Chitosan-adjuvanted Mycoplasma gallisepticum bacterin via intraocular administration enhances Mycoplasma gallisepticum protection in commercial layers

Chitosan-adjuvanted Mycoplasma gallisepticum bacterin via intraocular administration enhances... ABSTRACT Mycoplasma gallisepticum (MG) causes respiratory signs and economic losses in the poultry industry. MG vaccination is one of the effective prevention and control measures that have been used around the world. Our previous study demonstrated that chitosan-adjuvanted MG bacterin could effectively reduce pathological lesions induced by MG and that chitosan could be used as an adjuvant in MG bacterin. The present study determining the efficacy of MG bacterins against the Thai MG strain was based on vaccine programs. Seven groups (25 layers/group) were received MG bacterins containing 0.5% chitosan or a commercial bacterin via intramuscular (IM) or intraocular (IO) route at 6 and 10 wk of age. Sham-negative and sham-positive controls were groups 1 and 2, respectively. Group 3: IM route of chitosan bacterin followed by IM route of chitosan bacterin; group 4: commercial bacterin via IM route followed by chitosan bacterin via IO route; group 5: commercial bacterin via IM route followed by commercial bacterin via IM route; group 6: chitosan bacterin via IM followed by chitosan bacterin via IO route; and group 7: chitosan bacterin via IO route followed by chitosan bacterin via IO route were determined. At 16 wk of age, all groups, excluding group 1, were challenged intratracheally with 0.1 mL containing Thai MG strain 107 colony-forming unit. At 17, 18, and 20 wk of age, 5 birds in each group were bled for serological testing and swabbed at the choanal cleft for the quantitative real-time PCR assay, the euthanized and necropsied. The results showed that birds vaccinated with a commercial intramuscular bacterin followed by an intraocularly chitosan adjuvant bacterin showed the best protection against the MG challenge. The study indicated that chitosan could be the effective mucosal adjuvant and increased the effectiveness of MG bacterin. INTRODUCTION Mycoplasma gallisepticum (MG) is one of the important pathogens of poultry around the world. MG causes chronic respiratory disease (CRD) leading to respiratory symptoms, including rales, coughs, sneezing, ocular discharge, and nasal discharge. In addition, MG infection increases condemnation in processing plants and degradation of carcasses, causing economic losses in the poultry industry (Raviv and Ley, 2013). To reduce the economic impact of MG infection in chickens, there are 3 commercial vaccines that have been used for the prevention program. Live, inactivated, and recombinant MG poultry (rFP-MG) vaccines are commonly used in the poultry industry. The efficacy of each type of vaccine in the protection of clinical signs and induction of the systemic immune response is different; however, these vaccines can reduce post-challenge production losses with the MG field strain (Carpenter et al., 1981; Whithear et al., 1990; Evans and Hafez, 1992; Zhang et al., 2010). Commercial MG vaccines are strains: F, ts-11, and 6/85. F strain vaccine is more virulent compared to other live vaccines (Raviv and Ley, 2013). It may induce mild respiratory signs in turkeys and broilers (Rodríguez and Kleven, 1980). Other vaccine strains, including ts-11 and 6/85, showed less protection against the MG challenge compared to the F strain (Abd-el-Motelib and Kleven, 1993). The inactivated vaccine is safe for use in turkeys and may reduce production losses in chickens and turkeys, but the inactivated vaccine is more expensive and cannot protect against MG infection in the respiratory tract (Panigraphy et al., 1981). For the recombinant vaccine, rFP-MG, the MG surface protein is encoded by the recombinant avian poxvirus (Vectormune® FP-MG vaccine, CEVA Santé Animale, Libourne, France). Although FP-MG is safe to vaccinate in chickens (Zhang et al., 2010), it did not show good protection against the MG challenge (Ferguson-Noel et al., 2012). Recently, a novel vaccine adjuvant has been developed to improve the efficacy of the vaccine. Chitosan is one of the potential adjuvants for the inactivated vaccine and is a polysaccharide made from deacetylation of chitin. Several studies found that chitosan could enhance the humoral and cell-mediated immune response in mice (Zaharoff et al., 2007) and chickens (Rauw et al., 2010a). From our previous study, chitosan could be used as an adjuvant for MG bacterin (Limsatanun et al., 2016). The chitosan-adjuvanted MG bacterin provided better protection of the tracheal lesion and caused less tissue reaction at the site of injection, compared to the commercial bacterin MG. But it has produced a low level of systemic immune response. The experimental groups receiving bacterin with chitosan adjuvant had lower numbers of antibody-positive birds compared to a commercial MG bacterin group (Limsatanun et al., 2016). However, the efficacy of the vaccine from the previous study was determined only by the intramuscular route, which could provoke tissue reaction and low immune response to the mucosa for vaccination with MG from poultry. To improve MG-induced immunity, different administrations, including intraocular routes and vaccination protocols, which included only MG bacterin adjuvanted with chitosan and/or commercial MG bacterin, were explored in the present study. MATERIALS AND METHODS Vaccine MG bacterin with chitosan adjuvant was prepared and described by Limsatanun et al. (2016). Briefly, Thai MG strain AHRL 20/52 was propagated in Frey broth supplemented with 15% porcine serum (FMS) (Kleven, 2008) and incubated at 37°C until the color of the broth changed from pink to yellow-orange or phase log. The culture broth was centrifuged at 12,000 xg at 4°C for 30 min to collect MG cells. MG cells were inactivated with 0.01% thimerosal (Sigma-Aldrich Corp., St. Louis, MO) in phosphate buffered saline (PBS) and incubated at 4°C for 7 d (Chukiatsiri et al., 2009). Chitosan solution (Sigma-Aldrich Corp., St. Louis, MO) was added to the MG cells to a final 0.5% (w/v) concentration. Two concentrations of MG organisms were determined in the bacterin with chitosan adjuvant. Bacteria were used with chitosan adjuvant containing approximately 5 × 108 MG microorganisms and 5 × 1011 colony-forming units (CFU)/mL for intramuscular and intraocular administration, respectively. All the chitosan-adjuvanted MG bacterins were stored at 4°C until use. Experimental Design And Sample Collection Protocol One-hundred-seventy-five one-day-old commercial female layer chickens, Isa brown breed, were obtained from an MG- and Mycoplasma synoviae (MS)-free breeder farm and raised in wire cages in an isolated room. All birds received food and drinking water ad libitum. At 6 wk of age, 30 birds were bled at random for MG antibody titers and swabbed at the choanal cleft. Each cotton swab was placed in 2 mL of FMS broth for standard PCR to confirm it was MG free. All birds were divided into 7 groups, 25 birds each. Groups 1 and 2 served as sham negative control and