A not pregnant 4-year-old Jersey cow was presented with the sudden appearance of respiratory noise, nasal discharge and moderate respiratory difficulty. Upon physical examination a snoring-like noise, extended head and neck position, exaggerated abdominal effort, bilateral nasal discharge and left prescapular lymph node enlargement were noted. Sub-occlusion of the initial portion of the respiratory tract was suspected. Radiographic and endoscopic examinations revealed a pedunculate mass on the dorsal aspect of the rhinopharynx, which was removed with endoscopically assisted electrosurgery. Histologic examination revealed a chronic pyogranulomatous inflammation with eosinophilic club-like bodies surrounding small colonies of rod-shaped bacteria. Results of histochemical staining were consistent with Actinobacillus-like bacteria and a diagnosis of respiratory actinobacillosis was reached. Surgery and antibiotic therapy were resolutive, as demonstated by an endoscopic check at the second month after surgery, even without the association of the traditional iodine cure, which is regarded as the treatment of choice for actinobacillosis.
Actinobacillosis/*diagnosis/drug therapy/microbiology/surgery ; Actinobacillus/physiology ; Animals ; Cattle ; Cattle Diseases/*diagnosis/drug therapy/pathology/surgery ; Female ; Respiratory Tract Infections/drug therapy/pathology/surgery/*veterinary ; Treatment Outcome

Apx IV, a forth RTX toxin indentified in Actionbacillus pleuropneumoniae recently, is expressed by all A. pleuropneumoniae regardless the serotypes and inducible only in vivo toxin, so it is the optimal to develop species-specific and differentiated diagnostic assay. Here the 2445bp DNA fragment of apxIVA gene of A. pleuroneumoniae was amplified and fused in-frame to the downstream of the T7 promoter and 6 His Tag of the prokaryotic expression vector pET-28b. The construct was transformed into E. coli BL21(DE3). After induction by 1.0 mol/L IPTG, a recombinant protein about 90 kD in size, designed as ApxIVAN, was detected, which was present as inclusion bodies and reacted specifically with swine antisera to the APP-serotype-1 by dot-blot. An indirect ELISA (ApxIVA-ELISA) was developed using purified recombinant ApxIVAN from the inclusion bodies as described previously, which had excellent specificity to A. pleuroneunoniae. Using the ApxIVA-ELISA, the ApxIV antibodies were not detected in the inactivated APP bacterins vaccinated pigs, but were detected in A. pleuropneumoniae serotype 1, 2 and 7 infected pigs and mice. These results suggested that ApxIVA-ELISA can be used not only to detect all serotypes of APP, but also to differentiate the naturally infected and inactivated vaccine immunized pigs.
Actinobacillus Infections ; diagnosis ; microbiology ; veterinary ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; metabolism ; Bacterial Proteins ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; veterinary ; Gene Expression ; Genes, Bacterial ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.
Actinobacillus Infections/microbiology/*prevention & control ; Actinobacillus pleuropneumoniae ; Animals ; Antigens, Bacterial/immunology ; Bacterial Proteins/*immunology ; Bacterial Vaccines/*immunology ; Cholera Toxin/*chemistry ; Female ; Hemolysin Proteins/*immunology ; Immunization, Secondary ; Mice ; Mice, Inbred ICR ; Plants, Genetically Modified ; Zea mays/*genetics

Actinobacillus pleuropneumoniae (A. pleuropneumoniae), the causative agent of porcine contagious pleuropneumonia (PCP), is a significant pathogen of the world pig industry, vaccination is potentially an effective tool for the prevention of PCP. The purpose of present study was to enhance the immunogenicity of A. pleuropneumoniae live vaccine strain HB04C- (serovar 7), which was unable to express ApxIA, and to develop effective multivalent vaccines for the respiratory pathogens based on the attenuated A. pleuropneumoniae. We introduced a shuttle vector containing intact apxIA gene into HB04C-, generating HB04C2, an A. pleuropneumoniae serovar 7 live attenuated vaccine strain co-expressing ApxIA. Then we investigated the biological characteristics of HB04C2. We found that the shuttle vector expressing ApxIA was stable in HB04C2, and the growth ability of HB04C2 was not affected by the shuttle vector. We observed that HB04C2 elicited detectable antibodies against ApxIA and ApxIIA when it was administrated intratracheally as a live vaccine in pigs, and all immunized pigs were protected from heterologous virulent A. pleuropneumoniae (serovar 1) challenge. In conclusion, we demonstrated that A. pleuropneumoniae live vaccine could be used as a vector for expression of heterologous antigens.
Actinobacillus Infections ; prevention & control ; veterinary ; Actinobacillus pleuropneumoniae ; classification ; immunology ; Animals ; Bacterial Proteins ; biosynthesis ; genetics ; Bacterial Vaccines ; biosynthesis ; immunology ; Hemolysin Proteins ; biosynthesis ; genetics ; Pleuropneumonia ; microbiology ; prevention & control ; Swine ; Swine Diseases ; microbiology ; prevention & control ; Vaccines, Attenuated ; biosynthesis ; immunology

OBJECTIVEThe aim of this study was to investigate effects of immunodeficiency on the periodontal infection characters of the specific pathogens of juvenile periodontitis.

