An evaluation of inactivated infectious coryza vaccines containing a double-emulsion adjuvant system.

The efficacy of experimental inactivated infectious coryza vaccines produced by a commercial vaccine manufacturer was evaluated. The vaccines, containing as the adjuvant phase either a double-emulsion mineral oil system or aluminum-hydroxide gel, were administered to 6-week-old chickens as a single dose. Some vaccines were a monovalent product containing a Page serovar C Haemophilus paragallinarum strain, and others were a bivalent product containing both Page serovar A and serovar C strains. After 3 weeks, all chickens were challenged by infraorbital sinus inoculation of virulent H. paragallinarum, either Page serovar C (strain HP31) or Page serovar A (strain HP14). The monovalent serovar C double-emulsion-based vaccines gave significant protection against a serovar C challenge, with the level of protection varying from 60% to 100%. The monovalent serovar C aluminum-hydroxide-gel vaccine also gave significant protection (94%) against a serovar C challenge. The bivalent double-emulsion vaccine gave significant protection against challenge from both serovars (100% for serovar C and 83% for serovar A). Although no major adverse reactions were detected, some chickens receiving both the double-emulsion vaccines and the aluminum-hydroxide vaccine developed relatively minor granulomatous reactions at the site of injection.     

Serotyping of Haemophilus paragallinarum isolates from Mexico by the Kume hemagglutinin scheme

A total of 42 isolates of Haemophilus paragallinarum from Mexico were serotyped by the Kume hemagglutinin scheme. Serovars A-1, A-2, B-1, and C-2 were recognized among 11 (26.2%), 7 (16.6%), 4 (9.5%), and 14 (33.3%) isolates, respectively. A further six isolates (14.3%) showed hemagglutinating activity but could not be classified into any serovar. Commercial vaccines containing Kume serovars A-1, A-2, B-1, and C-2 may provide better protection than those bi- or trivalent infectious coryza vaccines currently used in Mexico.     

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.     

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.     

Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).     

Comparison of adjuvants for an inactivated infectious coryza vaccine

Differently formulated inactivated infectious coryza vaccines were administered to 6-week-old chickens as a single dose of 10(8) colony-forming units of Haemophilus paragallinarum HP31. After 3 weeks, all chickens were challenged by intrasinus inoculation of HP31. Two vaccines, one containing an aluminum-hydroxide adjuvant and the other a combined aluminum-hydroxide + mineral-oil adjuvant, gave the best protection (means of 80% and 90%, respectively). Two vaccines that contained mineral oil as the sole adjuvant gave less protection (50% and 35%). The Quil A vaccine gave no significant protection. Granulomatous swellings developed at the site of injection in birds given mineral-oil adjuvant but not in those that received other adjuvants.     

ERIC-PCR genotyping of emergent serovar C-1 isolates of Avibacterium paragallinarum from Mexico

Between 2008 and 2010, 14 isolates of Avibacterium paragallinarum were identified as serovar C-1 in Mexico. All isolates were obtained from commercial laying hens suffering infectious coryza despite a history of vaccination. The enterobacterial repetitive intergenic consensus-based PCR genotyping showed that all isolates had a common pattern. Until recently, serovars A-1, A-2, B-1, and C-2 were the serovars prevalent in Mexico. Serovar C-1 has been identified in Japan and recently in the Americas in Ecuador. Our current study suggests that Av. paragallinarum serovar C-1 is an emerging serovar in Mexico. Our results also indicate that the Mexican isolates of Av. paragallinarum serovar C-1 may have a clonal relationship. Knowledge of the genetic diversity of Av. paragallinarum may be of value in understanding vaccine performance and identifying the best combination to achieve broader protection.     

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.     

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.     

The presence of nicotinamide adenine dinucleotide-independent Haemophilus paragallinarum in México

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent-growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3-wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NAD-independent H. paragallinarum of serovar B has been reported.     

Further comparison of adjuvants for an inactivated infectious coryza vaccine

Inactivated infectious coryza vaccines containing different adjuvants were administered to 6-week-old chickens as a single dose containing 10(8) colony-forming units of Haemophilus paragallinarum HP31. After 3 weeks, all chickens were challenged by intrasinus inoculation of HP31. Two vaccines, one containing an aluminum-hydroxide adjuvant and the other a combined aluminum-hydroxide + avridine (a lipoidal amine) adjuvant, were effective. The three remaining vaccines--containing mineral-oil double-emulsion, avridine, or a combination of mineral-oil double-emulsion + avridine--gave much lower levels of protection. No adverse reactions were seen with any vaccine.     

鸡传染性鼻炎三价灭活疫苗安全性试验和效力检验初报

利用PageA、B、C型副鸡禽杆菌,研制鸡传染性鼻炎(IC)三价灭活疫苗。用6批IC疫苗进行了大剂量单次接种和单剂量多次接种的安全性试验,结果表明,IC三价灭活疫苗安全无副作用;用3批疫苗进行SPF鸡和普通鸡的免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥80%;商品鸡42日龄首免,110日龄二免,二免后约9个月对A、B、C型副鸡禽杆菌攻毒的保护率≥80%;用4℃～8℃保存一年的3批疫苗进行了SPF鸡的近期免疫效力试验,结果对A、B、C型副鸡禽杆菌攻毒的保护率≥80%。     

Isolation of serovar C-3 Haemophilus paragallinarum from Zimbabwe: A further indication of the need for the production of vaccines against infectious coryza containing local isolates of H. paragallinarum

Various isolates of Haemophilus paragallinarum, collected from a severe outbreak of infectious coryza in poultry from Zimbabwe, were serotyped and were found to belong to serovar C-3. Previously, isolates were serotyped using polyclonal antiserum produced against serogroup reference strains (0083 for serogroup A, 0222 for serogroup B and Modesto, or H-18 for serogroup C) of H. paragallinarum. In this case, polyclonal antiserum produced against these reference isolates were used, as well as polyclonal antiserum that has been raised specifically against the serovar C-3 isolate 46 C-3. When using the latter serum at a 1 in 50 dilution, no cross-reaction with other members of serogroup C were found. The severity of the disease outbreak in Zimbabwe, the vaccination history of the infected flocks on the sites and the isolation of the uniquely southern African serovar C-3, further highlights the need for vaccines composed of local isolates to control infectious coryza in regions where vaccination failures occur.     

