Dissertations / Theses on the topic 'Pleuropneumonia vaccines'

Contagious bovine pleuropneumonia (CBPP) is a bovine bacterial disease of major economic importance in sub-Saharan Africa. Vaccination has been recommended to control the disease in endemic areas such as the Maasai ecosystems of Kenya and Tanzania; however, the currently used live attenuated vaccine has been reported to have poor vaccine safety and efficacy. To compare standard (current) and an improved (buffered) version of the live CBPP-vaccine, several epidemiological studies were carried out in Maasai cattle in Kenya between 2006 and 2008. Specifically, the aims were to estimate CBPP seroprevalence at herd and animal level; to identify risk factors for seroprevalence at both levels; to investigate the spatial distribution of seroprevalence; to compare post vaccination adverse events in cattle vaccinated with a standard and a buffered vaccine, and finally to compare efficacy of the two vaccines to induce seroconversion and to prevent development of clinical signs suggestive of CBPP. A cross-sectional study was carried out in 6872 cattle in 175 randomly selected herds from Loita and Mara divisions. A competitive ELISA revealed that 85% of the herds in the area had at least one seropositive animal and that seropositive herds were harbouring 11% seropositive cattle. A complement fixation test revealed that 46% of the herds had at least one seropositive animal and that seropositive herds were harbouring 4% seropositive cattle. A multivariable logistic regression analysis of the seroprevalence indicated that previous vaccination against CBPP, a history of CBPP outbreaks in the herd, animal age and the location of the herd in the division of Mara were positively correlated to seroprevalence. To investigate the observed difference in herd seroprevalence between the two divisions further, a spatial analysis was conducted. A SatScan test revealed clusters in Mara in areas identified by veterinary personnel as CBPP ‘hot spots’. A logistic regression using spatial information identified that location in the midland agro-ecological zone or close to a river and vaccination were positively associated with seroprevalence. To compare safety and efficacy of a standard and a buffered vaccine, two cohorts of approximately 40,000 cattle were used. The study showed that within 100 days post vaccination, 6.2 cattle per 1000 vaccinates developed adverse events, 4.1 of which were specifically attributable to vaccination and ranging from swelling of the tail to the tail sloughing off. This study revealed a slightly higher incidence of adverse events in cattle vaccinated with the buffered vaccine compared to the standard vaccine. A comparison of the efficacy of the two vaccines revealed that cattle vaccinated with the buffered vaccine had higher odds of seroconversion and lower odds of developing symptoms of CBPP, three and twelve months post vaccination respectively. The epidemiological studies conducted clearly show wide spread seroprevalence in the Maasai cattle. Given the (spatial) heterogeneity observed, control measures should probably be targeted in areas of increased risk (clusters). However, positive association of vaccination and seropositivity call for better diagnostics tests that can differentiate vaccinated from infected animals. Vaccination with buffered vaccine resulted in increased seroconversion, decreased clinical signs indicative of CBPP post vaccination and low seroprevalence post ‘outbreak’. Nevertheless, the increase in adverse events related to the buffered vaccine calls for further research into safer CBPP vaccines.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP.