METHODSA total of 36 immunodeficient guinea pigs produced by twice whole-body irradiation with 60Co were divided randomly into four groups, in which Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and A. actinomycetemcomitans with P. gingivalis were inoculated into the gingival sulcus of two mandibular incisors respectively. The pigs in the control group did not receive any inoculation. At 2, 3 and 6 weeks after inoculation, three animals in each group were sacrificed successively. Clinical and histological examinations were used to examine the changes in the periodontal tissues. The other 36 normal guinea pigs were divided into four groups and treated in a similar way described above.

RESULTSSignificant periodontal damages were noted in immunodeficient pigs inoculated with A. actinomycetemcomitans, P. gingivalis or A. actinomycetemcomitans and P. gingivalis in 2 and 3 weeks after bacterial inoculation. The damages were more severe than in the normal groups. The immunodeficient groups demonstrated larger numbers of osteoclasts than the normal groups (P < 0.05).

CONCLUSIONThe loss of periodontal tissue in immunodeficient hosts is much serious than those with normal defence system, after they are infected with A. actinomycetemcomitans and P. gingivalis. Abnormal defence system in hosts may play an important role in onset and development of juvenile periodontitis.

Actinobacillus Infections ; immunology ; Aggregatibacter actinomycetemcomitans ; Aggressive Periodontitis ; immunology ; microbiology ; Animals ; Bacteroidaceae Infections ; immunology ; Female ; Immunocompromised Host ; immunology ; Male ; Porphyromonas gingivalis ; Random Allocation

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.
Actinobacillus Infections/prevention & control ; Actinobacillus pleuropneumoniae/genetics/*immunology ; Animals ; Antibodies, Bacterial/blood ; Bacterial Proteins/analysis/*immunology ; Cytokines/analysis/blood ; Disease Models, Animal ; Female ; Hemolysin Proteins/analysis/*immunology ; Immunoglobulin A/blood/immunology ; Intestines/immunology ; Lung/cytology/immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/*immunology ; Saccharomyces cerevisiae/*genetics/immunology ; Survival Analysis ; Time Factors ; Vaccination ; Vaccines, Synthetic/administration & dosage/*immunology

Acute toxicity and immunoprotection of Actinobacillus pleuropneumoniae (APP) ApxI toxin recombinant proteins (include crude inclusion bodies and refolded recombinant protein) were evaluated in mice, and compared with the natural ApxI extracted from culture supernatant of APP serotype 10. In the acute toxicity experiment, the three proteins were intraperitoneally injected into Kunming mice in a dose of 200microg per mouse. The body and organ weight, heamatological and biochemical indexes were examined at 24h, 7 days and 14 days post administration. There was no death after the intraperitoneal administration of the three proteins, and no significant change was found in the body weight, organ indexes, heamatological and biochemical indexes. To study their immunoprotection, the three proteins were emulsified with Freund's adjuvant respectively and vaccinated the mice twice with a 2-week of interval. Two weeks after the second vaccination, the mice were challenged intraperitoneally with a lethal dose of APP serotype 10 (1.09 x 10(8) cfu), and serums were examined by an ApxI-specific ELISA. The results revealed that the recombinant protein had a good immunogenicity and could induce protection immune reaction.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; metabolism ; Animals ; Bacterial Proteins ; genetics ; immunology ; Bacterial Vaccines ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Hemolysin Proteins ; genetics ; immunology ; Immunization ; Male ; Mice ; Random Allocation ; Recombinant Proteins ; genetics ; immunology ; Toxicity Tests, Acute

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; Animals ; Antibodies ; blood ; Bacterial Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Cytotoxins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hemolysin Proteins ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

OBJECTIVETo establish a 16S rDNA multiplex polymerase chain reaction (PCR) for simultaneously detecting P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens of chronic periodontitis and to study the correlation between different modes of infection and severity of the disease.

METHODSPeriodontal pocket specimens from 152 patients with mild, moderate or advanced chronic periodontitis and gingival sulcus specimens from 30 periodontally healthy individuals were collected and placed in 200 microl lysis buffer. The specimens were incubated in 100 degrees C for 10 min and 10 microl of the supernatant was directly used as PCR template. DNAs from P. gingivalis strain ATCC33277, A. actinomycetemcomitans strain Y4, T. denticola strain FM and E. coli strain DH5alpha were used as positive and negative controls in PCR with all of which were prepared by routing phenol-chloroform method. A multiplex PCR assay, using three sets of primers specific to 16S rDNA genes of the three anaerobes, was developed to detect the specimens. The target amplification fragments from 3 cases of PCR products positive for all the three anaerobes were sequenced after T-A cloning. Chi-square test was applied to analyze the correlation between different coinfection of the three anaerobes and severity of the disease.