sham positive control, respectively. Groups 3 and 6 received 0.5 mL of adjuvant bacterin with 0.5% chitosan containing approximately 2.5 × 108 CFU MG organisms via the intramuscular route. Groups 4 and 5 received commercial MG bacterin (MG-Bac®, Zoetis, Florham Park, NJ) intramuscularly. Group 7 received 0.1 mL of bacterin with 0.5% chitosan containing approximately 5 × 1010 CFU MG organisms, which were divided into both eyes intraocularly. At 10 wk of age, groups 3 and 5 received 0.5 mL of 0.5% chitosan-adjuvanted bacterin containing approximately 2.5 × 108 CFU MG microorganisms and commercial MG bacterin intramuscularly, respectively. Groups 4, 6, and 7 received 0.1 mL of adjuvant bacterin with 0.5% chitosan containing approximately 5 × 1010 CFU MG microorganisms via the intraocular route as described above (Table 1). Clinical signs, including nasal and ocular discharges, were observed in the groups receiving intraocular administration. At 16 wk of age, all birds were bled for MG serology by means of the serum plate agglutination (SPA) test and ELISA and then inoculated with 0.1 mL of FMS or 0.1 mL of Thai MG organisms strain AHRL 58/46 containing approximately 1 × 107 CFU intratracheally. At 1, 2, and 4 wk after inoculation, the remaining birds were bled for MG serology as previously described. Five birds in each group at 1, 2, and 4 wk post inoculation were randomly selected, swabbed at the choanal cleft, euthanized, and assayed to blindly assess the gross thoracic air sacs and histopathological tracheal lesions. The cotton swab was placed in 1 mL of PBS for the quantitative real-time PCR assay. For histopathological tracheal lesion scores, each trachea preserved in 10% formalin was cut into 4 sections as follows: 1 proximal end, 2 middle sections, and 1 distal end, and subjected to histopathological examination. The protocol for the use of animals in this study was approved by Institutional Animal Care and Use (IACUC), Faculty of Veterinary Sciences, Chulalongkorn University (Protocol No. 13310081). Table 1. Experimental designs of chickens of each group at 6, 10, and 16 wk of age (n = 25). Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 1Challenged by intratracheal route. 2(IM) = intramuscular route. 3(IO) = intraocular route. View Large Table 1. Experimental designs of chickens of each group at 6, 10, and 16 wk of age (n = 25). Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 1Challenged by intratracheal route. 2(IM) = intramuscular route. 3(IO) = intraocular route. View Large MG Serology Serum Plate Agglutination Test. In this study, the commercial MG antigen, Soleil® (Biovac Animal Health, Beaucouze, France) was used. The test was followed by the procedure described in the manufacturer's instructions. Briefly, 30 μL of serum were mixed with 30 μL of MG antigen on a glass slide at room temperature for 2 minutes. The positive reaction was identified by clumping reaction. If the serum tested showed a positive reaction, the sera were diluted to a 1:8 dilutions ratio with PBS and then re-analyzed as described above. Enzyme Linked Immunosorbent Assay. MG-ELISA test kits (Synbiotics Corporation, Kansas City, MO) were used to detect MG-specific antibody titers following the manufacturer's instructions. Briefly, the diluted serum was placed on the MG antigen-coated plate, incubated, and washed, followed by addition of anti-chicken immunoglobulin G labeled with horseradish peroxidase. After 30 min of the incubation period, the plate was washed extensively in solution. The plate was read in an ELISA reader at the optical density (OD) at 450 nm. The result was considered negative, suspect, and positive when the antibody titers ranged from 0 to 148, 149 to 743, and above 744, respectively. Polymerase Chain Reaction Assay. The FMS broth samples were incubated at 37°C for 2 h and then subjected to DNA extraction. Target DNA amplified by PCR followed the protocol described by Lauerman (1998). Briefly, the culture broth was centrifuged at 15,000 × g for 6 min. The MG pellets were washed with distilled water and centrifuged twice. The pellets were re-suspended with approximately 30 μL of distilled water, depending on the size of the pellets. Samples were boiled for 10 min, then immediately placed on ice for 5 min and centrifuged at 15,000 × g for 2 min. The supernatant containing the DNA template was collected and maintained at –20°C until use. The PCR mixture was prepared at 25 μL volume containing 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 1.25 mM MgCl2, 1 mM dNTP (Thermo Scientific, Vilnius, Lithuania), 10 pmole each of primer F 5΄-GAGCTAATCTGTAAAGTTGGTC-3΄ and primer R 5΄-GCTTCCTTGCGGTTAGCAAC-3΄) (Qiagen®, Valencia, CA), 1.25 U Taq polymerase (Promega, Madison, WI), and 2.5 μL (250 ng) of the DNA template. MG S6 strain (ATCC 15,302) was used as the positive control. PCR mixtures were amplified in a DNA thermal cycler, Sprint® PCR (Thermo Electron Corporation, Beverly, MA) at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s for 40 cycles, followed by 72°C for 5 minutes. The PCR product was analyzed by 2% agarose gel electrophoresis (Pharmacia Biotech AB, Uppsala, Sweden), stained with 0.20 μg/mL ethidium bromide and visualized by the E-BOX VXII UV transilluminator (Vilber Lourmat, Eberhardzell, Germany). Quantitative Real-Time Pcr Assay. DNA Extraction. The DNA samples were extracted using the QIAamp DNA Mini Kit (Qiagen®, Valencia, CA) according to the manufacturer's instructions. After elution, the genomic DNA was collected and stored at –20°C until use. Preparation of Standard DNA And Standard Curve. The standard DNA was prepared from the DNA of the partial mgc2 gene from Thai MG strain AHRL58/46, which was amplified by PCR as previously described (Ferguson-Noel et al., 2005). The amplified MG DNA was gel purified using a gel extraction kit and a PCR cleaning kit (GenepHlow™ Gel, Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. The purified DNA was measured at 260 nm using the nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA) and stored at –80°C until use. The purified mgc2 gene was diluted in 10-fold dilution to perform real-time PCR and constructed a standard curve of Ct value vs. copies/μL. Primer And Probe Design. Probes for the mgc2 gene of MG consisted of oligonucleotides with the 5 “6-carboxyfluorescein (FAM) reporter dye and the 3” quencher dye Black Hole Quencher-1 (BHQ1). The real-time TaqMan probe sequence of the mgc2 gene was 5΄6-FAMTGATGATCCAAGAACGTGAAGAACACC3΄-BHQ1 (Integrated DNA Technologies, Coralville, IA). The primers of the mgc2 gene sequences are as follow: forward 5΄-ATCGGCAGAAGGGGCAAAGTAG-3; reverse 5΄-GCAACGCAGACTTCTCATCTTCAAG-3΄ (Qiagen®, Valencia, CA). The reaction followed the previously described protocol (Raviv and Kleven, 2009). The 25 μL PCR mixture contained 12.5 μL 2X QuantiFast Probe PCR Mix without ROX dye (Qiagen®, Valencia, CA). The final concentration probe was 1.25 μL of each primer (0.5 μM), 0.5 μL of the probe (0.2 μM), 4.5 μL of nuclease-free water, and 5 μL of DNA template solution. The PCR reaction was performed using Rotor-Gene®RG-3000 (Corbett Research, Sydney, Australia) under the following conditions: 95°C for 3 min and 40 denaturation cycles at 94°C for 3 s and annealing at 60°C for 30 s (Raviv and Kleven, 2009). For each amplification, fluorescence emission was measured at 518 nm for the FAM indicator dye and 534 nm for the quencher BHQ-1 dye. All data were analyzed using the Rotor-Gene software, v.6.0.19. The sample with a Ct value less than or equal to 40 was considered positive. Evaluation Of Lesion Scores. The score of the gross thoracic injury was determined as described by Kleven et al. (1972). Scores include: 0: no injury; 1: slight cloudiness of the air sac membrane; 2: slightly thicker air sac membrane with small accumulations of cheese exudates; 3: air sac membrane obviously thick and fleshy, with large accumulations of cheese exudates in an air sac; 4: same as 3, but 2 or more air sacs were involved. The histopathological lesion score of the trachea was evaluated after the previously described (Yagihashi and Tajima, 1986) as follows: 0: no significant changes; 1: small aggregate of cells (mainly lymphocytes); 2: moderate thickening of the wall due to cellular infiltration and edema commonly accompanied by epithelial degeneration and exudation; 3: extensive thickening of the wall due to cellular infiltration with or without exudation. Statistical Analysis. Gross air sac lesion scores and histopathological lesions of the trachea were analyzed using the Kruskal–Wallis test and the Mann–Whitney U test with a 95% confidence level (P < 0.05). Analysis of unidirectional variance (ANOVA) was performed on antibody titers in order to identify statistical significance at a 95% confidence level (P < 0.05). SPSS® version 22 was used for statistical analysis. RESULTS Clinical Signs After receiving the intraocular vaccination, all birds were normal, and no inflammatory reaction or eye irritation was observed. After the challenge, respiratory signs were not observed in any groups, excluding group 2 (positive control). Some birds in group 2 showed mild respiratory signs, including sneezing and respiratory rales for 3 to 7 days. There was no death in any group. Serology SPA Test. At 6 wk of age, all birds were seronegative. At 16 wk of age, seroconversions were observed in groups receiving chitosan-agglutinated MG bacterin and/or commercial MG bacterin including groups 3, 4, 5, 6, and 7 ranging from 4 to 20 birds of 24 to 25 birds; however, group 7 had the lowest number of positive birds. One wk after the challenge, 15 to 20 of 19 to 20 birds in only MG challengeed groups were positive. At 2 and 4 wk post challenge, all birds in the MG challenge groups were positive. Group 1 did not show positive birds at any age (Table 2). Table 2. Serology determined by SPA and ELISA antibody titers of each group at 16, 17, 18, and 20 wk of age. SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Number of MG positive birds/total tested birds. 3Expressed as mean ± SD. The number of tested birds was the same as that of tested birds of SPA. 4One bird died during this wk; no remarkable lesion was observed. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large Table 2. Serology determined by SPA and ELISA antibody titers of each group at 16, 17, 18, and 20 wk of age. SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Number of MG positive birds/total tested birds. 3Expressed as mean ± SD. The number of tested birds was the same as that of tested birds of SPA. 4One bird died during this wk; no remarkable lesion was observed. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large ELISA. At 6 wk of age, all birds were seronegative. At 16 wk of age, the antibody titer of birds in groups 3, 4, 5, and 6 ranged from 91 to 4717 (n = 24 or 25), while group 7 received only MG bacterin with the chitosan-adjuvant intraocular route at 6 and 10 wk of age and were seronegative. The highest number of bird antibodies was found in groups 4 and 5 (receiving commercial MG bacterin), and group 5 had the highest antibody titers (P < 0.05). At one wk after exposure, the antibody titer of groups 3, 4, 6, and 7 were found in the range 303 to 5,277 (n = 19 or 20); however, the antibody titer of group 5 was greater than that of groups 3, 4, 6, and 7. After 2 and 4 wk after stimulation, the antibody titer of groups 2, 3, 4, 5, 6, and 7 ranged from 774 to 7,774 (n = 14 or 15) and 2266 to 9787 (n = 9 or 10), respectively. Group 2 (positive control) had significantly lower antibody titer at 2 and 4 wk post challenge, respectively (P < 0.05). Group 1 was seronegative at all ages (Table 2). Lesion Score Evaluation. Gross Thoracic Air Sac Lesion Scores. At 16 wk of age and 1, 2, and 4 wk after inoculation, mean lesion scores of the gross thoracic air sacs of 5 birds ranged from 0 to 0.7. At 2 wk after inoculation, group 2 (positive control) showed higher air sac lesion scores than the other groups. There were no significant differences among groups of the same age (P > 0.05) (Table 3). Table 3. Airsac and tracheal lesion scores of each group at 16, 17, 18, and 20 wk of age. Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Expressed as mean ± SD and n = 5. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large Table 3. Airsac and tracheal lesion scores of each group at 16, 17, 18, and 20 wk of age. Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Expressed as mean ± SD and n = 5. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large Histopathological Tracheal Lesion Scores. At 16 wk of age and 1, 2, and 4 wk after inoculation, the histopathological scores of the tracheal lesions of 5 birds ranged from 0 to 1.7. No significant differences were observed in any groups at 16 wk of age. After the inoculation, the mean histopathological scores of the tracheal lesion of group 1 (negative control) were significantly lower than the other groups (P < 0.05). At one wk after the inoculation, the histopathological scores of mean tracheal lesion of groups 2 and 5 were significantly higher than those of groups 3, 4, and 6 (P < 0.05). At 4 wk after inoculation, the mean histopathological scores of tracheal lesion of group 4 (which received commercial MG bacterin and chitosan-adjuvanted MG bacterin) were significantly lower than those of other inoculated groups (P < 0.05). Conventional PCR And Quantitative Real-Time PCR Assays. At 6 wk of age (prior to vaccination), no MG DNA was detected in any FMS broth samples (data not shown). For the quantitative real-time PCR assay, the result was obtained from the standard curve between the number of copies and the cycle threshold. The linear equation of the standard curve was y = -2.789X + 38.916 with R2 = 0.998. At 4 wk after inoculation, birds in groups 4 and 6 (receiving chitosan-adjuvanted MG bacterin or MG commercially injected intramuscularly followed by chitosan-adjuvanted MG bacterin via intraocular administration) gave fewer copy numbers of DNA of the mgc2 gene, while birds in groups 3 and 5 were the second groups that had low DNA numbers. The MG DNA positive samples were found in all groups, excluding group 1, ranging from 4 to 5 samples from 5 