Resident bacteria contribute to opportunistic infections of the respiratory tract.

Opportunistic pathogens frequently cause volatile infections in hosts with compromised immune systems or a disrupted normal microbiota. The commensalism of diverse microorganisms contributes to colonization resistance, which prevents the expansion of opportunistic pathogens. Following microbiota disruption, pathogens promptly adapt to altered niches and obtain growth advantages. Nevertheless, whether and how resident bacteria modulate the growth dynamics of invasive pathogens and the eventual outcome of such infections are still unclear. Here, we utilized birds as a model animal and observed a resident bacterium exacerbating the invasion of Avibacterium paragallinarum (previously Haemophilus paragallinarum) in the respiratory tract. We first found that negligibly abundant Staphylococcus chromogenes, rather than Staphylococcus aureus, played a dominant role in Av. paragallinarum-associated infectious coryza in poultry based on epidemic investigations and in vitro analyses. Furthermore, we determined that S. chromogenes not only directly provides the necessary nutrition factor nicotinamide adenine dinucleotide (NAD+) but also accelerates its biosynthesis and release from host cells to promote the survival and growth of Av. paragallinarum. Last, we successfully intervened in Av. paragallinarum-associated infections in animal models using antibiotics that specifically target S. chromogenes. Our findings show that opportunistic pathogens can hijack commensal bacteria to initiate infection and expansion and suggest a new paradigm to ameliorate opportunistic infections by modulating the dynamics of resident bacteria.

Virulence of Serovar C-1 Strains of Avibacterium paragallinarum.

The bacterium Avibacterium paragallinarum is the etiologic agent of infectious coryza of chickens. There are nine serovars of A. paragallinarum , and serovar C-1 has emerged in outbreaks of infectious coryza in layer hens in the Americas, with all isolates having been obtained from infectious coryza-vaccinated chickens. In the current study, the clinical and histopathologic outcomes of experimental infections in chickens with A. paragallinarum of serovar C-1 were investigated. The Japanese serovar reference strain, H-18, and a Mexican isolate, ESV-135, were included in the study. No differences in clinical sign scores or morbidity were observed between the two strains. The two bacterial strains caused microscopic lesions of lymphoplasmacytic inflammation in the mucosa of the nasal cavity, infraorbital sinus, and trachea. Similar severe lesions were observed in birds inoculated with both H-18 and ESV-135 strains. The lesions were present 48 hr after inoculation and persisted until day 10 after inoculation. Slight to severe, extensive hemorrhages were observed in the lumen, mucous membranes, and lamina propria of the nasal cavity and infraorbital sinus in most of the chickens inoculated with either the reference strain H-18 or the ESV-135 isolate. Hemorrhages in the upper respiratory tract of chickens experimentally infected with A. paragallinarum are reported here for the first time. The results have confirmed the high virulence of the reference strain H-18 as previously reported and have shown that the Mexican isolate was as virulent as the reference strain. The virulence of A. paragallinarum isolates may play a role in explaining why severe infectious coryza outbreaks are being seen in both vaccinated and nonvaccinated chicken flocks.

The Fimbrial Protein is a Virulence Factor and Potential Vaccine Antigen of Avibacterium paragallinarum.

Fimbriae are recognized as virulence factors and potential vaccine antigens of several pathogenic bacteria, but the function of the fimbriae from Avibacterium paragallinarum is not well known. In this study, a gene encoding the fimbrial protein FlfA was identified in A. paragallinarum . Sequencing analysis of the putative promoter region of flfA suggests that flfA expression in A. paragallinarum might be controlled by phase variation. The flfA gene from A. paragallinarum was expressed as a recombinant protein (r-FlfA) in Escherichia coli . Immunization with r-FlfA conferred chickens protection against challenge infection with A. paragallinarum . Virulence assays showed that the flfA-deficient mutants of A. paragallinarum were less virulent than their parental wild-type strains. These results indicated that the fimbrial protein FlfA is a virulence factor and potential vaccine antigen from A. paragallinarum .

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.

Study on the efficacy and safety of different antigens and oil formulations of infectious coryza vaccines containing an NAD-independent strain of Avibacterium paragallinarum.

