Infectious Coryza: Persistence, Genotyping, and Vaccine Testing.

The reemergence of infectious coryza (IC) caused by Avibacterium paragallinarum (AP) as an acute and occasionally chronic respiratory disease in domestic poultry has caused severe losses in several U.S. states. The disease is also associated with decreased egg production in layers and increased condemnations from air sac infections in broilers. A series of applied experiments were performed to elucidate the persistence of AP in infected broiler flocks, to genotype AP strains isolated from field cases, and to evaluate commercial and autogenous vaccine protection in commercial and specific-pathogen-free (SPF) chickens. Experimental evaluation of environmental persistence suggests that AP did not persist more than 12 hr in a hypothetically contaminated environment. Additionally, other detected potential pathogens such as Gallibacterium anatis and infectious bronchitis virus caused mild respiratory signs in the exposed birds. The HMTp210 and HagA genes of four IC field strains were sequenced and compared with published sequences of HMTp210 and HagA. The HMTp210 phylogeny showed a marginally imperfect clustering of the sequences in genogroups A, B, and C. Although not definitive, this phylogeny provided evidence that the four field strains aligned with previously characterized serovar C strains. Moreover, the base pair homology of the four strains was 100% identical to serovar C reference strains (H-18 and Modesto). HagA phylogeny was unclear, but interestingly, the IC field strains were 100% homologous to C-1 strains reported from Mexico and Ecuador. Finally, vaccine protection studies in commercial hens indicate that clinical signs are induced by a combination of IC and other concomitant pathogens infecting commercial birds. Additionally, vaccine protection experiments performed in SPF hens indicated that protection provided by the two commercial vaccines tested provided a reduction in clinical signs and bacterial shedding after two applications.

Coriza infecciosa: Persistencia, genotipificación y pruebas para vacunas. El resurgimiento de la coriza infecciosa (CI) causada por Avibacterium paragallinarum (AP) como una enfermedad respiratoria aguda y ocasionalmente crónica en aves domésticas ha causado graves pérdidas en varios estados de los Estados Unidos. La enfermedad también se asocia con una disminución en la producción de huevo en gallinas de postura y al incremento de decomisos por infecciones de los sacos aéreos en pollos de engorde. Se realizó una serie de experimentos aplicados para dilucidar la persistencia de A. paragillanarum en parvadas de pollos de engorde infectados, para genotipificar las cepas de A. paragallinarum aisladas de casos de campo y para evaluar la protección de vacunas comerciales y autógenas en pollos comerciales y en aves libres de patógenos específicos (SPF). La evaluación experimental de la persistencia ambiental sugiere que A. paragallinarum no persistió más de doce horas en un ambiente hipotéticamente contaminado. Además, otros patógenos potenciales detectados como Gallibacterium anatis y el virus de la bronquitis infecciosa causaron signos respiratorios leves en las aves expuestas. Los genes HMTp210 y HagA de cuatro cepas de campo de coriza infecciosa se secuenciaron y compararon con las secuencias publicadas de HMTp210 y HagA. La filogenia de HMTp210 mostró una agrupación marginalmente imperfecta de las secuencias en los genogrupos A, B y C. Aunque no es definitiva, esta filogenia proporcionó evidencia de que las cuatro cepas de campo se alinearon con cepas del serovar C previamente caracterizadas. Además, la homología de pares de bases de las cuatro cepas fue 100% idéntica a las cepas de referencia del serovar C (H-18 y Modesto). La filogenia de HagA no fue clara, pero curiosamente, las cepas de campo de coriza infecciosa fueron 100% similares con las cepas C-1 reportadas en México y Ecuador. Finalmente, los estudios de protección de vacunas en gallinas comerciales indican que los signos clínicos son inducidos por una combinación de coriza infecciosa y otros patógenos concomitantes que infectan a las aves comerciales. Además, los experimentos de protección de vacunas realizados en aves libres de patógenos específicos indicaron que la protección proporcionada por las dos vacunas comerciales analizadas proporcionó una reducción en los signos clínicos y en la eliminación bacteriana después de dos aplicaciones.

