Presence of <i>Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae</i>in upper respiratory tract of swine in farms from Aguascalientes, Mexico

Respiratory diseases are one of the most important health problems in pig herds. The porcine respiratory disease complex (PRDC) is the term used to describe pneumonic diseases caused by multiple infectious agents that provoke weight loss in animals or death. In the PRDC multiple pathogens (bacteria and/or viruses) work in combination to induce this respiratory disease. Within this complex, Actinobacillus pleuropneumoniae, Streptococcus suis, Pasteurella multocida, Bordetella bronchiseptica, Haemophilus parasuis and Mycoplasma hyopneumoniae are the main bacterial pathogens involved in great economic losses to the swine industry. The aim of this work was to estimate the presence of A. pleuropneumoniae, S. suis, P. multocida, B. bronchiseptica, H. parasuis and M. hyopneumoniae in the upper respiratory tract of pigs in representative swine farms inAguascalientes,Mexico, using PCR technique. The study was performed in 14 swine farms. We obtained a total of 212 nasal swabs. Near 20% of samples were positive for A. pleuropneumoniae (located in the 79% of farms);17% were positive for S. suis (in 86% of farms), of these, 3% were S. suis serovar 2;30% were positive for H. parasuis (93% of farms);23% of the samples to P. multocida (in 79% of farms);and 19% to M. hyopneumoniae (in 64% of farms). B. bronchiseptica was not detected in this study. The results obtained show that bacterial pathogens of PRDC were present in the upper respiratory tract of pigs in all farms studied;therefore, these pathogens are widely disseminated in pig farms of Aguascalientes, Mexico.

Establishment of an Indirect ELISA with the Major Epitope Domain of ApxⅡ of Actinobacillus pleuropneumoniae Expressed in Prokaryotic Cells

[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.

Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.

Cloning, Expression of apxl Gene of Actinobacillus pleuropneumoniae and Development of ELISA

Based on the published nucleotide sequence of the apxICA of Actinobacillus pleuropneumoniae in Genbank(S4074), a pair of primers were designed. A 3 640 bp(4 687 - 8 326 bp)gene fragment was amplified by PCR from the isolated strain of A. pleuropneumoniae serovar 1. Then, it was cloned into pMD18-T, identified by both restriction endonuclease and sequence analysis, and inserted into pET-28a expression vector to yield the expression plasmid. SDS-PAGE result indicated expression of apxICA in BL21 (DE3), Western blot analysis showed the protein's immunogenicity. Using the expressed protein, ELISA was established to detect serum antibody against ApxI. The feature of ELISA to detect highly virulent A. pleuropneumoniae strains infection was proved by primary clinical application.

ArcA Gene Expression Level of Actinobacillus pleuropneumoniae in Anaerobic Culture

In the present study,mRNA levels of the ArcA gene of Actinobacillus pleuropneumoniae serotype1 were measured during response to stress by growing under anaerobic conditions.Expression levels were measured by semi-quantitative RT-PCR using the housekeeping gene recF as an internal standard.The expression of ArcA was undetectable until about 3 hours under standard culture conditions,but it was promptly and highly expressed in anaerobic culture.The results are consistent with ArcA being a potential virulence gene of Actinobacillus pleuropneumoniae,and likely playing an important role in pathogenesis caused by this organism.

Study on Formation and Inhibition Mechanism of Biofilm of Pig Actinobacillus pleuropneumoniae

Pig Actinobacillus pleuropneumoniae(App) could induce chronic respiratory tract infection in pigs, which causes major economic losses on pig industry. Bacterial biofilm(BBF) is bacterial community adsorbed on the surface of biomaterials or body cavity, to protect bacteria escape, and recurrent outbreaks of related infectious diseases and chronic infections resulting therefrom are called bacterial biofilm diseases. App BF belongs to polymers with spatial structure in vitro, and its formation is regulated by multiple genes. Among them, gene deletion of the key component TolC of multidrug efflux pumps and type I secretion systems causes that App BF adhesion weakens; gene deletion of catalytic core ClpP of Clp proteolytic complex induces the inhibition of BF formation; outer membrane lipoprotein VacJ of App promotes BF formation; gene deletion of active enzyme LuxS enhances the formation of App BF and decreases bacterial adhesion ability; gene deletion of Adh obviously declines bacterial accumulation, BF formation and adhesion to host cells. In this paper, BF formation or inhibition mechanism in App is elaborated from molecular level, which could provide reference basis for exploring the prevention of its biofilm diseases.