samples (Table 4). Table 4. MG DNA from choanal cleft samples expressed as DNA load and number of MG DNA positive samples at 17, 18, and 20 wk of age. DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2DNA load (copies/μL) and expressed as mean ± SD and n = 5, (number of MG DNA positive samples/total tested samples). No significant difference was observed in the same ages (P > 0.05). View Large Table 4. MG DNA from choanal cleft samples expressed as DNA load and number of MG DNA positive samples at 17, 18, and 20 wk of age. DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2DNA load (copies/μL) and expressed as mean ± SD and n = 5, (number of MG DNA positive samples/total tested samples). No significant difference was observed in the same ages (P > 0.05). View Large DISCUSSION Chitosan is a potential mucosal adjuvant (He et al., 1998). There were several studies demonstrating that chitosan is an effective adjuvant, which can stimulate local immunity through mucosal immunization (Zaharoff et al., 2007; Ghendon et al., 2009; Sui et al., 2010). From the previous study, chitosan-adjuvanted MG bacterin could reduce the tracheal lesion score, while the antibody response was lower compared to the commercial MG bacterin (Limsatanun et al., 2016). To improve the efficacy of the vaccine, the different administrations and protocols of MG bacterin vaccination with chitosan adjuvant alone and/or commercial MG bacterin were explored in the present study. Birds that received commercial MG bacterin showed significantly higher antibody titers similar to the previous study bacterin (Limsatanun et al., 2016). The MG bacterin with chitosan adjuvant did not improve systemic antibody response as compared to commercial MG bacterin, which was possibly caused by the efficacy of chitosan following intramuscular vaccination. As chitosan showed a prominent effect as a mucosal adjuvant (Illum et al., 2001; Moschos et al., 2005; Wang et al., 2012), the results of the present study are incompatible with other studies. Chitosan is a potential substance to use as mucosal and humoral immunostimulation (Jabbal-Gill et al., 1998; Sui et al., 2010). Jabbal-Gill et al. (1998) demonstrated that mice vaccinated with recombinant toxin containing chitosan as an adjuvant via intranasal route could stimulate a higher level of serum IgG and IgA secreting against Bordetella pertussis than those receiving only recombinant toxin. In other studies, the M2 influenza subunit vaccine using chitosan as an adjuvant could increase the higher systemic IgG and secretory IgA titers in mice after intranasal immunization compared to mice receiving the M2 subunit alone (Carpenter et al., 1981). In contrast, the live mixture of Newcastle disease vaccine in the chitosan-PBS diluent, which was vaccinated in birds via the oculonasal route, slightly stimulated the systemic and local immune system, but effectively measured the cellular immune response (Rauw et al., 2010a). The low concentration of pathogenic cells in a distinct animal model probably caused the low level of systemic immune response (Rauw et al., 2010a). Taking into account lesion scores, birds receiving intramuscular commercial bacterin or chitosan-adjuvanted bacterin followed by intraocular chitosan adjuvant showed better protection against the tracheal lesion score than that of other groups. In particular, birds immunized with commercial bacterin at 6 wk of age and chitosan-adjuvanted bacterin via the intraocular route at 10 wk of age had a significantly lower mean tracheal lesion score at 20 wk of age. This study suggested that the chitosan-adjuvanted bacterin could enhance the local immunity of the birds, which can protect the trachea against MG challenge. In addition, the result of the real-time PCR assay suggested that the chitosan-adjuvanted bacterin reduced the number of MG microorganisms colonizing the trachea after administration via the intramuscular route followed by the intraocular route. Although the mucosal immune response was not investigated in this study, there is strong evidence from several studies that chitosan adjuvant vaccines promote the mucosal immune response (McNeela et al., 2000; Svindland et al., 2012; Rauw et al., 2010b; Wang et al., 2012). In a previous study, intranasal administration of live influenza vaccine with chitosan adjuvant in mice stimulated IgA, IgG antibody, and virus-specific CD4+ T cells, producing cytokines that received the adjuvant influenza subunit vaccine (Wang et al., 2012). In some studies, chitosan increased the immune response mediated by the nasal passages, especially IgA after mucosal administration of the subunit vaccine in mice (Van der Lubben et al., 2001). However, the thoracic air sac lesion scores were fairly low in the MG inoculated groups because the number of microorganisms in the MG inoculum was quite low or the MG strain used in this study was not virulent. However, the number of MG microorganisms prepared in the bacterin with chitosan adjuvant for intraocular administration was greater than that of intramuscular administration in the present study because the volume of intraocular administration was lower than that of intramuscular administration. Although the largest number of MG microorganisms was used in the intraocular bacterin with chitosan adjuvant, no eye or nasal irritation was observed in the vaccinated birds, suggesting that the chitosan adjuvant bacterin was safe and non-toxic for chickens. In this study, the results suggested that chitosan-adjuvanted bacterin could be used as a mucosal adjuvant vaccine. Chitosan showed the potential to improve protection against MG infection through the mucosal route. Chitosan could be apromising promoter to improve the commercial MG bacterin. Therefore, birds receiving one injection of the oil emulsion bacterin and one injection of the chitosan MG bacterin could protect MG organisms. ACKNOWLEDGMENTS This study was supported by grants from the Thailand Research Fund through the Royal Golden Jubilee Ph.D. (No.Ph.D./0047/2553) to Limsatanun and Pakpinyo, Ratchadaphiseksomphot 2013 Endowment Fund of Chulalongkorn University (CU-56–347-FW), the 90th Anniversary of the Ratchadaphiseksomphot Endorsement Fund, in the Fund for Microbial Food Safety and Prof. Dr. Alongkorn Amonsin. (Senior Academic Award RTA6080012). We also thank Prof. Dr. Sanipa Suradhat of the Faculty of Veterinary Sciences of the University of Chulalongkorn for having reviewed the manuscript. REFERENCES Abd-el-Motelib T. Y. , Kleven S. H. . 1993 . A comparative study of Mycoplasma gallisepticum vaccines in young chickens . Avian Dis. 37 : 981 – 987 . Google Scholar CrossRef Search ADS PubMed Carpenter T. E. , Mallinson E. T. , Miller K. F. , Gentry R. F. , Schwartz L. D. . 1981 . Vaccination with F-strain Mycoplasma gallisepticum to reduce production losses in layer chickens . 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Chitosan-adjuvanted Mycoplasma gallisepticum bacterin via intraocular administration enhances Mycoplasma gallisepticum protection in commercial layers

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
D.O.I.
