Differentiation of Haemophilus strains isolated from chickens. IV. Studies on the dissociation of Haemophilus paragallinarum

Bacteria of 2 phenotypic forms of one strain of Haemophilus paragallinarum were morphologically, culturally, biochemically and serologically investigated. On transparent solid media bacteria of the M variant (mucoid) were capsulated and showed iridescence in oblique transmitted light throughout the first 8-14 hours of incubation. Iridescence disappeared completely after 36 hours incubation. Bacteria of this variant formed stable homogeneous suspensions in 0.15 M NaCl and an homogeneous cloudiness after growth in liquid media. A type specific soluble substance was found in fluid culture filtrates and in bacterial washings using the indirect haemagglutination test. Bacteria of the R variant (rough) derived by dissociation of iridescent colonies were not capsulated and showed no iridescence throughout 8-14 hours incubation (minus variant). This bacteria showed spontaneous agglutination in 0.15 M NaCl and a granular growth in liquid media. There were no biochemical differences between M and R variant. A type specific substance was not found in the R bacteria using the indirect haemagglutination test. In the plate agglutination test no serological reaction between anti-K-sera and M antigen were observed. R to M variation occurred in SPF chickens following intranasal inoculation of organisms of R cultures grown for 6 or 20 passages on solid medium.     

Proposal of a new serovar and altered nomenclature for Haemophilus paragallinarum in the Kume hemagglutinin scheme.

By using the Kume hemagglutinin serotyping scheme, 13 Australian isolates of Haemophilus paragallinarum were shown to constitute a new serovar within the presently termed serogroup II. Because of the likelihood that new serovars will continue to be established, we propose a rationalization of the nomenclature of the Kume scheme. Under this altered scheme, the three recognized serogroups I, II, and III are renamed A, C, and B, respectively. Within each of the serogroups, the serovars are numbered sequentially, allowing new serovars to be added in numerical order. Thus, the nine currently recognized Kume serovars are termed A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4.     

Differentiation of Haemophilus strains isolated from chickens. III. Pathogenicity tests in chickens

Pathogenicity tests of 6 isolates of the Haemophilus-group I (H. paragallinarum) and 8 isolates of the Haemophilus-group II isolated from chickens in the Federal German Republic and one strain of H. parainfluenzae isolated from man were carried out in 6-week old SPF-chickens. Infectious coryza (Coryza contagiosa gallinarum) could be produced in chickens with all strains of group I after experimental inoculation and by contact exposure. At necropsy 3 weeks post inoculation (p.i.) organisms of group I were recovered from the sinus and nose of chickens with and without symptoms. The Haemophilus-bacteria however could not be reisolated from lungs and air sacs. No clinical and pathological symptoms were observed in chickens inoculated with isolates : of group II. In another trial these strains were not pathogenic even though chickens were pre-infected with an attenuated infectious bronchitis virus (IBV; Massachusetts type). However, these Haemophilus-bacteria were able to spread from inoculated to non-inoculated chickens by direct contact, and airborne transmission over a distance of 1.5 metres also occurred. Haemophilus organisms of group II were reisolated up to the end of the observation period of 35 days p.i. from the nose, sinus and trachea, but not from the lungs and air sacs. In chickens pre-infected with Mycoplasma gallisepticum and IBV, one strain of group I and of group II could be recovered from the lower respiratory tract (lungs and air sac exudate). Sera from chickens inoculated with the group II strain contained agglutinins to Haemophilus and the titre increased in 8 out of 10 chickens. Sera from chickens without pre-infection with both M. gallisepticum and IBV showed no antibody response to the inoculated group II Haemophilus strain. The H. parainfluenzae strain was non pathogenic and could not be isolated from the respiratory tract of the inoculated chickens at 48 hours p.i.     

Isolation and identification of NAD-independent bacteria from chickens with symptoms of infectious coryza.