Identification and Detection of Actinobacillus pleuropneumoniae in Infected and Subclinically Infected Pigs by Multiplex PCR Based on the Genes ApxIVA and OmlA

PCRs based on different genes of Actinobacillus pleuropneumoniae have been developed for detecting and identifying A. pleuropneumoniae. Some of them could amplify positive fragments from the phylogenetically closely related species bacteria. To improve veracity and specificity of PCR, a species-specific multiplex PCR assay was developed to identify and detect A. pleuropneumoniae, based on the 3＇-terminus of the species-specific apxlVA gene and the already existing species-specific primers in the omlA gene. Both 346-bp and 950-bp fragments could be simultaneously amplified from all A. pleuropneumoniae reference strains and isolates, and the species specificity of the assay was evaluated with a collection of ten strains representing eight different species bacteria including species normally found in the respiratory tracts of swine. All of these strains turned out negative in the multiplex PCR. All sequences of products of multiplex PCR randomly sampled were also correct. The sensitivity of the multiplex PCR was determined to be 10 pg of A. pleuropneumoniae DNA. The multiplex PCR and bacterial isolation were compared to determine their sensitivities by using experimentally infected pigs and clinical disease pigs. The multiplex PCR was more sensitive than bacterial isolation. The multiplex PCR was also evaluated on mixed bacterial cultures from clinical healthy pigs. 26/100 （26%） of the subclinically infected pigs were detected from clinical healthy pigs. The results indicate that the multiplex PCR assay is a sensitive, highly specific, and effective diagnostic tool for identification and detection of A. pleuropneumoniae.

Development of Multiplex-PCR for Identification of Pasteurella multocida,Haemophilus parasuis and Actinbacillus pleuropneumoniae

[ Objective] To develop multiplex-PCR for diagnosis of mixed infection caused by Pasteurella multocida （ PM）, Haomophilus parasuis （HPS） and Actinbaci/lus pleuropneumoniae （App）. [ Method ] PCR method was developed to detect single infection caused by PM, HPS or App. The conditions of amplification and primers were optimized, and the multiple-PCR was developed to detect mixed infection of PM, HPS and App. [ Result] A 457-bp band, a 821-bp band and a 342-bp band were simultaneously amplified in the one PCR reaction system. The method had high sensitivity and specificity. [ Conduslon] The multiple-PCR is successfully developed and can be used for differential diagnosis of PM, HPS and App.

Cloning and Expression of Actinobacillus pleuropneumoniae Gene Coding for TbpA and Development of an Indirect TbpA-ELISA

This study presents the cloning and expression of gene encoding transferrin-binding protein A from Actinobacillus pleuropneurnoniae in Escherichia coli expression system and the development of an indirect TbpA-ELISA. The gene coding TbpA was amplified from the A. pleuropneumoniae serotype 2 genome using polymerase chain reaction and cloned to pET-28b expression vector under the control of strong, inducible T7 promoter. The recombinant plasmid was expressed in E. coli BL21 （DE3）. The expressed fusion protein was analyzed using SDS-PAGE and Western blotting. The diagnostic potential of recombinant TbpA （rTbpA） was evaluated through an antibody-detection indirect ELISA based on the purified rTbpA. The TbpA antibodies were detectable in mice on day 7 after vaccination with purified rTbpA protein or infection with A. pleuropneumoniae serotype 10 with the TbpA-based ELISA. In addition, the TbpA-ELISA was able to detect 12 serotyping rabbit antisera postinoculation （PI） with A. pleuropneumoniae 12 serotypes experimentally. The comparable result was obtained by detecting the 117 clinical serum samples using, respectively, the TbpA-ELISA and indirect hemagglutination test （IHA） based on multiplex antigen. The result indicates that the TbpA-ELISA was the more sensitive method compared with the Mix-IHA method because of its consistent presence in A. pleuropneumoniae serotypes. In conclusion, the conserved TbpA of A. pleuropneumoniae can be used for the development of a cross-serotype diagnostic method for the detection of antibodies against A. pleuropneurnoniae.

Genetic Evolution Analysis of Actinobacillus pleuropneumoniae

In order to explore the genetic evolution of Actinobacillus pleuropneumoniae(App) in different countries and clarify the relationships among different App in each region, the 16 S rRNA gene of App in the NCBI nucleotide database was analyzed and compared by the bioinformatics method. The phylogenetic tree was constructed after tailoring alignment. The results showed that a stable genetic phenomenon was indicated in the evolutionary process of App. The isolates derived from China were clustered and showed a high degree of conservation. They had a certain genetic relationship with the British and American strains, but had far relationship with the strains from Japan which was a neighboring country of China. The isolates from different countries in the Eurasian continent shared high homology. The isolates of the two regions originated from common ancestors.