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.

Selection of Avibacterium paragallinarum Page serovar B strains for an infectious coryza vaccine.

Infectious coryza is an important respiratory disease of chickens around the world and is caused by Avibacterium paragallinarum. Among the three Page serovars currently recognized for this bacterium, serovar B is a major circulating serovar in China nowadays. The cross-protection ability of the Page serovar B reference strain (0222) and five local isolates was evaluated by a vaccination-challenge trial in SPF chickens. The clinical signs seen in control birds challenged by strain 0222 and isolate HB 01 were significantly different, with isolate HB 01 giving more severe clinical signs. In terms of cross-protection, the protection in the groups vaccinated with isolate HB 01 and BJ 02 was significantly higher than that in the groups vaccinated with 0222 and the other three isolates. In addition, an experimental oil adjuvant trivalent vaccine, containing field isolate HB 01 antigen, was compared for immune efficacy with two commercial trivalent infectious coryza vaccines containing internationally recognized serovar B strains. The experimental oil adjuvant trivalent vaccine elicited best protection (80%) among the three trivalent vaccines. In conclusion, the oil adjuvant vaccine, containing field isolate HB 01 may be a better choice in control of current serovar B Av. paragallinarum outbreaks in China under current circumstances.

Coinfection of Avibacterium paragallinarum and Gallibacterium anatis in Specific-Pathogen-Free Chickens Complicates Clinical Signs of Infectious Coryza, Which Can Be Prevented by Vaccination.

Avibacterium paragallinarum and Gallibacterium anatis are recognized bacterial pathogens both infecting the respiratory tract of chickens. The present study investigated outcomes of their coinfection by elucidating clinical signs, pathologic lesions, and bacteriologic findings. Additionally, the efficacy of a commercially available vaccine to prevent diseases caused by A. paragallinarum and G. anatis was evaluated. Birds inoculated with G. anatis alone did not present any clinical signs and gross pathologic lesions in the respiratory tract. However, clinical signs of infectious coryza were reproduced in nonvaccinated birds that were challenged with A. paragallinarum alone or together with G. anatis . Such clinical signs were more severe in the coinfected group, including the death of four birds. Some of the birds that were vaccinated and challenged showed mild clinical signs at 7 days postinfection (dpi). Inflammation of sinus infraorbitalis was the most prominent gross pathologic lesion found in the respiratory tract of nonvaccinated birds inoculated either with A. paragallinarum and G. anatis or A. paragallinarum alone. In the reproductive tract, hemorrhagic follicles were observed in nonvaccinated birds that were infected either with G. anatis alone or together with A. paragallinarum . In vaccinated birds, no gross pathologic lesions were found except in one bird that was coinfected with both the pathogens characterized by mucoid tracheitis. Bacteriologic investigations revealed that multiplication of G. anatis at 7 dpi was supported by the coinfection with A. paragallinarum . Altogether, it can be concluded that simultaneous infection of A. paragallinarum and G. anatis can increase the severities of disease conditions in chickens. In such a scenario, vaccination appears to be an effective tool for prevention of the disease, as protection was conferred based on clinical, pathologic, bacteriologic, and serologic data.

Evaluation of the ERIC-PCR as a probable method to differentiate Avibacterium paragallinarum serovars.