Since 1990, NAD-independent bacteria have been isolated in South Africa from poultry showing respiratory manifestations similar to infectious coryza. A total of 126 isolates was examined biochemically and serologically, using polyclonal as well as monoclonal antibodies. Forty isolates were identified as Ornithobacterium rhinotra-cheale, some of which agglutinated glutaraldehyde-fixed red blood cells. Furthermore, fourteen Pasteurella avium isolates, five P. volantium and three Pasteurella species A were isolated for the first time. The remaining 64 isolates were biochemically identified as NAD-independent H. paragallinarum. Of these, 37 were Page serovar A, while no Page serovar B isolates were found. The remaining 25 isolates were typed as Page serovar C. Two different haemagglutination inhibition reaction patterns were found among the Page serovar C isolates, i.e. isolates which reacted with a 1 in 100 dilution of the Page serovar C antiserum, and another larger group which did not react with this dilution of the serum, but did react with rabbit raised antiserum prepared against other Kume serogroup C isolates. This is the first recorded isolation of Page serovar C NAD-independent H. paragallinarum.     

Oral and nasal DNA vaccines delivered by attenuated Salmonella enterica serovar Typhimurium induce a protective immune response against infectious bronchitis in chickens

Several studies have reported that intramuscular injection of DNA vaccines against infectious bronchitis virus (IBV) induces protective immune responses. In the present study, we developed oral and nasal DNA vaccines that carried the S1 gene and N gene of IBV delivered by attenuated Salmonella enterica serovar Typhimurium strains SL/pV-S1 and SL/pV-N, respectively. The safety and stability of recombinant Salmonella vaccine were evaluated. Following oral and nasal administration to chickens, the serum and mucosal samples were collected and antibodies against IBV were measured. Chickens were then challenged with IBV strain M41 by the nasal-ocular route 3 weeks after boosting. The results showed that oral and nasal immunization with coadministered SL/pV-S1 and SL/pV-N elicited significant IBV-specific humoral and mucosal immune responses and conferred protective efficacy against IBV challenge higher than that in chickens immunized only with SL/pV-S1. The current study shows that novel DNA vaccines delivered by attenuated S. Typhimurium may be promising candidates for the prevention of infectious bronchitis (IB).These vaccines are efficacious, easily produced economically, and able to be delivered orally and nasally rather than injected. Coadministration of SL/pV-S1 and SL/pV-N may represent an effective mucosal vaccination regimen.     

Efficacy of inactivated infectious bursal disease (IBD) vaccines: Comparison of serology with protection of progeny chickens against IBD virus strains of varying virulence

The efficacy of inactivated infectious bursal disease vaccines was determined by measuring both the antibody response of vaccinated chickens and clinical protection of progeny chicks from vaccinated dams. Similar virus neutralizing (VN) antibody titres were obtained in 4-week-old chickens and mature hens after vaccination with one vaccine dose. VN titres below 10 log 2 increased considerably between the fourth and seventh week after vaccination in 4-week-old chickens as well as in mature chickens. All 2-week-old progeny chicks with serum VN antibody titres of at least 9 log 2 were clinically protected against the classical virulent 52/70 infectious bursal disease virus (IBDV) strain, as well as against the very virulent IBDV (vvIBDV) strain D6948. However, vaccination often did not prevent subclinical infection in these 2-week-old progeny chicks, which often resulted in severe lymphocyte depletion in the bursa of Fabricius. Even a serum VN titre of 11 log 2 was not always sufficient to prevent severe bursal damage. Although 52/70 IBDV and vvIBDV were equally pathogenic in 2-weekold specific pathogen free chickens, significant higher maternal antibody titres were required to prevent the adverse effects of vvIBDV in comparison with 52/70 IBDV. The relation between the serological response of chickens after application of inactivated IBD vaccines and the protection of progeny chicks of vaccinated dams depended on both the virulence of the IBDV challenge strain and the IBDV strain in the vaccine.     

Molecular characterization of isolates of Haemophilus paragallinarum from China by ribotyping.

Ribotyping profiles were determined for 12 Page serovar A isolates of Haemophilus paragallinarum from five outbreaks of infectious coryza in Hebei province in the People's Republic of China. Four enzymes were used to generate restriction digests of chromosomal DNA: HaeIII, HindIII, HpaII and SspI. The digests were probed with a digoxigenin-11-dUTP-labelled DNA probe produced by PCR amplification of the 16S rDNA of the type strain of H. paragallinarum. Only one profile was seen among the 12 isolates when using either HindIII or SspI. Three different ribotype profiles were seen with HpaII, while four different profiles were detected with HaeIII. Cluster analysis of the profiles generated by HpaII and HaeIII corresponded with the known epidemiological history of the isolates. These results are the first molecular characterization of isolates of H. paragallinarum from China and confirm the existence of genetic diversity in the H. paragallinarum population in that country.     