Epidemiological Investigation of Actinobacillus Pleuropneumoniae in Western Shandong

[ Objective ] The aim of this study was to investigate the epidemic status of porcine pleuropneumonia in western Shandong and establish the PCR method of actinobacillus pleuropneumoniae （APP）. [ Method] The epidemic status of APP in lesion tissues of 186 death pigs and 545 health pigs without clinical symptoms was analyzed by PCR method. [ Result] APP positive rate in 186 samples accounted for 43.0% （80/186）, while that in 545 porcine serums accounted for 9.4% （51/545）. [ Conclusion] This PCR method can be used as one of the important means for APP clinical diagnosis.

Effect Found in the Combination of Kitasamycin with Other Antibiotics Used against <i>Actinobacillus pleuropneumoniae</i>

Background: Combinations of antibiotics are used to increase the therapeutic options via the simultaneous activities of two compounds. The goals of combination therapy are to resolve the bacterial infection while reducing the treatment cost. The use of kitasamycin is used frequently and in addition to other antibiotics, the desired combinations were not found. Methods: Fields strains: we use serotypes 1, 3, 5 y 7. Microbial culturing: The serotypes 1, 3, 5 y 7 was identified. Antibiotics and Reagents: The antibiotics used were kitasamycin;enrofloxacin and oxolinic acid. The following combinations were tested: a) kitasamycin with enrofloxacin;b) kitasamycin with norfloxacin;and c) kitasamycin with oxolinic acid. Preparation of bacterial suspensions: The cultures were adjusted to a concentration of 5 colony-forming units per ml (CFU/ml) using the same sterile culture medium as the diluent. Determination of the fractional inhibitory concentrations (FICs): To test each combination and their interactions and to calculate the fractional inhibitory concentration (FIC) of each antibiotic. Calculation of the index of the fractional minimum inhibitory concentration (FMIC): The FMIC index was calculated by applying the following formula: FIC index = FIC of antibiotic A/FIC of antibiotic B. Results: What was found showed that: A combination of kitasamycin and norfloxacin has been shown to have an indifferent effect. A combination of kitasamycin and enrofloxacin had antagonistic effects against all of the Actinobacillus pleuropneumoniae serotypes studied. Finally, a combination of kitasamycin and oxolinic acid had antagonist effects against serotypes 1, 3 and 5 and a synergistic effect against serotype 7. Conclusion: The “in vitro” combination Kitasamycin-Norfloxacin showed an indifferent effect, and combination Kitasamycin and Enrofloxacin demonstrated an antagonistic interaction between these two antibiotics. A combination of Kitasamycin and Oxolinic Acid had synergistic effect against serotype 7.

The QseB/QseC two-component system contributes to virulence of Actinobacillus pleuropneumoniae by downregulating apf gene cluster transcription

Actinobacillus pleuropneumoniae(APP)is the major pathogen of porcine contagious pleuropneumoniae(PCP).The QseB/QseC two-component system(TCS)consists of the regulator QseB and the kinase QseC,which relates to quorum sensing(QS)and virulence in some bacteria.Here,we investigated the role of QseB/QseC in apf gene cluster(apfABCD)expression of APP.Our results have showed that QseB/QseC TCS can potentially regulate the expression of apf gene cluster.The△qseBC,△apfA,△apfB,△apfC and△apfD strains are more sensitive to acidic and osmotic stressful conditions,and exhibite lower biofilm formation ability than wild-type(WT)strain,whereas the complemented strains show similar phenotype to the wr strain.In additon,the mutants have defective antiphagocytosis,adhesion and invasion when they come into contact with the host cells.In experimental animal models of infection,mice infected with△qseBC,△apfA,△apfB,△apfC and △apfD strains showed lower mortality and bacterial loads in the lung and the blood than those infected with wr strain.In conclusion,our results suggest that QseB/QseC TCS contributes to stress resistance,biofilm formation,phagocytosis,adhesion,invasion and virulence by downregulating expression of apf gene cluster in A.pleuropneumoniae.