10.3382/ps/pey051
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Abstract

ABSTRACT Mycoplasma gallisepticum (MG) causes respiratory signs and economic losses in the poultry industry. MG vaccination is one of the effective prevention and control measures that have been used around the world. Our previous study demonstrated that chitosan-adjuvanted MG bacterin could effectively reduce pathological lesions induced by MG and that chitosan could be used as an adjuvant in MG bacterin. The present study determining the efficacy of MG bacterins against the Thai MG strain was based on vaccine programs. Seven groups (25 layers/group) were received MG bacterins containing 0.5% chitosan or a commercial bacterin via intramuscular (IM) or intraocular (IO) route at 6 and 10 wk of age. Sham-negative and sham-positive controls were groups 1 and 2, respectively. Group 3: IM route of chitosan bacterin followed by IM route of chitosan bacterin; group 4: commercial bacterin via IM route followed by chitosan bacterin via IO route; group 5: commercial bacterin via IM route followed by commercial bacterin via IM route; group 6: chitosan bacterin via IM followed by chitosan bacterin via IO route; and group 7: chitosan bacterin via IO route followed by chitosan bacterin via IO route were determined. At 16 wk of age, all groups, excluding group 1, were challenged intratracheally with 0.1 mL containing Thai MG strain 107 colony-forming unit. At 17, 18, and 20 wk of age, 5 birds in each group were bled for serological testing and swabbed at the choanal cleft for the quantitative real-time PCR assay, the euthanized and necropsied. The results showed that birds vaccinated with a commercial intramuscular bacterin followed by an intraocularly chitosan adjuvant bacterin showed the best protection against the MG challenge. The study indicated that chitosan could be the effective mucosal adjuvant and increased the effectiveness of MG bacterin. INTRODUCTION Mycoplasma gallisepticum (MG) is one of the important pathogens of poultry around the world. MG causes chronic respiratory disease (CRD) leading to respiratory symptoms, including rales, coughs, sneezing, ocular discharge, and nasal discharge. In addition, MG infection increases condemnation in processing plants and degradation of carcasses, causing economic losses in the poultry industry (Raviv and Ley, 2013). To reduce the economic impact of MG infection in chickens, there are 3 commercial vaccines that have been used for the prevention program. Live, inactivated, and recombinant MG poultry (rFP-MG) vaccines are commonly used in the poultry industry. The efficacy of each type of vaccine in the protection of clinical signs and induction of the systemic immune response is different; however, these vaccines can reduce post-challenge production losses with the MG field strain (Carpenter et al., 1981; Whithear et al., 1990; Evans and Hafez, 1992; Zhang et al., 2010). Commercial MG vaccines are strains: F, ts-11, and 6/85. F strain vaccine is more virulent compared to other live vaccines (Raviv and Ley, 2013). It may induce mild respiratory signs in turkeys and broilers (Rodríguez and Kleven, 1980). Other vaccine strains, including ts-11 and 6/85, showed less protection against the MG challenge compared to the F strain (Abd-el-Motelib and Kleven, 1993). The inactivated vaccine is safe for use in turkeys and may reduce production losses in chickens and turkeys, but the inactivated vaccine is more expensive and cannot protect against MG infection in the respiratory tract (Panigraphy et al., 1981). For the recombinant vaccine, rFP-MG, the MG surface protein is encoded by the recombinant avian poxvirus (Vectormune® FP-MG vaccine, CEVA Santé Animale, Libourne, France). Although FP-MG is safe to vaccinate in chickens (Zhang et al., 2010), it did not show good protection against the MG challenge (Ferguson-Noel et al., 2012). Recently, a novel vaccine adjuvant has been developed to improve the efficacy of the vaccine. Chitosan is one of the potential adjuvants for the inactivated vaccine and is a polysaccharide made from deacetylation of chitin. Several studies found that chitosan could enhance the humoral and cell-mediated immune response in mice (Zaharoff et al., 2007) and chickens (Rauw et al., 2010a). From our previous study, chitosan could be used as an adjuvant for MG bacterin (Limsatanun et al., 2016). The chitosan-adjuvanted MG bacterin provided better protection of the tracheal lesion and caused less tissue reaction at the site of injection, compared to the commercial bacterin MG. But it has produced a low level of systemic immune response. The experimental groups receiving bacterin with chitosan adjuvant had lower numbers of antibody-positive birds compared to a commercial MG bacterin group (Limsatanun et al., 2016). However, the efficacy of the vaccine from the previous study was determined only by the intramuscular route, which could provoke tissue reaction and low immune response to the mucosa for vaccination with MG from poultry. To improve MG-induced immunity, different administrations, including intraocular routes and vaccination protocols, which included only MG bacterin adjuvanted with chitosan and/or commercial MG bacterin, were explored in the present study. MATERIALS AND METHODS Vaccine MG bacterin with chitosan adjuvant was prepared and described by Limsatanun et al. (2016). Briefly, Thai MG strain AHRL 20/52 was propagated in Frey broth supplemented with 15% porcine serum (FMS) (Kleven, 2008) and incubated at 37°C until the color of the broth changed from pink to yellow-orange or phase log. The culture broth was centrifuged at 12,000 xg at 4°C for 30 min to collect MG cells. MG cells were inactivated with 0.01% thimerosal (Sigma-Aldrich Corp., St. Louis, MO) in phosphate buffered saline (PBS) and incubated at 4°C for 7 d (Chukiatsiri et al., 2009). Chitosan solution (Sigma-Aldrich Corp., St. Louis, MO) was added to the MG cells to a final 0.5% (w/v) concentration. Two concentrations of MG organisms were determined in the bacterin with chitosan adjuvant. Bacteria were used with chitosan adjuvant containing approximately 5 × 108 MG microorganisms and 5 × 1011 colony-forming units (CFU)/mL for intramuscular and intraocular administration, respectively. All the chitosan-adjuvanted MG bacterins were stored at 4°C until use. Experimental Design And Sample Collection Protocol One-hundred-seventy-five one-day-old commercial female layer chickens, Isa brown breed, were obtained from an MG- and Mycoplasma synoviae (MS)-free breeder farm and raised in wire cages in an isolated room. All birds received food and drinking water ad libitum. At 6 wk of age, 30 birds were bled at random for MG antibody titers and swabbed at the choanal cleft. Each cotton swab was placed in 2 mL of FMS broth for standard PCR to confirm it was MG free. All birds were divided into 7 groups, 25 birds each. Groups 1 and 2 served as sham negative control and sham positive control, respectively. Groups 3 and 6 received 0.5 mL of adjuvant bacterin with 0.5% chitosan containing approximately 2.5 × 108 CFU MG organisms via the intramuscular route. Groups 4 and 5 received commercial MG bacterin (MG-Bac®, Zoetis, Florham Park, NJ) intramuscularly. Group 7 received 0.1 mL of bacterin with 0.5% chitosan containing approximately 5 × 1010 CFU MG organisms, which were divided into both eyes intraocularly. At 10 wk of age, groups 3 and 5 received 0.5 mL of 0.5% chitosan-adjuvanted bacterin containing approximately 2.5 × 108 CFU MG microorganisms and commercial MG bacterin intramuscularly, respectively. Groups 4, 6, and 7 received 0.1 mL of adjuvant bacterin with 0.5% chitosan containing approximately 5 × 1010 CFU MG microorganisms via the intraocular route as described above (Table 