The infraorbital sinus was swabbed with cotton sticks and incubated on chocolate agar (Hanil Komed, Korea) with 5–10% CO₂ for 48 h at 37℃ [142028]. Selected clear, dew like colonies were further incubated for 24 h at 37℃ in chicken meat infusion broth for enrichment culture [16].

Av. paragallinarum cultured on chocolate agar was incubated in brain heart infusion (BHI) broth (BD, USA) with 5% (v/v) heat-inactivated swine serum (Sigma-Aldrich, USA) and 0.01% nicotinamide adenine dinucleotide (Sigma-Aldrich) at 37℃ for 16 h [14]. The enriched broth was centrifuged at 4,000 × g for 20 min, then washed 3 times with phosphate buffer solution (PBS) with 0.01% thimerosal (Sigma-Aldrich) and incubated at 4℃ overnight. The hemagglutination test was as follows. PBS (25 µL per well) was added to a 96 well microtiter plate, followed by 25 µL of antigen, and then diluted two-fold. A 25 µL aliquot of fresh chicken RBC made from Alsever's solution was added to each well, after which plates were incubated at room temperature for 40 min after mixing. The concentration of the antigen was diluted to 4 hemagglutinating units (HAU) for the HI test for serovar A.

The HI test for serovar A was performed with a 4 HAU antigen concentration from field strains and antibody of Page serovar A (221 strain). Positive control serum of serovar A (221 strain) for the HI test was generously supplied by ChoongAng Vaccine Laboratories (Korea), and serum from specific pathogen-free (SPF) chickens was used as the negative control. Twenty five µL of positive serum (221 strain) and negative serum were added to a 96-well plate and diluted two-fold with PBS. An equal volume of serum with an antigen concentration of 4 HAU was added to each well, followed by 45 min incubation at room temperature for 20 min after mixing [14]. Next, 25 µL of fresh chicken RBC produced from Alsever's solution were added to each well, after which the plate was incubated at room temperature for 40 min. Among serum samples submitted to the Chungbuk National University Avian Disease Laboratory from 2009 to 2012, those obtained from flocks with a history of respiratory distress or decreased egg production were chosen. Flocks with one or more positive results from the five serum samples were considered positive.

To determine if the isolated pathogen was Av. paragallinarum, all isolated field strains were subjected to HP-2 PCR [89]. Viral Gene-spin (iNtRON Biotechnology, Korea) was used for DNA extraction. The PCR mixture consisted of Maxime PCR PreMix (iNtRON Biotechnology), 1 µL of each forward and reverse primer, 1 µL of extracted template gene, and 17 µL of distilled water. The primer used was the N1/R1 primer set for HP-2 PCR described by Chen et al. [8]. The PCR protocol consisted of an initial step at 94℃ for 2 min followed by 40 cycles of 94℃ for 20 sec, 65℃ for 10 sec and 72℃ for 40 sec and then a final step at 72℃ for 2 min. The PCR products were examined by agarose gel electrophoresis, with the band at 0.5 kbp signifying Av. paragallinarum [8]. After confirming the identity of the pathogen as Av. paragallinarum, multiplex PCR was conducted to distinguish between Page serovar A, B, and C. The primer set used to distinguish regions of the outer membrane protein HMTp210 was used to confirm band sizes. Multiplex PCR mixture consisted of Maxime PCR PreMix, 1 µL of each A, B, and C serotype forward and reverse primer sets, 1 µL of template, and 13 µL of distilled water. The PCR protocol consisted of an initial step at 94℃ for 2 min followed by 30 cycles of 94℃ for 20 sec, 56℃ for 12 sec and 72℃ for 60 sec, and then a final step at 72℃ for 3 min. The PCR products were examined by agarose gel electrophoresis. Bands at 0.8, 1.1, and 1.6 kbp correspond to Page scheme serovars A, B, and C, respectively [21].