QC 20100719

碩士
國立高雄大學
生物科技研究所
94
Actinobacillus pleuropneumoniae can cause haemorrhagic, fibrinous and necrotic pleuropneumonia in pigs that causes critical economic losses in pig farm. Virulence factors of A. pleuropneumoniae include the capsular polysaccharides, lipopolysaccharides, outer membrane proteins, adhesion factors and exotoxins. Virulence is strongly correlated with the Apx toxins. ApxI and ApxⅡ are exotoxins of A. pleuropneumoniae. ApxI is strongly hemolytic and strongly cytotoxic. ApxⅡ is weakly hemolytic and weakly cytotoxic. Pretoxins ApxIA and ApxⅡA are structural proteins of ApxI and ApxⅡ, respectively. ApxIA and ApxⅡA are non-toxic before they are acetylated by ApxIC and ApxⅡC, respectively. Outer membrane proteins are exposed on the surface of pathogen, which are more accessible for immune system to induce immune responses than other proteins and usually be used as candidates for vaccine development. A. pleuropneumoniae needs to obtain iron from iron-containing proteins of the host to express full virulence. These iron-containing proteins include transferrin, haem, and haemoglobin. In this study, ApxIA, ApxⅡA, OmlA and HgbA were chosen as the target antigens for DNA vaccine development against A. pleuropneumoniae. These genes were cloned to vectors that can express antigens in mammalian system and inserted genes were confirmed by DNA sequencing analysis. The swine kidney cell line LLC-PK1 was transfected with these DNA constructs and detected the expressed proteins by Western blot analysis to demonstrate these proteins can be expressed in mammalian cells. To evaluate the potency of these DNA vaccines, each DNA vaccine and a mixture vaccine containing above four DNA constructs (4 in 1 vaccine) were administrated into mice by intramuscular injection. A commercial inactivated A. pleuropneumoniae vaccine also used as positive control, empty vector and PBS as negative control. The titers of antisera increased significantly except empty vector and PBS group after second immunization. These mice were challenged with 5x108 colony-forming unit (cfu) of serotype 1 of A. pleuropneumoniae after 24 days of first immunization. The survival rate of each group after 5 days of challenge was as following: 67% for 4 in 1 vaccine; 50% for ApxIA vaccine; 25% for ApxIIA, OmlA and commercial inactivated vaccine; 0% for HgbA, empty vector and PBS group. The results showed that the 4 in 1 DNA vaccine could obtain the best protective efficacy and better than the commercial inactivated vaccine. It indicates that these DNA vaccines used in this study could be a new strategy against infection by A. pleuropneumoniae.

碩士
東海大學
畜產學研究所
84
The objectives of this study were to investigate the best growth condition for App and to evaluate the efficiency of App vaccine in pig. Among these four medium, TS 1%NAD, TS 5%Yeast extract (YE), BHI 1%NAD and BHI 5%YE, App had the highest bacterial count and hemolycin titer (256h.U.) when cultured in TS 1%NAD. The changes of pH curve were similar in four different medium. After 4 hours incubation, the pH value decreased from 7 to 6. To investigate the allergic component of App, bacterial culture, bacteria -free culture medium, bacterial pellet, exotoxin, endotoxin and alumina hydroxide (adjuvent) were injected into mice. The results showed that bacterial pellet, exotoxin and endotoxin induced anaphylaxis in mice. After treating these three allergic components, bacterial pellet and exotoxin failed to induced anaphylaxis whereas endotoxin remained the capacity of inducing anaphylaxis in mouse. To evaluate the efficiency of the App vaccine in the pig, four different methods of administration, oral 10ml, oral 20 ml, I. M. 20ml and oral and I.M. combination, were used to immunize pigs. After challenged with App, all of the immunized pigs died within 24 hours. The antibody titers did not increase in serum and intestinal mucus of immunized pigs. These results imply that this App vaccine fails to protect pigs from App infection.

