independent H. paragallinarum has previously been tested in unvaccinated chickens. In this study, the disease profiles caused by the NAD-dependent isolates of H. paragallinarum in vaccinated chickens were studied. It was shown that the clinical signs induced in the vaccinated chickens were substantially less severe than were those in unvaccinated chickens, as was expected. However, due to the high virulence of the serovar C-3 isolates, clinical signs in the vaccinated chickens challenged with this isolate were still detected. These were as severe as those occurring in unvaccinated chickens challenged with serovar B-1 isolates. Although the clinical signs induced in unvaccinated birds challenged with serovar A-1 were more severe than those occurring when vaccinated birds were challenged with serovar C-3, the overall disease profiles were similar. Substantial clinical signs were recorded in vaccinated birds challenged with serovar C-3. This could be interpreted as vaccination failure if the disease profile obtained in unvaccinated birds is not considered. It was found that a high level of protection was provided by this vaccine against challenge by serovar C-3. The high virulence of this serovar resulted in the development of clinical signs in vaccinated birds. These findings could possibly explain the large number of so-called vaccination failures that are reported in South Africa.

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).

Evaluation of the protection conferred by a disinfectant against clinical disease caused by Avibacterium paragallinarum serovars A, B, and C from Argentina.

This work evaluates the efficiency of the administration of the disinfectant N-alkyl dimethyl benzyl ammonium chloride (TIMSEN) in the prevention of the horizontal transmission of serovars A, B, and C of Avibacterium paragallinarum, the causative agent of avian infectious coryza. This disinfectant was administered in drinking water (50 ppm) and once or twice per day by coarse spray (800 ppm, 8 ml per m3 during 3 seconds). In three trials conducted with vaccinated birds, the disinfectant reduced the clinical signs of infectious coryza significantly (P < 0.05). There was no significant effect when the product was used in a fourth trial with unvaccinated birds. Furthermore, the application of only one daily environmental spraying was sufficient to significantly reduce clinical signs. According to these results, in order to diminish the clinical signs of infectious coryza in birds vaccinated against A. paragallinarum, it is recommended to administer this disinfectant in drinking water and by environmental spraying.

Protection conferred by bivalent and trivalent infectious coryza bacterins against prevalent serovars of Avibacterium (Haemophilus) paragallinarum in Mexico.

The protection and level of hemagglutination-inhibition (HI) antibodies conferred in infectious coryza bivalent- and trivalent-immunized chickens against Avibacterium (Haemophilus) paragallinarum field isolates of the prevalent serovars in Mexico (A-1, A-2, B-1, and C-2) were investigated. The bivalent bacterin (A-1 and C-1) conferred significant protection and increased HI antibodies against isolates of serovars A-1, A-2, and C-2, but not against a serovar B-1 isolate. The trivalent bacterin (A-1, B-1, and C-2) conferred protection and increased HI antibodies against all four of the isolates. The results confirmed that in poultry areas where serovar B-1 is prevalent, the inclusion of this serovar in bacterins is needed to confer protection against infectious coryza caused by A. (H.) paragallinarum isolates of serovar B-1.

Cross-protection study of the nine serovars of Haemophilus paragallinarum in the Kume haemagglutinin scheme.

The cross-protection and haemagglutination-inhibition antibodies present in chickens vaccinated with one of the nine currently recognized Kume haemagglutinin serovars of Haemophilus paragallinarum were investigated. The results confirmed the widely accepted dogma that serogroups A, B, and C represent three distinct immunovars. Within Kume serogroup A, there was generally good cross-protection among all four serovars. However, within Kume serogroup C, there was evidence of a reduced level of cross-protection between some of the four serovars. The haemagglutination-inhibition antibody levels generally showed the same trend as with the cross-protection results. This study suggests that some apparent field failures of infectious coryza vaccines may be due to a lack of cross-protection between the vaccine strains and the field strains. Our results will help guide the selection of strains for inclusion in infectious coryza vaccines.

The testing and modification of a commercially available transport medium for the transportation of pure cultures of Haemophilus paragallinarum for serotyping.

Haemophilus paragallinarum, the causative agent of infectious coryza in poultry, is an extremely fastidious organism requiring specific growth conditions for isolation. For complete control of the disease in regions where more that one of the serovars of the different serogroups occurs, it is essential that the bacterium causing the problem be isolated and serotyped. This work describes the modification and testing of transport media, which will ensure the survival of the causative agents in suspected infectious coryza cases for transport to a laboratory where the bacterium can be isolated and serotyped. The various transport media used are based on commercially available Amies Transport Medium supplemented with the different supplements used for the growth of H. paragallinarum. It was established that the bacterium remains viable for up to 18 days in Amies Transport Medium containing all the supplements when stored at 4 degrees C or 37 degrees C. At room temperature or 25 degrees C, there was no difference in the survival of H. paragallinarum in commercial Amies Transport Medium (without charcoal) and Amies Transport Medium with supplements.

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.