The disk diffusion method was used to test antibiotic sensitivity in field isolates of Av. paragallinarum. The disk diffusion test was performed as recommended by the Clinical and Laboratory Standards Institute [12], with some modification by Chukiatsiri et al. [11]. The recommended reference strain (Escherichia coli ATCC 25922) was used as the quality control strain. Twelve antibiotics were chosen based on those commonly used in farms, ampicillin, ceftiofur, cloxacillin, enrofloxacin, erythromycin, gentamicin, lincomycin, neomycin, oxytetracycline, sulfamethoxazole-trimethoprim, spectinomycin, and tylosin. All of the antimicrobial disks were from Liofilchem (Italy). The isolated Av. paragallinarum was incubated at 37≧ for 16 h in BHI broth with 5% (v/v) heat-inactivated swine serum and 0.01% nicotinamide adenine dinucleotide. The enriched medium was spread on modified TM/SN agar plates at a concentration of 10⁸ CFU/mL. Antibiotic disks were dispensed on the plates and incubated at 37℃ for 24 h. Diameters of the regions where growth was inhibited were measured with Vernier calipers in millimeters [6]. These measurements were grouped into categories of sensitive, intermediate, or resistant based on the values in Table 1.

Chicks were hatched and grown from SPF eggs (Namduk SPF, Korea) until postnatal 40 days old under identical conditions in separate sterile isolators (Three-Shine, Korea). At 40 days of age, 55 chickens were randomly divided into five groups of 11 subjects. The details describing the groups are listed in Table 2, with two vaccinated groups (group A and C), two non-vaccinated groups (group B and D), and a negative control group (group E). Group A and C were administered 1 mL intramuscular injections of the vaccine PoulShot Coryza (ChoongAng Vaccine Laboratories) at 40 days of age. Among of isolated field strains, ADL121286 and ADL121500 were selected for challenge. Because the ADL121286 field strain caused mild clinical signs and there were no secondary infections. Field strain ADL121500 produced severe clinical signs and infectious bronchitis. At 54 days of age, each group was challenged by nasal inoculations of 0.2 mL. Specifically, group A and B were inoculated with ADL121286, group C and D were inoculated with ADL121500, and group E was inoculated with medium. The concentrations of the inoculated field strains were adjusted to 1 × 10⁸ CFU/mL and the actual count was confirmed by spread plating of 10 fold dilutions. After inoculation, three, three, and five chickens from each group were euthanatized at 5 days post challenge (dpc), 10 dpc, and 15 dpc, respectively. If Av. paragallinarum was re-isolated from either infraorbital sinus, the animal was considered positive for infection. Samples were also collected from both infraorbital sinuses and the choanal cleft with cotton swabs and diluted in 1 mL PBS, after which the diluted mixture was used for HP-2 PCR. If more than one of the three PCR samples of each chickens was positive the animal was considered positive for infection. Additionally, HI mean titer and HI titer ranges were determined via HI tests for serovar A, which were carried out using chicken serum obtained from all chickens at pre-vaccinated 40 days of age and pre-challenged 54 days of age, as well as from the chicken serum of euthanatized animals at 5 dpc, 10 dpc, and 15 dpc. The HP211 strain was used as the antigen in these HI tests for serovar A. Throughout the experiment, organisms were grown in a high efficiency particulate air filtered isolate system maintained at room temperature. Antibiotics-free feed was provided by NongHyup feed (Seoul, Korea) and water was provided by Laboratory Animal Research Center (Chungbuk, Korea). All processes were conducted in accordance with the guidelines of the Institute of Laboratory Animal Resources (confirmation No. CBNUA-637-13-01).

From 2 to 15 dpc, clinical signs were scored as follows: 0, no signs; 1, nasal discharge or slight facial swelling; 2, nasal discharge and moderate facial swelling; 3, abundant nasal discharge and severe facial swelling; and 4, the same as 3, but with swelling of wattles [2428].

Significant differences between serological surveys based on the year the survey was conducted and the age of the animals were identified by the chi-square test and Fisher's exact test. To compare the clinical sign scores of experimental groups, one-way ANOVA was used to compare the means of each group and the Bonferroni correction was employed for multiple comparisons. A p < 0.05 was considered to indicate statistical significance for all analyses.