Evaluation of a PCR test for the detection of Haemophilus paragallinarum in China

The application of a recently described PCR test for the detection of Haemophilus paragallinarum in China is described. The test was used to examine a total of 127 chickens sourced from a challenge trial (38 chickens), a respiratory disease-free experimental chicken farm (50 chickens) and eight farms with suspect infectious coryza (IC) outbreaks (39 chickens). The PCR results were compared with traditional culture. The PCR detected 14/14 infected birds in the challenge trial as compared with 13/14 for culture. The 50 chickens from the disease-free experimental farm were all negative by both PCR and culture. PCR yielded 15/39 birds and 6/8 commercial farms positive as compared with 8/39 birds and 4/8 farms positive by culture. All farms positive by PCR had chickens showing the typical clinical signs of IC, indicating that culture failed to confirm coryza on two farms that had the typical clinical signs of the disease. Although chickens on two commercial farms were thought initially to be suffering from coryza, detailed clinical examination yielded no birds with typical clinical signs. The 12 chickens examined from these two farms were negative by both PCR and culture. The results suggest that the PCR test for H. paragallinarum is a suitable alternative to culture even under the typical field and laboratory conditions that operate in China.     

An upper respiratory disease of commercial chickens resembling infectious coryza, but caused by a V factor-independent bacterium

From early 1989 the emergence of an infectious bacterial disease resembling infectious coryza was seen in several commercial chicken flocks in Natal Province of South Africa. Clinical signs were facial swelling and nasal discharge. An organism was routinely isolated from the infra-orbital sinus or trachea of infected chickens. The organism was found to be a Gram-negative rod, non-motile, V factor (nicotinamide adenine dinucleotide, NAD)-independent, catalase negative, oxidase positive and urease and indole negative. No gas was produced from carbohydrates and acid was produced from glucose, mannitol, inositol and sorbitol. Experimental inoculation of this organism into the infraorbital sinus of SPF chickens and conventional broilers produced an acute upper respiratory disease. The organism could be recovered for up to 7 days post-inoculation. The organism is closely related to Haemophilus paragallinarum, the cause of infectious coryza, but because it is NAD-independent it cannot be classed as an Haemophilus species.     

Experimental induction of chicken amyloid A amyloidosis in white layer chickens by inoculation with inactivated vaccines

We investigated the amyloidogenic potential of inactivated vaccines and the localized production of serum amyloid A (SAA) at the injection site in white layer chickens. Hens in the treated group were injected intramuscularly three times with high doses of inactivated oil-emulsion Salmonella Enteritidis vaccine and multivalent viral and bacterial inactivated oil-emulsion vaccines at two-week intervals. Chickens in the control group did not receive any inoculum. In the treated group, emaciation and granulomas were present, while several chickens died between 4 and 6 weeks after the first injection. Hepatomegaly was seen at necropsy, and the liver parenchyma showed inconsistent discolouration with patchy green to yellowish-brown areas, or sometimes red-brown areas with haemorrhage. Amyloid deposition in the liver, spleen, duodenum, and at injection sites was demonstrated using haematoxylin and eosin staining, Congo red, and immunohistochemistry. The incidence of chicken amyloid A (AA) amyloidosis was 47% (28 of 60) in the treated group. In addition, RT-PCR was used to identify chicken SAA mRNA expression in the liver and at the injection sites. Furthermore, SAA mRNA was detected by in situ hybridization in fibroblasts at the injection sites, and also in hepatocytes. We believe that this is the first report of the experimental induction of systemic AA amyloidosis in white layer chickens following repeated inoculation with inactivated vaccines without the administration of amyloid fibrils or other amyloid-enhancing factors.     

Retrospective evidence that the MHC (B haplotype) of chickens influences genetic resistance to attenuated infectious bronchitis vaccine strains in chickens.