Infectious coryza, an upper respiratory tract disease in chickens, caused by Avibacterium paragallinarum, leads to huge economic losses. The disease is controlled through vaccination; but vaccination efficacy is dependent on correct identification of the infecting serovar, as limited cross-protection is reported amongst some serovars. Current identification methods include the heamagglutination inhibition test, which is demanding and could be subjective. To overcome this, molecular typing methods proposed are the Multiplex polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism-PCR, but low reproducibility is reported. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR has been suggested for molecular groupings of various bacterial species. This study focuses on evaluating the ERIC-PCR as a probable method to differentiate between different Av. paragallinarum serovars by grouping with reference isolates, based on clonal relations. The ERIC-PCR was performed on 12 reference isolates and 41 field isolates originating from South Africa and South America. The data indicate that the ERIC-PCR is not ideal for the differentiation or for molecular typing of Av. paragallinarum serovars, as no correlation is drawn upon comparison of banding patterns of field isolates and reference strains. However, the results do indicate isolates from the same origin sharing unique banding patterns, indicating potential clonal relationship; but when compared to the reference isolates dominant in the specific area, no correlation could be drawn. Furthermore, although the ERIC-PCR serves a purpose in epidemiological studies, it has proved to have little application in differentiating amongst serovars of Av. paragallinarum and to group untyped field strains with known reference strains.

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 .

The current epidemiological status of infectious coryza and efficacy of PoulShot Coryza in specific pathogen-free chickens.

Infectious coryza (IC) is an infectious disease caused by Avibacterium (Av.) paragallinarum. IC is known to cause economic losses in the poultry industry via decreased egg production in layers. Between 2012 and 2013, Av. paragallinarum was isolated from seven chicken farms by Chungbuk National University. We identified Av. paragallinarum, the causative pathogen of IC by polymerase chain reaction (PCR) and serovar serotype A, by multiplex PCR. Antibiotic sensitivity tests indicated that a few field-isolated strains showed susceptibility to erythromycin, gentamicin, lincomycin, neomycin, oxytetracycline, spectinomycin, and tylosin. A serological survey was conducted to evaluate the number of flocks that were positive for Av. paragallinarum by utilizing a HI test to determine the existence of serovar A. Serological surveys revealed high positivity rates of 86.4% in 2009, 78.9% in 2010, 70.0% in 2011, and 69.6% in 2012. We also challenged specific pathogen-free chickens with isolated domestic strains, ADL121286 and ADL121500, according to the measured efficacy of the commercial IC vaccine, PoulShot Coryza. We confirmed the effectiveness of the vaccine based on relief of clinical signs and a decreased re-isolation rate of ADL121500 strain. Our results indicate IC is currently prevalent in Korea, and that the commercial vaccine is effective at protecting against field strains.

The haemagglutinin of Avibacterium paragallinarum is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities.

The haemagglutinin (HA) protein plays a key role in the immunogenicity and pathogenicity of Avibacterium paragallinarum. A 210-kDa protein (HMTp210) was previously reported to be the HA of Av. paragallinarum, but the biological function of HMTp210 is not well defined. In this study, mutant strains that lacked HMTp210 were constructed using the TargeTron(®) gene knockout system. Haemagglutination and haemagglutination-inhibition (HI) assays showed that the HMTp210-deficient mutants exhibited no HA activity and failed to elicit HI antibodies in immunized chickens. Additionally, HMTp210-deficient mutants exhibited reduced ability to adhere to HeLa cells and to form biofilms on abiotic surfaces. Virulence assays showed that HMTp210-deficient mutants are less virulent than their isogenic wild-type strains. HMTp210 bears significant similarity to proteins of the trimeric autotransporter adhesin (TAA) family, and recombinant HMTp210 expressed in E. coli formed a trimeric structure. Taken together, these results indicated that HMTp210 is a trimeric autotransporter adhesin that confers haemagglutination, cell adherence and biofilm formation activities. These results should prove valuable to further elucidate the biological function of HA and the mechanism of pathogenicity of Av. 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.

Development of a recombinant vaccine against infectious coryza in chickens.