As depicted in Table 3, attempts to isolate Av. paragallinarum have been conducted by swabbing the infraorbital sinus of the chickens submitted from poultry farms with a history of decreased egg production to the Avian Disease Laboratory, Chungbuk National University. Common observations from the submitted cases include a swelling of the infraorbital sinus, edema of the surrounding tissues, sometimes extending to the wattles, and hyperemia of the conjunctiva. Edema in the margins of eyelids and wattle varied from severe to mild.

Field strain of ADL121154, ADL121286, ADL121314, ADL121500, ADL121610, ADL131215, and ADL131961 were identified by the presence of clear dew-like colonies in chocolate agar. In HP-2 PCR, seven field strains and positive control strain 221 were also confirmed as Page serovar A based on the presence of bands at 0.5 kbp. The strains were also confirmed as Page serovar A by multiplex PCR based on the presence of bands at 0.8 kbp.

We evaluated the susceptibility of the seven field strains of Av. paragallinarum to 12 antibiotics, as described in Table 1. The strains were shown to be the lowest susceptible against enrofloxacin (57.1%). The strains were relatively more susceptible against ampicillin (71.4%), cloxacillin (85.7%), gentamicin (85.7%) and sulfamethoxazole-trimethoprim (85.7%). The strains were shown to be sensitive against erythromycin, gentamicin, lincomycin, neomycin, oxytetracycline, spectinomycin, and tylosin.

The results of the serological survey are summarized in Table 4 according to tested year and age. Groups were divided by age, with the first group consisting of birds prior to egg laying age (1–19 weeks). Groups were further divided into separate age groups about 10 weeks apart for each group. The positivity rate of the 1–19 weeks group was 33.3%, which was relatively lower than that of other groups. The positivity rate of groups above 20 weeks of age ranged from 70.4% to 88.2% with no significant difference among groups. Overall positivity was highest in 2009 (86.4%) and lowest in 2012 (69.6%), but the differences between years were not significant.

Average clinical sign score: The clinical sign scores for challenged groups are depicted in Table 5. The average clinical sign scores for groups challenged with ADL121286 were 0.38 ± 0.23 for group A and 0.24 ± 0.14 for group B. There was no statistical difference among groups. However, the average clinical sign score of groups challenged with ADL121500 strain was 0.80 ± 0.13 for group C and 1.34 ± 0.41 for group D, with vaccinated group C exhibiting significantly lower clinical sign scores. No clinical signs were observed in negative control group E.

Re-isolation rate: Re-isolation rates based on media and PCR from samples obtained from animals euthanized on 5 dpc, 10 dpc, and 15 dpc are depicted in Table 5. Re-isolation was conducted by culture in media and confirmed by PCR. While re-isolation rate was relatively low in group A (33%) in media, it was shown to be 100% by PCR at 5 dpc. The re-isolation rates of groups B, C and D were shown to be 100% in both media and PCR at 5 dpc. Re-isolation rates decreased in both media and PCR in groups A, B, and C at 10 dpc. However, the re-isolation rate of group D was relatively high throughout the experiment, showing isolation rates of 100% in media and 80% for PCR, even at 15 dpc. Strains were not re-isolated from vaccinated group A and C at 15 dpc. The re-isolation rate was moderate in group B and relatively high in D group at 15 dpc. Overall, the media and PCR re-isolation rates were shown to be similar. Strains were not isolated from negative control E group by media or PCR.

HI test: Chickens were vaccinated at 40 days of age prior to challenge with field strain. Chickens were challenged at 54 days of age and HI titers were obtained at 5 dpc, 10 dpc and 15 dpc after challenge. The titers are displayed in Table 6. Both vaccinated and non-vaccinated groups showed negative titers prior to vaccination at 40 days of age. At 54 days of age in 0 dpc, the mean titers of the vaccine groups were 22.4 and 14.2, respectively, showing positive titers. Both vaccine groups showed positive titers at 54 days of age, with highest being observed at 59 days of age. Both groups showed decreases in titer at 64 days of age. After challenge with field strain, group B and D showed positive titers at 64 days of age, resulting in all groups showing positive titers with the exception of control group E at 64 days of age.