Infectious bronchitis is a respiratory disease of chickens that is caused by the coronavirus infectious bronchitis virus (IBV). Virtually all broiler and layer breeder flocks are routinely vaccinated against IBV. Two hatches of 1-day-old chicks from four lines were mistakenly vaccinated for infectious bronchitis using a moderately attenuated vaccine designed for chicks of an older age. The vaccination resulted in high mortality, and chicks from three of four lines died with signs typical of infectious bronchitis. The mortality that occurred using this less-attenuated vaccine was significantly influenced by the genetic line, and the MHC (B) haplotype in chickens of three B congenic lines. B congenic chickens possessing the B*15 haplotype were resistant in contrast to chickens possessing the B*13 or B*21 haplotypes. Chicks from two further hatches of the four lines were vaccinated appropriately with a more attenuated IBV vaccine, and only limited chick mortality was seen. These retrospective data from two repeated hatches confirm earlier data indicating chicken genes influence resistance to IBV, and indicate for the first time that genes tightly linked to the B haplotype are relevant in resistance to IBV. Due to extenuating circumstances it was not possible to verify results with chicks from F2 matings. Factors that may enhance definition of the role of the B haplotype in immune response to IBV, and the desirability for further analysis of a B haplotype-linked influence on immunity to IBV are discussed.     

Rapid selection in chickens of subpopulations within ArkDPI-derived infectious bronchitis virus vaccines

We examined spike (S) gene sequences of the virus populations of four different commercial ArkDPI-derived infectious bronchitis coronavirus vaccines before and during a single passage in specific pathogen free chickens. We found different degrees of genetic heterogeneity among the four vaccines before passage in chickens, ranging from no apparent heterogeneity to heterogeneity in 20 positions in the S gene. In all except one position, nucleotide differences were non-synonymous. The majority of amino acid differences were in the S1 subunit of the protein. For three of the four ArkDPI-derived vaccines, a single subpopulation with an S gene sequence distinct from the vaccine majority consensus at 5 to 11 codons was selected in chickens within 3 days after ocular vaccination. In contrast, we obtained no evidence for selection of specific subpopulations of the fourth ArkDPI-derived vaccine or Massachusetts or DE072 serotype vaccines. The virus subpopulations within each vaccine selected by chickens are similar in their S1 gene sequences, but distinct in the 3′ portion of the S2 subunit gene for each of the three vaccines. In the S1 gene, the selected subpopulations are more similar to the virulent parental ArkDPI isolate than to the predominant vaccine population. The different proportions of distinct subpopulations in Ark vaccines apparently more fit for replication in the respiratory tract of chickens might cause different degrees of damage to respiratory epithelium and/or immune responses in vaccinated chickens. Sequence comparisons provided no evidence to support that ArkDPI-like field isolates were derived directly from host-selected vaccine virus subpopulations.     

NAD (V-factor)-independent and typical Haemophilus paragallinarum infection in commercial chickens: A five year field study

An unusual bacterium causing respiratory disease in chickens emerged in South Africa in February 1989. The disease resembled infectious coryza but the organism differed from typical Haemophilus paragallinarum especially in that it did not require V-factor for growth. It has been termed an NAD-independent H. paragallinarum. A study of avian haemophili isolated from diseased chickens in Kwazulu-Natal over the past five years revealed the presence of typical H. paragallinarum, NAD-independent H. paragallinarum and H. avium (now transferred to the genus Pasteurella). Before the end of 1989 the NAD-independent H. paragallinarum had become the predominate isolate and thereafter was isolated from commercial chickens in other regions of South Africa. The disease affected all strains of chickens in an overall age range of 14 days to 64 weeks. The organism was responsible for upper respiratory disease of broilers and layers and implicated in lower respiratory disease of broilers. It was commonly isolated from diseased adult birds previously vaccinated against typical H. paragallinarum. Broilers were most commonly infected from 3 weeks of age and layers within the placement to peak production period. Whole cell protein profiles of NAD-independent H. paragallinarum isolates from five different commercial poultry units were identical but differed from that of a typical isolate.     