1). Clinical signs, including nasal and ocular discharges, were observed in the groups receiving intraocular administration. At 16 wk of age, all birds were bled for MG serology by means of the serum plate agglutination (SPA) test and ELISA and then inoculated with 0.1 mL of FMS or 0.1 mL of Thai MG organisms strain AHRL 58/46 containing approximately 1 × 107 CFU intratracheally. At 1, 2, and 4 wk after inoculation, the remaining birds were bled for MG serology as previously described. Five birds in each group at 1, 2, and 4 wk post inoculation were randomly selected, swabbed at the choanal cleft, euthanized, and assayed to blindly assess the gross thoracic air sacs and histopathological tracheal lesions. The cotton swab was placed in 1 mL of PBS for the quantitative real-time PCR assay. For histopathological tracheal lesion scores, each trachea preserved in 10% formalin was cut into 4 sections as follows: 1 proximal end, 2 middle sections, and 1 distal end, and subjected to histopathological examination. The protocol for the use of animals in this study was approved by Institutional Animal Care and Use (IACUC), Faculty of Veterinary Sciences, Chulalongkorn University (Protocol No. 13310081). Table 1. Experimental designs of chickens of each group at 6, 10, and 16 wk of age (n = 25). Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 1Challenged by intratracheal route. 2(IM) = intramuscular route. 3(IO) = intraocular route. View Large Table 1. Experimental designs of chickens of each group at 6, 10, and 16 wk of age (n = 25). Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 Group 6 wk of age 10 wk of age 16 wk of age1 1 0.5% chitosan (IM)2 0.5% chitosan (IM) FMS broth 2 0.5% chitosan (IM) 0.5% chitosan (IM) MG strain AHRL 58/46 3 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IM) MG strain AHRL 58/46 4 MG-Bac® (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 5 MG-Bac® (IM) MG-Bac® (IM) MG strain AHRL 58/46 6 Chitosan-adjuvanted vaccine (IM) Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 7 Chitosan-adjuvanted vaccine (IO)3 Chitosan-adjuvanted vaccine (IO) MG strain AHRL 58/46 1Challenged by intratracheal route. 2(IM) = intramuscular route. 3(IO) = intraocular route. View Large MG Serology Serum Plate Agglutination Test. In this study, the commercial MG antigen, Soleil® (Biovac Animal Health, Beaucouze, France) was used. The test was followed by the procedure described in the manufacturer's instructions. Briefly, 30 μL of serum were mixed with 30 μL of MG antigen on a glass slide at room temperature for 2 minutes. The positive reaction was identified by clumping reaction. If the serum tested showed a positive reaction, the sera were diluted to a 1:8 dilutions ratio with PBS and then re-analyzed as described above. Enzyme Linked Immunosorbent Assay. MG-ELISA test kits (Synbiotics Corporation, Kansas City, MO) were used to detect MG-specific antibody titers following the manufacturer's instructions. Briefly, the diluted serum was placed on the MG antigen-coated plate, incubated, and washed, followed by addition of anti-chicken immunoglobulin G labeled with horseradish peroxidase. After 30 min of the incubation period, the plate was washed extensively in solution. The plate was read in an ELISA reader at the optical density (OD) at 450 nm. The result was considered negative, suspect, and positive when the antibody titers ranged from 0 to 148, 149 to 743, and above 744, respectively. Polymerase Chain Reaction Assay. The FMS broth samples were incubated at 37°C for 2 h and then subjected to DNA extraction. Target DNA amplified by PCR followed the protocol described by Lauerman (1998). Briefly, the culture broth was centrifuged at 15,000 × g for 6 min. The MG pellets were washed with distilled water and centrifuged twice. The pellets were re-suspended with approximately 30 μL of distilled water, depending on the size of the pellets. Samples were boiled for 10 min, then immediately placed on ice for 5 min and centrifuged at 15,000 × g for 2 min. The supernatant containing the DNA template was collected and maintained at –20°C until use. The PCR mixture was prepared at 25 μL volume containing 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 1.25 mM MgCl2, 1 mM dNTP (Thermo Scientific, Vilnius, Lithuania), 10 pmole each of primer F 5΄-GAGCTAATCTGTAAAGTTGGTC-3΄ and primer R 5΄-GCTTCCTTGCGGTTAGCAAC-3΄) (Qiagen®, Valencia, CA), 1.25 U Taq polymerase (Promega, Madison, WI), and 2.5 μL (250 ng) of the DNA template. MG S6 strain (ATCC 15,302) was used as the positive control. PCR mixtures were amplified in a DNA thermal cycler, Sprint® PCR (Thermo Electron Corporation, Beverly, MA) at 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s for 40 cycles, followed by 72°C for 5 minutes. The PCR product was analyzed by 2% agarose gel electrophoresis (Pharmacia Biotech AB, Uppsala, Sweden), stained with 0.20 μg/mL ethidium bromide and visualized by the E-BOX VXII UV transilluminator (Vilber Lourmat, Eberhardzell, Germany). Quantitative Real-Time Pcr Assay. DNA Extraction. The DNA samples were extracted using the QIAamp DNA Mini Kit (Qiagen®, Valencia, CA) according to the manufacturer's instructions. After elution, the genomic DNA was collected and stored at –20°C until use. Preparation of Standard DNA And Standard Curve. The standard DNA was prepared from the DNA of the partial mgc2 gene from Thai MG strain AHRL58/46, which was amplified by PCR as previously described (Ferguson-Noel et al., 2005). The amplified MG DNA was gel purified using a gel extraction kit and a PCR cleaning kit (GenepHlow™ Gel, Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. The purified DNA was measured at 260 nm using the nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA) and stored at –80°C until use. The purified mgc2 gene was diluted in 10-fold dilution to perform real-time PCR and constructed a standard curve of Ct value vs. copies/μL. Primer And Probe Design. Probes for the mgc2 gene of MG consisted of oligonucleotides with the 5 “6-carboxyfluorescein (FAM) reporter dye and the 3” quencher dye Black Hole Quencher-1 (BHQ1). The real-time TaqMan probe sequence of the mgc2 gene was 5΄6-FAMTGATGATCCAAGAACGTGAAGAACACC3΄-BHQ1 (Integrated DNA Technologies, Coralville, IA). The primers of the mgc2 gene sequences are as follow: forward 5΄-ATCGGCAGAAGGGGCAAAGTAG-3; reverse 5΄-GCAACGCAGACTTCTCATCTTCAAG-3΄ (Qiagen®, Valencia, CA). The reaction followed the previously described protocol (Raviv and Kleven, 2009). The 25 μL PCR mixture contained 12.5 μL 2X QuantiFast Probe PCR Mix without ROX dye (Qiagen®, Valencia, CA). The final concentration probe was 1.25 μL of each primer (0.5 μM), 0.5 μL of the probe (0.2 μM), 4.5 μL of nuclease-free water, and 5 μL of DNA template solution. The PCR reaction was performed using Rotor-Gene®RG-3000 (Corbett Research, Sydney, Australia) under the following conditions: 95°C for 3 min and 40 denaturation cycles at 94°C for 3 s and annealing at 60°C for 30 s (Raviv and Kleven, 2009). For each amplification, fluorescence emission was measured at 518 nm for the FAM indicator dye and 534 nm for the quencher BHQ-1 dye. All data were analyzed using the Rotor-Gene software, v.6.0.19. The sample with a Ct value less than or equal to 40 was considered positive. Evaluation Of Lesion Scores. The score of the gross thoracic injury was determined as described by Kleven et al. (1972). Scores include: 0: no injury; 1: slight cloudiness of the air sac membrane; 2: slightly thicker air sac membrane with small accumulations of cheese exudates; 3: air sac membrane obviously thick and fleshy, with large accumulations of cheese exudates in an air sac; 4: same as 3, but 2 or more air sacs were involved. The histopathological lesion score of the trachea was evaluated