Infectious coryza is an acute respiratory disease of chickens caused by Avibacterium paragallinarum, and this infection is associated with growth retardation and reduced egg production. Previous studies have shown that HMTp210, a 210-kDa outer-membrane protein, is the major protective antigen of Av. paragallinarum both serovars A and C. Region 2 is a serovar-specific domain in the HMTp210 protein. Although the serovar C region 2 has been reported to be an effective vaccine antigen for infectious coryza, there have been no reports on the efficacy of region 2 from serovar A. In the current study, region 2 from serovars A and C was expressed as a fusion peptide. Chickens inoculated with vaccine consisting of 0.6 µg of the fusion peptide showed no clinical signs of disease after challenge with either serovar A or C, and there were no side effects such as swelling at the injection site. These results demonstrate that the recombinant fusion peptide derived from HMTp210 could be useful for producing effective and safe vaccines against infectious coryza in chickens.

Development of an enzyme-linked immunosorbent assay for the measurement of antibodies against infectious coryza vaccine.

Infectious coryza is an acute respiratory disease caused by infection with Avibacterium (Haemophilus) paragallinarum. It is characterized by nasal discharge and facial swelling and is associated with growth retardation and a reduction in egg production. Hemagglutination inhibition (HI) tests are used to estimate vaccine-induced immunity against infectious coryza in vitro; however, these procedures are complicated and their sensitivity is insufficient. To address these problems, an enzyme-linked immunosorbent assay (ELISA) technique using serovar-specific regions of HMTp210 (210 kDa), an outer-membrane protein of A. paragallinarum, was developed to measure the antibodies against infectious coryza. Chickens with an ELISA titer of 0.3 or more did not exhibit clinical signs of infectious coryza against challenge with A. paragallinarum, although their HI antibody titers were negative. On the other hand, chickens with an ELISA titer below 0.3 exhibited clinical signs of the disease with one exception. Antibody prevalence rates on ELISA were 80% and 60% against infection with serovars A and C, respectively, and ELISA also detected antibodies in chickens infected with A. paragallinarum with a sensitivity higher than that of HI tests. Taken together, the ELISA technique developed in this study is a valuable tool for the measurement of antibodies produced against the infectious coryza vaccine or in response to an infection with A. paragallinarum.

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.

Prevalence of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella multocida, and Streptococcus suis in representative Ontario swine herds.

Tonsillar and nasal swabs were collected from weanling pigs in 50 representative Ontario swine herds and tested for the presence of 5 important bacterial upper respiratory tract pathogens. All but 1 herd (2%) tested positive for Streptococcus suis by polymerase chain reaction (PCR); 48% of herds were S. suis serovar 2, 1/2 positive. In all but 2 herds there was evidence of Haemophilus parasuis infection. In contrast, toxigenic strains of Pasteurella multocida were detected by a P. multocida--enzyme-linked immunosorbant assay (PMT-ELISA) in only one herd. Seventy-eight percent of the herds were diagnosed positive for Actinobacillus pleuropneumoniae by apxIV PCR. Sera from finishing pigs on the same farms were also collected and tested by ELISA for the presence of A. pleuropneumoniae antibodies. Seventy percent of the herds tested had evidence of antibodies to A. pleuropneumoniae including serovars 1-9-11 (2%), 2 (4%), 3-6-8-15 (15%), 5 (6%), 4-7 (26%), and 12 (17%). This likely represents a shift from previous years when infection with A. pleuropneumoniae serovars 1, 5, and 7 predominated. At least 16% and possibly as many as 94% of the herds tested were Actinobacillus suis positive; only 3 of the 50 herds were both A. pleuropneumoniae and A. suis negative as judged by the absence of a positive PCR test for apxII. Taken together, these data suggest that over the past 10 years, there has been a shift in the presence of pathogenic bacteria carried by healthy Ontario swine with the virtual elimination of toxigenic strains of P. multocida and a move to less virulent A. pleuropneumoniae serovars. As well, there appears to be an increase in prevalence of S. suis serovar 2, 1/2, but this may be a reflection of the use of a more sensitive detection method.

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.

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.

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.

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.

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.