碩士
國立高雄大學
生物科技研究所
98
Adjuvant is an immune stimulator which can enhance the immunogenicity of vaccine. The function of adjuvant is to enhance the protective immunity of vaccine or to change the type of immune response. The development of mucosal adjuvant was major focused in the subunit protein of E. coli heat labile toxin (LT) and cholera toxin (CT). In this study, a novel mucosal adjuvant was developed using the structural protein of respiratory track toxin produced by Actinobacillus pleuropneumoniae. It was known that ApxII has the lower hemolytic and cytotoxic activity in all Apx toxins of A. pleuropneumoniae, and ApxIIA is the structural protein of ApxII. According to the functional domain of RTX (repeats in toxin) family toxin, the full length, N terminal and C terminal fragment of ApxIIA gene was cloned, expressed and purified, respectively. In the MTT assay for the toxicity analysis of these recombinant proteins to macrophage, revealed that ApxIIA-N has lowest cytotoxic activity. The viability of 100 μg/ml ApxIIA-N treatment is still about 45 %. In the ApxIIA-F or ApxIIA-C treatment, all cells could not survive in the concentration of 50 μg/ml. The toxicity is 10-fold less than E. coli heat labile toxin B subunit (LTB) and 200-fold less than LPS of A. pleuropneumoniae. These recombinant proteins have the superiority to develop as adjuvant in safety concerned issue. According to the cytokine expression profile induced by these recombinant proteins, the data indicated that ApxIIA-N could induce Th1 pathway, and both Th1 and Th2 pathway could be induced by ApxIIA-F and ApxIIA-C. Using ovalbumin as antigen and combined with these recombinant proteins to evaluate their adjuvant effect in mice. The antibody titers specific to ovalbumin of ApxIIA adjuvant-containing groups could reach 51200-102400, which are 8- to 16-fold than adjuvant-free group. Analysis the expression of IgG1 and IgG2a subclass revealed that all ApxIIA adjuvant-containing groups could significantly enhance IgG1 and limited IgG2a titer. It indicates that the major induced pathway was Th2. The analysis of IgA titer in bronchoalveolar lavages fluid (BALF) of all ApxIIA adjuvant-containing groups showed that was 10-fold than adjuvant-free group. The body weight of mice was measured to evaluate the influence of adjuvant administered. After third immunization, the mice body weight of ApxIIA-F and ApxIIA-C groups did not increase normally as adjuvant-free group. It indicates that ApxIIA-F and ApxIIA-C may have higher cytotoxic activity as previously in vitro results shown. In summary, ApxIIA protein could enhance the antibody titer and has the potential for the development of mucosal adjuvant. However, ApxIIA-F and ApxIIA-C have higher cytotoxic activity. Therefore, ApxIIA-N is thebest candidate for the development of novel mucosal adjuvant.

Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia. Transmission of the disease occurs through direct contact or aerosols. The bacteria possess many virulence factors, namely capsular polysaccharides, lipopolysaccharides, four Apx toxins and iron acquisition mechanisms. To this day, an efficient cross-serotype vaccine has yet to be developed. In order to investigate regulation mechanisms in A. pleuropneumoniae and to identify new potential targets for the synthesis of subunit vaccines, the transcriptomic profile of the bacteria under conditions that simulate the infection and following a natural acute infection in vivo were studied. The experiences relied on first and second generation microarrays (AppChip1 and AppChip2) designed using 2025 ORFs of the draft version of the A. pleuropneumoniae serotype 5b strain L20 genome and 2033 ORFs of the final and annotated version of the same genome respectively. First, experiments were conducted under iron-restricted conditions and 210 genes were deemed differentially expressed, 92 of which were up-regulated. Some new putative iron acquisition mechanisms were identified, including genes homologous to those of the Yersinia pestis YfeABCD chelated-iron acquisition system, as well as other genes homologous to components of the HmbR iron uptake from hemoglobin system of Neisseria meningitidis. When cultured in conditions promoting biofilm production, genes tadC and tadD from a putative tad (« tight adherence locus ») operon, genes pgaABC involved in the biosynthesis of a polysaccharide of the biofilm matrix as well as two ORFs encoding a putative autotransporter adhesins similar to the Haemophilus influenzae Hsf adhesin were all significantly overexpressed. Many of these genes were also overexpressed when lung epithelial cells were infected with A. pleuropneumoniae. While 170 genes were differentially expressed after planktonic growth in the culture medium above the cells, another 131 were identified following direct adhesion to the cells. Genes tadB and rcpA of the tad locus, as well as genes pgaBC and an ORF coding for the Hsf homolog where all found among overexpressed genes. When A. pleuropneumoniae was cultured in contact with broncho-alveolar lavage fluids (BALF), 156 genes were significantly differentially expressed and gene apxIVA, which was up-regulated, was detected for the first time during in in vitro growth conditions. Finally, experiments were conducted in vivo in animals naturally infected with A. pleuropneumoniae in the late stage of the acute phase in order to identify genes that are expressed during the infection of the natural host. While 150 genes were deemed differentially expressed, genes apxIVA as well as two genes from an operon coding for a putative type IV fimbriae were up-regulated. Out of those 72 genes that were overexpressed, 3 encode proteins or lipoproteins of the outer membrane which are conserved among all serotypes of the bacteria. Overall, we were able to identify several new potential virulence factors for A. pleuropneumoniae in the course of our experiments, as well as several new potential targets for the elaboration of an efficient cross-serotype vaccine.