after the previously described (Yagihashi and Tajima, 1986) as follows: 0: no significant changes; 1: small aggregate of cells (mainly lymphocytes); 2: moderate thickening of the wall due to cellular infiltration and edema commonly accompanied by epithelial degeneration and exudation; 3: extensive thickening of the wall due to cellular infiltration with or without exudation. Statistical Analysis. Gross air sac lesion scores and histopathological lesions of the trachea were analyzed using the Kruskal–Wallis test and the Mann–Whitney U test with a 95% confidence level (P < 0.05). Analysis of unidirectional variance (ANOVA) was performed on antibody titers in order to identify statistical significance at a 95% confidence level (P < 0.05). SPSS® version 22 was used for statistical analysis. RESULTS Clinical Signs After receiving the intraocular vaccination, all birds were normal, and no inflammatory reaction or eye irritation was observed. After the challenge, respiratory signs were not observed in any groups, excluding group 2 (positive control). Some birds in group 2 showed mild respiratory signs, including sneezing and respiratory rales for 3 to 7 days. There was no death in any group. Serology SPA Test. At 6 wk of age, all birds were seronegative. At 16 wk of age, seroconversions were observed in groups receiving chitosan-agglutinated MG bacterin and/or commercial MG bacterin including groups 3, 4, 5, 6, and 7 ranging from 4 to 20 birds of 24 to 25 birds; however, group 7 had the lowest number of positive birds. One wk after the challenge, 15 to 20 of 19 to 20 birds in only MG challengeed groups were positive. At 2 and 4 wk post challenge, all birds in the MG challenge groups were positive. Group 1 did not show positive birds at any age (Table 2). Table 2. Serology determined by SPA and ELISA antibody titers of each group at 16, 17, 18, and 20 wk of age. SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Number of MG positive birds/total tested birds. 3Expressed as mean ± SD. The number of tested birds was the same as that of tested birds of SPA. 4One bird died during this wk; no remarkable lesion was observed. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large Table 2. Serology determined by SPA and ELISA antibody titers of each group at 16, 17, 18, and 20 wk of age. SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c SPA2 ELISA antibody titers3 Group1 16 17 18 20 16 17 18 20 1 0/25 0/20 0/15 0/10 0 ± 0a 0 ± 0a 0 ± 0a 0 ± 0a 2 0/25 20/20 15/15 10/10 0 ± 0a 0 ± 0a 774 ± 568a 2266 ± 1528a 3 17/25 19/20 15/15 10/10 680 ± 620a,b 1533 ± 1166a,b 6484 ± 1636b 6947 ± 2170b,c 4 16/25 15/194 14/14 9/9 1606 ± 151b 1951 ± 1901b 7774 ± 1354b 9072 ± 957b 5 20/244 18/19 14/14 9/9 4717 ± 276c 5277 ± 3706c 7691 ± 2343b 9532 ± 1300b 6 18/25 17/20 15/15 10/10 91 ± 343a 1187 ± 1672a,b 6295 ± 2117b 9787 ± 2385b,c 7 4/25 19/20 14/144 9/9 0 ± 0a 303 ± 854a,b 4246 ± 1773c 5895 ± 3055c 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Number of MG positive birds/total tested birds. 3Expressed as mean ± SD. The number of tested birds was the same as that of tested birds of SPA. 4One bird died during this wk; no remarkable lesion was observed. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large ELISA. At 6 wk of age, all birds were seronegative. At 16 wk of age, the antibody titer of birds in groups 3, 4, 5, and 6 ranged from 91 to 4717 (n = 24 or 25), while group 7 received only MG bacterin with the chitosan-adjuvant intraocular route at 6 and 10 wk of age and were seronegative. The highest number of bird antibodies was found in groups 4 and 5 (receiving commercial MG bacterin), and group 5 had the highest antibody titers (P < 0.05). At one wk after exposure, the antibody titer of groups 3, 4, 6, and 7 were found in the range 303 to 5,277 (n = 19 or 20); however, the antibody titer of group 5 was greater than that of groups 3, 4, 6, and 7. After 2 and 4 wk after stimulation, the antibody titer of groups 2, 3, 4, 5, 6, and 7 ranged from 774 to 7,774 (n = 14 or 15) and 2266 to 9787 (n = 9 or 10), respectively. Group 2 (positive control) had significantly lower antibody titer at 2 and 4 wk post challenge, respectively (P < 0.05). Group 1 was seronegative at all ages (Table 2). Lesion Score Evaluation. Gross Thoracic Air Sac Lesion Scores. At 16 wk of age and 1, 2, and 4 wk after inoculation, mean lesion scores of the gross thoracic air sacs of 5 birds ranged from 0 to 0.7. At 2 wk after inoculation, group 2 (positive control) showed higher air sac lesion scores than the other groups. There were no significant differences among groups of the same age (P > 0.05) (Table 3). Table 3. Airsac and tracheal lesion scores of each group at 16, 17, 18, and 20 wk of age. Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Expressed as mean ± SD and n = 5. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large Table 3. Airsac and tracheal lesion scores of each group at 16, 17, 18, and 20 wk of age. Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b Airsac lesion scores2 Tracheal lesion scores2 Group1 16 17 18 20 16 17 18 20 1 0 ± 0 0.1 ± 0.2 0 ± 0 0.1 ± 0.2 0.3 ± 0.3 0.1 ± 0.2a 0.4 ± 0.2a 0.1 ± 0.1a 2 0 ± 0 0.4 ± 0.5 0.7 ± 0.6 0.2 ± 0.3 0.2 ± 0.1 1.7 ± 0.6c 1.0 ± 0.5b 1.4 ± 0.7b 3 0 ± 0 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0 ± 0.1 1.0 ± 0.4b 1.4 ± 0.4b 1.5 ± 0.5b 4 0 ± 0 0.1 ± 0.2 0.1 ± 0.2 0.2 ± 0.4 0 ± 0 1.0 ± 0.2b 1.0 ± 0.9b 0.8 ± 0.3c 5 0 ± 0 0 ± 0 0.4 ± 0.4 0.2 ± 0.4 0 ± 0.1 1.7 ± 0.4c 1.4 ± 0.4b 1.4 ± 0.6b 6 0 ± 0 0.1 ± 0.2 0.4 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.0 ± 0.4b 1.0 ± 0.5b 1.3 ± 0.3b 7 0 ± 0 0.5 ± 0.4 0.3 ± 0.4 0.1 ± 0.2 0.1 ± 0.1 1.3 ± 0.5b, c 1.5 ± 0.6b 1.5 ± 0.5b 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2Expressed as mean ± SD and n = 5. a,b,cDifferent superscripts in the same column mean statistically significant difference (P < 0.05). View Large Histopathological Tracheal Lesion Scores. At 16 wk of age and 1, 2, and 4 wk after inoculation, the histopathological scores of the tracheal lesions of 5 birds ranged from 0 to 1.7. No significant differences were observed in any groups at 16 wk of age. After the inoculation, the mean histopathological scores of the tracheal lesion of group 1 (negative control) were significantly lower than the other groups (P < 0.05). At one wk after the inoculation, the histopathological scores of mean tracheal lesion of groups 2 and 5 were significantly higher than those of groups 3, 4, and 6 (P < 0.05). At 4 wk after inoculation, the mean histopathological scores of tracheal lesion of group 4 (which received commercial MG bacterin and chitosan-adjuvanted MG bacterin) were significantly lower than those of other inoculated groups (P < 0.05). Conventional PCR And Quantitative Real-Time PCR Assays. At 6 wk of age (prior to vaccination), no MG DNA was detected in any FMS broth samples (data not shown). For the quantitative real-time PCR assay, the result was obtained from the standard curve between the number of copies and the cycle threshold. The linear equation of the standard curve was y = -2.789X + 38.916 with R2 = 0.998. At 4 wk after inoculation, birds in groups 4 and 6 (receiving chitosan-adjuvanted MG bacterin or MG commercially injected intramuscularly followed by chitosan-adjuvanted MG bacterin via intraocular administration) gave fewer copy numbers of DNA of the mgc2 gene, while birds in groups 3 and 5 were the second groups that had low DNA numbers. The MG DNA positive samples were found in all groups, excluding group 1, ranging from 4 to 5 samples from 5 samples (Table 4). Table 4. MG DNA from choanal cleft samples expressed as DNA load and number of MG DNA positive samples at 17, 18, and 20 wk of age. DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2DNA load (copies/μL) and expressed as mean ± SD and n = 5, (number of MG DNA positive samples/total tested samples). No significant difference was observed in the same ages (P > 0.05). View Large Table 4. MG DNA from choanal cleft samples expressed as DNA load and number of MG DNA positive samples at 17, 18, and 20 wk of age. DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) DNA load and number of MG DNA positive samples2 Group1 17 18 20 1 0 (0/5) 0 (0/5) 0 (0/5) 2 932.0 ± 0.1 (5/5) 678.0 ± 3.9 (4/5) 398.0 ± 3.2 (5/5) 3 1179.0 ± 2.0 (5/5) 49.0 ± 3.0 (5/5) 38.0 ± 2.2 (5/5) 4 411.0 ± 2.3 (5/5) 504.0 ± 0.8 (5/5) 1.1 ± 0.4 (4/5) 5 812.0 ± 4.4 (5/5) 267.0 ± 0.6 (5/5) 31.2 ± 1.3 (5/5) 6 1448.0 ± 2.3 (5/5) 595.0 ± 3.4 (4/5) 5.0 ± 0.2 (4/5) 7 469.0 ± 3.5 (4/5) 131.0 ± 2.8 (5/5) 204.0 ± 2.6 (5/5) 1Group 1, negative control; group 2, positive control; group 3, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 and 10 wk of age; group 4, birds vaccinated with MG-Bac® (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 5, birds vaccinated with MG-Bac® (IM) at 6 and 10 wk of age; group 6, birds vaccinated with chitosan-adjuvanted vaccine (IM) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age; group 7, birds vaccinated with chitosan-adjuvanted vaccine (IO) at 6 wk of age and chitosan-adjuvanted vaccine (IO) at 10 wk of age. 2DNA load (copies/μL) and expressed as mean ± SD and n = 5, (number of MG DNA positive samples/total tested samples). No significant difference was observed in the same ages (P > 0.05). View Large DISCUSSION Chitosan is a potential mucosal adjuvant (He et al., 1998). There were several studies demonstrating that chitosan is an effective adjuvant, which can stimulate local immunity through mucosal immunization (Zaharoff et al., 2007; Ghendon et al., 2009; Sui et al., 2010). From the previous study, chitosan-adjuvanted MG bacterin could reduce the tracheal lesion score, while the antibody response was lower compared to the commercial MG bacterin (Limsatanun et al., 2016). To improve the efficacy of the vaccine, the different administrations and protocols of MG bacterin vaccination with chitosan adjuvant alone and/or commercial MG bacterin were explored in the present study. Birds that received commercial MG bacterin showed significantly higher antibody titers similar to the previous study bacterin (Limsatanun et al., 2016). The MG bacterin with chitosan adjuvant did not improve systemic antibody response as compared to commercial MG bacterin, which was possibly caused by the efficacy of chitosan following intramuscular vaccination. As chitosan showed a prominent effect as a mucosal adjuvant (Illum et al., 2001; Moschos et al., 2005; Wang et al., 2012), the results of the present study are incompatible with other studies. Chitosan is a potential substance to use as mucosal and humoral immunostimulation (Jabbal-Gill et al., 1998; Sui et al., 2010). Jabbal-Gill et al. (1998) demonstrated that mice vaccinated with recombinant toxin containing chitosan as an adjuvant via intranasal route could stimulate a higher level of serum IgG and IgA secreting against Bordetella pertussis than those receiving only recombinant toxin. In other studies, the M2 influenza subunit vaccine using chitosan as an adjuvant could increase the higher systemic IgG and secretory IgA titers in mice after intranasal immunization compared to mice receiving the M2 subunit alone (Carpenter et al., 1981). In contrast, the live mixture of Newcastle disease vaccine in the chitosan-PBS diluent, which was vaccinated in birds via the oculonasal route, slightly stimulated the systemic and local immune system, but effectively measured the cellular immune response (Rauw et al., 2010a). The low concentration of pathogenic cells in a distinct animal model probably caused the low level of systemic immune response (Rauw et al., 2010a). Taking into account lesion scores, birds receiving intramuscular commercial bacterin or chitosan-adjuvanted bacterin followed by intraocular chitosan adjuvant showed better protection against the tracheal lesion score than that of other groups. In particular, birds immunized with commercial bacterin at 6 wk of age and chitosan-adjuvanted bacterin via the intraocular route at 10 wk of age had a significantly lower mean tracheal lesion score at 20 wk of age. This study suggested that the chitosan-adjuvanted bacterin could enhance the local immunity of the birds, which can protect the trachea against MG challenge. In addition, the result of the real-time PCR assay suggested that the chitosan-adjuvanted bacterin reduced the number of MG microorganisms colonizing the trachea after administration via the intramuscular route followed by the intraocular route. Although the mucosal immune response was not investigated in this study, there is strong evidence from several studies that chitosan adjuvant vaccines promote the mucosal immune response (McNeela et al., 2000; Svindland et al., 2012; Rauw et al., 2010b; Wang et al., 2012). In a previous study, intranasal administration of live influenza vaccine with chitosan adjuvant in mice stimulated IgA, IgG antibody, and virus-specific CD4+ T cells, producing cytokines that received the adjuvant influenza subunit vaccine (Wang et al., 2012). In some studies, chitosan increased the immune response mediated by the nasal passages, especially IgA after mucosal administration of the subunit vaccine in mice (Van der Lubben et al., 2001). However, the thoracic air sac lesion scores were fairly low in the MG inoculated groups because the number of microorganisms in the MG inoculum was quite low or the MG strain used in this study was not virulent. However, the number of MG microorganisms prepared in the bacterin with chitosan adjuvant for intraocular administration was greater than that of intramuscular administration in the present study because the volume of intraocular administration was lower than that of intramuscular administration. Although the largest number of MG microorganisms was used in the intraocular bacterin with chitosan adjuvant, no eye or nasal irritation was observed in the vaccinated birds, suggesting that the chitosan adjuvant bacterin was safe and non-toxic for chickens. In this study, the results suggested that chitosan-adjuvanted bacterin could be used as a mucosal adjuvant vaccine. Chitosan showed the potential to improve protection against MG infection through the mucosal route. Chitosan could be apromising promoter to improve the commercial MG bacterin. Therefore, birds receiving one injection of the oil emulsion bacterin and one injection of the chitosan MG bacterin could protect MG organisms. ACKNOWLEDGMENTS This study was supported by grants from the Thailand Research Fund through the Royal Golden Jubilee Ph.D. (No.Ph.D./0047/2553) to Limsatanun and Pakpinyo, Ratchadaphiseksomphot 2013 Endowment Fund of Chulalongkorn University (CU-56–347-FW), the 90th Anniversary of the Ratchadaphiseksomphot Endorsement Fund, in the Fund for Microbial Food Safety and Prof. Dr. Alongkorn Amonsin. (Senior Academic Award RTA6080012). We also thank Prof. Dr. Sanipa Suradhat of the Faculty of Veterinary Sciences of the University of Chulalongkorn for having reviewed the manuscript. REFERENCES Abd-el-Motelib T. Y. , Kleven S. H. . 1993 . A comparative study of Mycoplasma gallisepticum vaccines in young chickens . Avian Dis. 37 : 981 – 987 . Google Scholar CrossRef Search ADS PubMed Carpenter T. E. , Mallinson E. T. , Miller K. F. , Gentry R. F. , Schwartz L. D. . 1981 . Vaccination with F-strain Mycoplasma gallisepticum to reduce production losses in layer chickens . 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Poultry ScienceOxford University Press

Published: Feb 15, 2018

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