RESULTSThe established 16S rDNA multiplex PCR assay was able to detect P. gingivalis, A. actinomycetemcomitans and T. denticola at a minimum of 10, 20 and 20 cells, respectively. In comparison with the reported corresponding sequences, similarities of the nucleotide sequences from the three anaerobes 16S rDNA amplification fragments were as high as 99.45%, 97.08% and 96.59%, respectively. Of the 30 periodontally healthy gingival sulcus specimens, only one (3.3%) positive for P. gingivalis and two (6.7%) for A. actinomycetemcomitans could be identified but all of the rest were negative. In the 152 CP periodontal pocket specimens, 147 cases (96.7%) were positive for P. gingivalis, A. actinomycetemcomitans and/or T. denticola, respectively, and 5 cases (3.3%) were negative for all the three anaerobes. The positive rate of P. gingivalis detection (91.5%, 139/152) was significantly higher than those of A. actinomycetemcomitans (72.4%, 110/152) and T. denticola (80.9%, 123/152) (chi(2) = 7.07, 18.67; P < 0.01). 89.8% of the specimens from patients showed coinfections with two (26.5%) or three anaerobes (63.3%), and the coinfection rates in the specimens from moderate and advanced CP were remarkably higher than that from mild CP (chi(2) = 10.43, P < 0.01).

CONCLUSIONThe 16S rDNA multiplex PCR established in this study showed high sensitivity and specificity, which could be used to detect P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens. CP was an disease caused by multiple pathogenic microbes while the synergistic pathopoiesis of the three microbes was closely related to the severity of the disease.

Actinobacillus Infections ; epidemiology ; microbiology ; Adult ; Aged ; Aggregatibacter actinomycetemcomitans ; isolation & purification ; Bacteroidaceae Infections ; epidemiology ; microbiology ; China ; epidemiology ; Chronic Disease ; Female ; Humans ; Male ; Middle Aged ; Periodontitis ; microbiology ; Polymerase Chain Reaction ; methods ; Porphyromonas gingivalis ; isolation & purification ; RNA, Ribosomal, 16S ; Treponema denticola ; isolation & purification ; Treponemal Infections ; microbiology

OBJECTIVEThe aim of this study was to investigate subgingival infection frequencies of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans strains with genetic variation in Chinese chronic periodontitis (CP) patients and to evaluate its correlation with clinical parameters.

METHODSTwo multiplex polymerase chain reaction (PCR) assays were developed to detect the 16SrDNA, collagenase (prtC) and fimbria (fimA) genes of P. gingivalis and the 16SrDNA, leukotoxin (lktA) and fimbria-associated protein (fap) genes of A. actinomycetemcomitans in 60 sulcus samples from 30 periodontal healthy subjects and in 122 subgingival plaque samples from 61 patients with CP. The PCR products were further T-A cloned and sent for nucleotide sequence analysis.

RESULTSThe 16SrDNA, prtC and fimA genes of P. gingivalis were detected in 92.6%, 85.2% and 80.3% of the subgingival plaque samples respectively, while the 16SrDNA, lktA and fap genes of A. actinomycetemcomitans were in 84.4%, 75.4% and 50.0% respectively. Nucleotide sequence analysis showed 98.62%~100% homology of the PCR products in these genes with the reported sequences. P. gingivalis strains with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ were predominant in deep pockets (>6 mm) or in sites with attachment loss > or =5 mm than in shallow pockets (3~4 mm) or in sites with attachment loss < or =2 mm (P<0.05). P. gingivalis strains with prtC+/fimA+ also showed higher frequency in gingival index (GI)=3 than in GI=1 group (P<0.05).

CONCLUSIONInfection of P. gingivalis with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ correlates with periodontal destruction of CP in Chinese. Nonetheless P. gingivalis fimA, prtC genes and A. actinomycetemcomitans lktA gene are closely associated with periodontal destruction, while A. actinomycetemcomitans fap gene is not.

Actinobacillus Infections ; epidemiology ; microbiology ; Adult ; Aged ; Aggregatibacter actinomycetemcomitans ; classification ; genetics ; isolation & purification ; Bacteroidaceae Infections ; epidemiology ; microbiology ; China ; epidemiology ; Chronic Disease ; Dental Plaque ; epidemiology ; microbiology ; Female ; Gingivitis ; epidemiology ; microbiology ; Humans ; Male ; Middle Aged ; Periodontitis ; epidemiology ; microbiology ; Porphyromonas gingivalis ; classification ; genetics ; isolation & purification ; Prevalence ; Risk Assessment ; methods ; Risk Factors ; Species Specificity ; Statistics as Topic