The present study was designed to assess and compare three different formulations of the new Onderstepoort infectious coryza (IC) quadrivalent vaccine, which contain an NAD-independent strain of Avibacterium paragallinarum (previously known as Haemophilus paragallinarum), and a commercial IC vaccine, not containing an NAD-independent strain, for their safety and ability to protect chickens of varying ages against virulent challenges with four different serovars of A. paragallinarum, including the NAD-independent strain of the C-3 serovar. Four groups of 140 chickens each were vaccinated at the age of 17 weeks and revaccinated at the age of 19 weeks with each of the four vaccine formulations. A similar sized group of non-vaccinated chickens was used as control. Two rounds of challenge were conducted: a group of chicken in each vaccination group was challenged between 31 and 35 weeks of age, while another group was challenged between 51 and 55 weeks of age. The "in-contact" challenge model was used in this experiment. For each vaccination group, the four challenge strains representing four local serovars were used in each challenge round. The efficacy of the vaccines was compared based on overall protection levels obtained and the duration of protection. The safety of the different vaccines was determined by the severity of post-vaccination reactions. The need for the incorporation of the NAD-independent strain in the vaccine was evidenced by the low protection level against NAD-independent challenge recorded in the group of birds vaccinated with the commercial vaccine. The results obtained confirmed not only the variation in virulence of different South African serovars, with serovar C-3 being the most virulent and serovar B having almost no virulence but also the age related increase in susceptibility. The importance of a suitable formulation of the vaccine is discussed.

Haemophilus paragallinarum 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.

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.

Haemophilus paragallinarum secretes metalloproteases.

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH(4))(2)SO(4)) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4-9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 degrees C, but its activity diminished at 70 degrees C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.

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.

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.

Limitation of the spread and impact of infectious coryza through the use of a continuous disinfection programme.

The effect of a continuous disinfection programme, using the non-toxic disinfectant Virukill, in layers, on the spread and impact of infectious coryza, caused by Haemophilus paragallinarum was evaluated. In this experiment, both unvaccinated layers and layers vaccinated against infectious coryza were used. Duplicate smaller groups of vaccinated and unvaccinated chickens were challenged with different serovars of both NAD-dependent as well as NAD-independent isolates of Haemophilus paragallinarum. One group of chickens challenged with each of the different becterial serovars was treated with the continuous disinfection programme, while the other group remained as the untreated controls. The clinical signs of infectious coryza were evaluated over a period of 20 days in each group. The egg production over this period was also evaluated. It was found in all experimental challenges, that the severity of the symptoms was reduced in the birds receiving the continuous disinfection programme. The drop in egg production was also found to be less severe in the treated groups when compared to the untreated control groups. The duration of infection was found to be either unchanged, or shorter in the birds treated with the continuous disinfection programme. In none of the experimental challenges was the duration or expression of clinical signs of IC increased due to the continuous disinfection programme.

Evidence of possible evasion of protective immunity by NAD-independent isolates of Haemophilus paragallinarum in poultry.

An indication of the ability of NAD-independent variants of Haemophilus paragallinarum to evade the immune system has been obtained from data obtained from several experiments. Firstly, it was noted that there was a difference in the serovar distribution between the NAD-dependent isolates in the 1990s and the NAD-independent isolates, as there was a significant decrease in the incidence of serogroup A NAD-dependent isolates. This can possibly be attributed to the extensive use of vaccines. On the other hand, most of the earlier NAD-independent isolates were serovar A. This is a possible indication of evasion of the protective immunity by the NAD-independent isolates. Further evidence of possible evasion of the protective immunity was obtained from results obtained when different isolates, both NAD dependent and NAD independent, were tested with a panel of monocional antibodies (Mabs). The V1 Mab reaction pattern was only seen in the reference strain 0083 among all of the NAD-dependent isolates tested in South Africa. This Mab was, however, found to react with some of the NAD-independent isolates. Furthermore, the isolation of NAD-dependent isolates in Australia which react with the V1 Mab also suggest possible evasion of the protective immunity by the NAD-independent isolates as no vaccines containing strain 0083 are used in Australia. In order to investigate the hypothesis of immune-evasion by NAD-independent H. paragallinarum, vaccinated and unvaccinated chickens were challenged with a NAD-independent serogroup C isolate. As a control, chickens were also challenged with NAD-dependent H. paragallinarum of the same serogroup. The results obtained indicate that there is no significnat difference in the disease profiles obtained in vaccinated and unvaccinated chickens challenged with the NAD-independent isolate, thus providing further evidence of evasion of the productivity immunity by the NAD-independent isolates. The ability of the NAD-independent isolates to evade the immune system suggests that a different vaccination strategy, or alternative control methods may be needed for the control of IC caused by these isolates.

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.