Efficacy of a commercially available coryza vaccine against challenge with recent South African NAD-independent isolates of Haemophilus paragallinarum in chickens

In South Africa the incidence of NAD-independent Haemnophilus paragallinarum isolation from clinical cases is increasing. This study was carried out to test whether a commercially available coryza vaccine (Nobilis Coryza, Intervet International BV) could protect chickens against challenge with recent NAD-independent isolates. SPF chickens were vaccinated twice at 3 and 7 weeks of age and were challenged at 9 weeks of age with 5 different NAD-independent isolates of serotype A or C-3. The results after challenge show that the coryza vaccine induces good protection against challenge with the different South African NAD-independent isolates of H. paragallinarum, including serotype C-3.     

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.     

Immunogenicity of Haemophilus paragallinarum serovar B strains.

Immunogenicity of three Haemophilus paragallinarum serovar B strains was investigated in cross-protection tests using monovalent vaccines prepared from the B strains, as well as one strain each of serovars A and C. A bivalent vaccine composed of the serovar A and C strains also was used. In the studies with the monovalent vaccines, the immunogenicity of serovar B strains was different from that of serovar A and C strains, although only partial serovar B-specific protection with the three strains was observed. Chickens vaccinated with the bivalent vaccine protected against challenge with one serovar B strain, as well as serovar A and C strains, but not against the other two serovar B strains.     

Immunogenicity and haemagglutination of recombinant Avibacterium paragallinarum HagA

Inactivated vaccines of Avibacterium paragallinarum provide protection and reduce the economic losses caused by infectious coryza. However, inactivated bacterins provide protection only against the Page serovars included in the vaccine. In this study, we investigated the immunological properties of a functional recombinant haemagglutinin protein (rHagA) derived from a Taiwan isolate strain A9 as the immunogen for vaccination. The rHagA subunit vaccine protected 71% of immunized chickens against 10(10) colony-forming units (CFU) of viable A9. Vaccinated chickens which showed no clinical signs of coryza developed haemagglutination inhibition (HI) titers of 1:10 or greater. Haemagglutination (HA) of serovars A and C was not affected by the presence of rHagA specific antiserum. The HA of rHagA could only be induced against formaldehyde-fixed chicken red blood cells (FA-RBCs). These results suggested that HagA is a moderate immunogen and might not be a major haemagglutinin in vivo. However, HagA might be involved in haemagglutination when treated serovar C aggregates fixed RBCs in vitro.     

Characterisation of isolates of Haemophilus paragallinarum from Indonesia

OBJECTIVE: To characterise 18 isolates of Haemophilus paragallinarum isolated from chickens in Indonesia. PROCEDURE: The isolates were identified to species level by traditional phenotypic methods. Six of the isolates were also identified by a species-specific polymerase chain reaction. Fourteen of the isolates were examined for resistance to a panel of seven antimicrobial agents using a disc diffusion method. All 18 isolates were serotyped according to the Page scheme using reference antisera in a haemagglutination inhibition test. RESULTS: Four of the 18 isolates were obtained from indigenous (kampung) chickens, with the remainder being from typical intensive poultry production systems. The 18 isolates were obtained from 11 outbreaks that showed the typical clinical signs of infectious coryza and 11 of the isolates were obtained from chickens that had been vaccinated with infectious coryza vaccines. All 18 isolates were confirmed as H paragallinarum by biochemical testing and six isolates were also identified as H paragallinarum by the polymerase chain reaction test. Eleven isolates were resistant to erythromycin and streptomycin, 10 to neomycin, eight to oxytetracycline, five isolates to doxycycline, three to sulphamethoxazoltrimethoprim but only one to ampicillin. Seven isolates were Page serovar A, four were Page serovar B and seven were Page serovar C. CONCLUSION: The presence of all three Page serovars (A, B and C) has been confirmed for the first time in Indonesian chickens. As the majority of the infectious coryza vaccines in use in Indonesia contain only serovar A and C, the presence of serovar B in chickens indicates that the protection by these bivalent vaccines would be reduced. The use of trivalent infectious coryza vaccines that contain serovars A, B and C is recommended for use in Indonesia.     

副鸡嗜血杆菌的分离鉴定及我国鸡传染性鼻炎的流行状况分析

从不同地区免疫失败鸡场的鸡传染性鼻炎疑似鸡体内共分离到19株细菌，经回归试验、PCR等方法鉴定为副鸡嗜血杆菌。又将分离株用血清平板凝集试验、血凝抑制试验和型特异性单抗进行了血清型的鉴定，结果表明分离菌有15株为A型，4株为B型，说明我国鸡传染性鼻炎的流行已经出现了新的特点，在疾病预防和控制中应加以注意。另外大多数鸡场的免疫失败是由于免疫效果不良所致，但也有相当一部分鸡场的免疫失败是由于B血清型的出现引起的。     

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.