Comparison of hemagglutinin and agglutinin schemes for the serological classification of Haemophilus paragallinarum and proposal of a new hemagglutinin serovar

Ninety-five isolates of Haemophilus paragallinarum were classified serologically by a hemagglutinin serotyping scheme, and the results were compared with results from agglutinin serotyping of the same isolates. Of the isolates, 65 were from Australia, 5 were from Japan, 6 were from the Federal Republic of Germany, 7 were from the United States, and 12 were from South Africa. Seven hemagglutinin serovars, HA-1 to HA-7, corresponding to agglutinin serovars A (HA-1, HA-2, and HA-3), C (HA-4, HA-5, and HA-6), and B (HA-7) have been described. Only one of the seven hemagglutinin serovars was found among the Australian isolates. This was serovar HA-5, comprising 49 isolates. Fifteen Australian isolates, all from southeast Queensland and northern New South Wales, were found to belong to a new hemagglutinin serovar. This was designated HA-8 and represented a fourth subgroup of agglutinin serovar A. Of the 95 isolates examined, only 1 did not produce a hemagglutinating antigen and was nonserotypeable by the hemagglutinin system. This compared favorably with the agglutinin scheme, which serotyped only 60 of the 95 isolates, with 29 nonserotypeable and 6 autoagglutinating.     

Efficacy of a new tetravalent coryza vaccine against emerging variant type B strains

Outbreaks of infectious coryza have been reported in vaccinated flocks in different countries, indicating that new serotype(s) of Haemophilus paragallinarum may have evolved. Several field isolates from vaccinated flocks in the US, Ecuador, Argentina and Zimbabwe were examined and, apart from one serotype C strain, all were typed as serotype B. An inactivated commercial trivalent vaccine, containing serotypes A, B and C, protected against challenge with the serotype C isolate but protection against challenge with serotype B isolates was weaker, suggesting that they might represent a new variant immunotype. An experimental tetravalent oil adjuvant vaccine, containing one of the serotype B isolates, appeared immunogenic against all isolates after one vaccination. Its efficacy and safety were further tested in layer chickens housed under field conditions. Chickens were vaccinated at 8 and 16 weeks of age while controls were unvaccinated. Vaccinates and controls were challenged with type A, B, C and variant type B at 25, 45 or 65 weeks of age. There was good protection (P < 0.05) against all four immunotypes after all challenges. No systemic reactions were observed and local reactions were similar to those found with the commercial trivalent vaccine. The tetravalent vaccine may therefore be a good choice for control of new field isolates.     

The vaccination-challenge trial: the gold standard test to evaluate the protective efficacy of infectious coryza vaccines

Infectious coryza is an upper respiratory tract disease of chickens with the major impact occurring in multi-age flocks. We investigated the relationship between the level of antibodies, as detected by a haemagglutination-inhibition (HI) assay, in infectious coryza-vaccinated chickens and the protection against challenge in those chickens. In one experiment, chickens given a single dose of either of two infectious coryza vaccines lacked a detectable HI response to vaccination but showed significant levels of protection 11 weeks after vaccination. In contrast, in chickens given two doses of an infectious coryza vaccine and challenged 3 weeks after the second vaccine dose, there was a strong serological response with 36/40 birds having a HI titre of 1/20 or greater. In this trial there was an apparent relationship between titre and subsequent protection, with none of the 32 chickens with a titre of 1/40 or 1/80 showing any clinical signs and only one of the same group yielding the challenge organism on culture. In contrast, three of the four vaccinated chickens with a HI titre less than 1/5 developed the typical clinical signs of coryza and yielded the challenge organism on culture. Overall, our results suggest that HI titres cannot be regarded as a definitive predictor of vaccine efficacy. We suggest that the vaccination-challenge trial is the gold standard for the evaluation of the immune response to infectious coryza vaccines.     

Relationships between virulence and morphological or serological properties of variants dissociated from serotype 1 Haemophilus paragallinarum strains

Relationships between morphological or serological properties and virulence were investigated in seven Haemophilus paragallinarum variants. The variants, originated from five serotype 1 strains, were classified into seven types on the basis of colony morphology and iridescence and the presence of variant-specific antigens. Smooth (S) and encapsulated organisms having variant-specific antigens and forming highly iridescent (ir+) colonies were highly virulent in vivo; slightly encapsulated organisms having variant-specific antigens and forming slightly iridescent (ir +/-) colonies were moderately virulent; and nonencapsulated or slightly encapsulated organisms with or without variant-specific antigens and forming noniridescent (ir-) or ir +/- colonies were avirulent. Virulence was well correlated with the amount of capsule substance containing hyaluronic acid. The evidence suggests that the presence of variant-specific agglutinogen L and hemagglutinin HA-L seem to be responsible for adherence or colonization, but not for virulence, of the organisms in chickens.