碩士
國立宜蘭大學
生物技術與動物科學系生物技術碩士班
106
Porcine pleuropneumonia caused by A. pleuropneumoniae (APP) infection is an infective and fatal respiratory disease that leads to severe loss of economy in the pig farming industry worldwide. To develop an effective vaccine is important. Vaccines against APP, including whole-cell bacterin vaccines, toxoid vaccines and subunit vaccines are developed for swine. The advantages of subunit vaccines are being able to retain only antigenic fragments and to remove the toxic fragments thus increase the efficacy and safety of the vaccines. Apx, outer membrane protein (OMP), fimbrial protein, transferrin-binding protein (Tbp), capsular, and lipopolysaccharide have been demonstrated to be candidates of the antigens for APP subunit vaccine. Type IV fimbrial of APP encodes a 15.9 kDa ApfA protein that plays an important role in adherence, colonization and growth. ApfA is expressed in many APP serotypes and is highly conserved among them, thus makes ApfA a potential antigen candidate. In this study, we predicted the fragments of ApfA epitopes by bioinformatics software and then further expressed by gene recombination that rApfA was added as a bioadjuvant to the subunit vaccine or fused with the rOMP into a novel subunit protein rOMP-rApfA and assessed whether it can promote the protection of subunit vaccines. In addition to ApfA, Apx, rOMP and flagellin were also indicated as good subunit vaccine components. In the present study, we have successfully cloned and highly expressed the recombinant proteins of ApfA, OMP and flagellin, which are further formulated as a recombinant subunit vaccine. The subunit vaccine was evaluated according to the Taiwan inspection of APP vaccine standards for its safety and protection effects. The vaccinated mice showed normal weight gain and survived to the end of the experiments. No adverse reactions were observed. The efficacy study showed that mice vaccinated with subunit vaccine had a higher survival rate than control group after APP whole bacteria challenge. Using rApfA or recombinant flagellin as adjuvants cannot further improve the protection effect of subunit vaccine.The antibodies against OMP and Apx were detected in serum from vaccinated mice by enzyme-linked immunosorbent assay (ELISA). The subunit vaccine can induce humoral immunity.

碩士
國立屏東科技大學
動物疫苗科技研究所
101
Actinobacillus pleuropneumoniae（App）is an acute and fatal pathogen that causes swine pleuropneumonia. Diseased pigs show hemorrhagic, necrotizing, fibrinous pleuropneumonia and acute septicemia. Previous study App-toxins are the exotoxins and important virulence factors with immunogenicity, is more efficient than other to induce immune response. Pasteurella multocida （Pm）is a common swine bacterial pathogen. The infected pigs reveal respiratory track pathology and hemorrhagic pneumonia. Salmonella choleraesuis （Sc） infected pigs show septicemia, enterocolitis pneumonia and polyserositis. The extensive population, environment, stress, the potential bacterium and virus factor make the disease more serious. They often cause greatly the serious economic losses. In our preliminary results to identify App serotype 1 and Apx I by utilizing multiplex PCR, then measured the growth curves and optimal time point purified toxin protein of Apx I in supernatant by ammonium sulfate and determined the size of 104 kDa by SDS-PAGE. In addition to, obtain Actinobacillus pleuropneumoniae, Pasteurella multocida and Salmonella choleraesuis peak period of growth. This vaccine is researched and developed under this foundation for screening bacterial strain and determine the protein concentration. The whole bacterial cell mix extracellular products with commercialized oil adjuvant and test by national regulation. The characteristic, Purity, Sterility and Safety test of this vaccine were all fit the nation regulation. The evalution of protective efficacy of prepared vaccine were done by mice protection test. The App protective index was 123, Pm survival rate was 80 % and Sc survival rate was 80 % and this vaccine was proved to be effective.

碩士
國立中興大學
獸醫病理生物學研究所
104
Vaccination is the most effective strategy to prevent the damage of Actinobacillus pleuropneumoniae (AP) infection, which induces fibrinohemorrhagic and necrotic pleuropneumonia with high mortality in growing to fattening pigs. However, 15 different serotypes of AP, based on various capsular polysaccharides, occur worldwide with varying incidence and severity. Therefore, variability of cross protection against heterologous AP serotypes inoculation is crucial for evaluation of vaccine efficacy in pigs. The objective of this study was to evaluate the cross protective efficacy of AP subunit vaccine by an intratracheal challenge model in pigs with heterologous AP serotypes. Challenge dose of serotype F (APF), G (APG), and H (APH), which are most prevalent in Taiwan pig farms, were determined in pigs before efficacy evaluation. In cross protection trials, six week-old pigs were vaccinated in a prime-boost regime three weeks apart either with AP subunit vaccine or placebo and were intratracheal challenged with APF, APG or APH respectively. After one-week observation, the clinical signs, rectal temperature, survival rate, lung lesion score were recapitulated to evaluate vaccine efficacy. In addition, the quantification of AP specific gene apxIVA were utilized to evaluate the residing AP in pulmonary tissue after necropsy. Results illustrated that AP subunit vaccine can minimize the lung lesions and reduce fever frequency with no mortality were noticed after heterologous AP inoculation. Moreover, significant decreasing of reisolating rate and less distribution of AP-conserved gene detecting from the contaminated lungs lesions were distinguished using quantitative real time PCR in vaccinated group. All results revealed that the AP subunit vaccine could provoke good protective immunity against heterologous AP infection. Further studies and animal trials are necessary to elucidate vaccine application in commercial pig farms and its protection for other heterologous AP infection.

碩士
國立高雄大學
生物科技研究所
96
Actinobacillus pleuropneumoniae, a Gram-negative bacterium that all of the 15 serotypes are known can cause infection and lead to lung lesion in pigs with high mortality. Several virulence factors are known including capsule, lipopolsaccharide, exotoxins (Apx toxins), fimbriae, and various outer membrane proteins. The Apx toxins are considered the most important virulence factors. ApxI has strongly hemolytic and cytolytic activity, and ApxII has weakly hemolytic and cytotoxic activity. However, ApxIII is not hemolytic but strongly cytotoxic to porcine neutrophils and pulmonary alveolar macrophages, and is secreted by serotypes 2, 3, 4, 6, and 8. There are 32 amino acids differences in the C terminus of ApxIII of serotype 2 comparing with other serotypes and they should influence the immunogenicity of ApxIII. Fimbriae are usually with highly antigenicity and mediated the attachment ability of bacteria to the host cell. The structural protein gene of A. pleuropneumoniae fimbria, apfA, was found with highly conserved in all serotypes. In the previous study of our laboratory showed that a tetravalent DNA vaccine containing ApxIA, ApxIIA and two outer membrane proteins can provide 70% protection against serotype 1 challenge. To extend the DNA vaccine against different serotypes infection, ApfA and ApxIIIA of serotypes 2 and 8 were chosen as the target antigens for DNA vaccine candidates. These genes were cloned into a mammalian cell expression vector and confirmed by DNA sequencing. The expression of these proteins was first verified in prokaryote by Western blot analysis and further confirmed their expression in NIH/3T3 cells by immunofluorescence assay. The ApfA and ApxIIIA DNA vaccine were injected intramuscularly into mice using monovalent or bivalent formulation. A commercial inactivated A. pleuropneumoniae vaccine was used as positive control as well as the empty vector and PBS were used as negative control. The survival rate of each group after challenged with A. pleuropneumoniae serotype 2 was as following: 30% for ApfA vaccine; 50% for commercial vaccine and ApxIIIA of serotype 2 vaccine; 60% for ApfA plus ApxIIIA of serotype 2 vaccine; 70% for ApfA plus ApxIIIA of serotype 8 vaccine; 0% for empty vector and PBS groups. These results showed that the bivalent vaccine provided better protective efficacy than monovalent vaccine or commercial inactivated vaccine. A tetravalent DNA vaccine containing ApxIIIA of serotype 8, ApfA, ApxIA and ApxIIA were used to evaluate the protective efficacy for different serotypes. The survival rate was 70% for serotype 2 and 30% for serotype 1 challenged, respectively. These results indicate that the protective efficacy of tetravalent vaccine against A. pleuropneumoniae serotype 2 is more significant than serotype 1. Taken together, the present study suggests that these DNA vaccines are promising candidates for prevention different serotypes infection and it is necessary to search the best antigens combination or through the adjuvant administration to improve the protective efficacy. Keywords: Actinobacillus pleuropneumoniae, ApfA, ApxIIIA, DNA vaccine

碩士
國立高雄大學
生物科技研究所
95
Actinobacillus pleuropneumoniae is the etiological agent that causes haemorrhagic, fibrinous and necrotic pleuropneumonia in pigs and makes critical economic losses in global pig farms. Cytokines play crucial roles in the regulation of immune responses, and different cytokines can stimulate the immune responses toward to Th1 or Th2 pathway. Interleukin-6 (IL-6) could be produced by variety of cells including monocytes, macrophages, fibroblasts, T cells and B cells. Using IL-6 as adjuvant can promote immune responses toward to Th2 pathway and enhance B cell to produce antibodies. CpG ODNs is a specific DNA sequence contains an unmethylated CpG motif. It plays a critical role in immune activity promotion and stimulating the differentiation of dendritic cells and inducing B cells, monocytes, natural killer cells to secrete cytokines. To improve the efficacy of DNA vaccine, swine IL-6 and various CpG ODNs containing different copy numbers of CpG motif (ATCGAT) which is suitable for swine were designed as the adjuvants for ApxIA DNA vaccine. The adjuvant effects of above designs were evaluated through a series of animal immunization assay. The swine IL-6 gene was amplified by RT-PCR from swine fibroblast cell line (ST) and cloned into eukaryotic expression vector to construct pcDNA-IL-6. The gene sequence was confirmed by DNA sequencing analysis. The pcDNA-IL-6 was transfected into swine kidney cell line (LLC-PK1) and detected the protein expression by Western blot analysis. The results showed that the cloned swine IL-6 can be expressed in eukaryotic cells. The IL-6 bioactivity was measured by MTT assay; the stimulation index (SI) of NS-1 cell line was 2.23 and 2.57 for 100 pg/ml cloned IL-6 and commercial IL-6, respectively. It indicated that both of IL-6 could stimulate NS-1 proliferation and revealed that the cloned IL-6 has bioactivity. To explore the antibody responses of DNA vaccine combine with adjuvant, mice were immunized by intramuscular injection. Comparison the adjuvant effect of 10 μg with 50 μg IL-6, the results showed that 50 μg IL-6 induced higher antibody titer. The results of different CpG ODNs adjuvant effect showed that CpG1 and CpG4 elicit the highest antibody titer. In IgG subtypes analysis indicated that IgG2a is the major subtype of the test groups. The IL-6 could induce higher IgG2a than IgG1, however, both of IgG2a and IgG1 were enhanced when administrated with CpG1 and CpG4. It indicated that Th1 and Th2 cells could be activated by CpG1 and CpG4 and promoting cellular and humoral immunity.