The dose regimen formulation of doxycycline hydrochloride and florfenicol injection based on ex vivo pharmacokinetic-pharmacodynamic modeling against the Actinobacillus pleuropneumoniae in pigs

Doxycycline hydrochloride and florfenicol combination(DoxHcl&FF)is an effective treatment for respiratory diseases.In the study,our objective Was to evaluate the activity of DoxHcl&FF against Actinobacillus pleuropneumoniae(APP)in porcine pulmonary epithelial lining fluid(PELF)and the optimal dosage scheme to avoid the development of resistance.The DoxHcl&FF Was administered intramuscularly(IM)at 20mg/kg,and the PELF was collected at differ-ent time points.The minimum inhibitory concentration(MIC)and time-mortality curves were also included in the study.Based on the sigmoid Emax equation and dose equations,the study integrated the in vivo pharmacokinetic data of infected pigs and ex vivo pharmacodynamic data to obtain the area under concentration time curve(AUCo-24h)MIC values in PELF and achieve bacteriostatic activity,bactericidal activity and the virtual eradication of bacteria.The study showed that the combination of DoxHcl and FF caused no significant changes in PK parameters.The peak concentration(Cmax)of FF in healthy and diseased pigs was 8.87±0.08 and 8.67±0.07μg/mL,the_AUCo-24h were.172.75±2.52 and 18022±3.13 h-μg/mL,the Cmax of DoxHcl was 7.91±0.09 and 7.99±0.05μg/mL,and the AUCo-24h was 129.96±3.70 h-μg/mL and 169.82±4.38 h-μg/mL.DoxHcl&FF showed strong concentra-tion-dependent tendencies.The bacteriostatic,bactericidal,and elimination activity were calculated as 5.61,18.83 and 32.68 h,and the doses were 1.37(bacteriostatic),4.59(bactericidal)and 7.99(elimination)mg/kg.These findings indicated that the calculated recommended dose could assist in achieving more precise administration,increasing the effectiveness of DoxHcl&FF treatment for APP infections.

Sero-Prevalence and Risk factors of Contagious Bovine Pleuropneumonia (CBPP) in Afgoye District Lower Shabelle Region, Somalia

Contagious bovine Pleuropneumonia (CBPP) is an infectious and highly contagious respiratory disease of cattle and water buffalo, caused by the Mycoplasma mycoides subspecies mycoides. It induces significant economic losses and leads to a severe livestock production problem, negatively influencing people’s livelihoods of affected countries. In Somalia, there is no updated data on the prevalence and distribution of the disease. Hence, a descriptive cross-sectional study was conducted from September 2022 to June 2023 in different villages under the Afgoye District of lower Shabelle region, Somalia. The main purpose of this study is to assess the sero-prevalence and identify the associated risk factors for the occurrence of the disease. In this study, villages, age, sex, breed, and body condition were considered as risk factors. A total of 90 blood samples were collected and tested in the laboratory using the Anti-CBPP Elisa kit test. Out of 90 serum samples from herd cattle, 32 were positive, resulting in an overall prevalence of 35.5%. In addition, we found a statistically significant variation between the prevalence of the disease and factors such as sex, age, body condition and breeds. In summary, the overall prevalence of Contagious bovine pleuropneumonia in this study area is worth to be considered because there is a low quality of health care and less awareness of the Contagious bovine Pleuropneumonia effects on herds, which warrants the official authorities to act and follow appropriate preventive and control measures to reduce the incidence of the disease and generate appropriate controlling and prevention measures in all regions of Somalia.

An abattoir survey of contagious bovine pleuropneumonia lesions in slaughtered cattle in selected districts in Northern Tanzania

Objective:To establish and estimate incidence of contagious bovine pleuropneumonia(CBPP),using abattoir survey as a diagnostic tool in slaughtered cattle in Northern Tanzania.Methods:A total of 4460 cattle were slaughtered in five abattoirs in 3 northern zone regions(Arusha,Kilimanjaro and Tanga)during the period of January to May 2004.They were examined ante-mortem for‘pneumonia signs',and‘characteristic contagious bovine pleuropneumonia(CBPP)lung lesions'.Results:Forty-one(0.91%)of the slaughtered cattle,the majority of which were Tanzania short horn zebu,had gross lung lesions suggestive of CBPP.The prevalence of lesions was significantly(P<0.05)higher in Karatu abattoir compared to others.No animal was detected to have lesion in Bomang'ombe abattoir.The most observed pneumonic signs included labored breathing(90%),dry cough(57%)and mucopurulent nasal discharge(47%).The gross characteristic CBPP pathological lesion,frequently encountered was left lung lesion(47%),pinkish lung(71%)and pleural adhesion(98%).Epidemiological reports show that the CBPP reported outbreaks increased from 19 in 2002,65 in 2003 and 18 in 2004(January-March).The corresponding number of reported deaths increased from 137 in 2002,269 in 2003 and 77 in 2004(January-March).Conclusions:It's concluded from this study that CBPP is a problem in spite of the extensive awareness and vaccination campaigns.Nevertheless,a continued surveillance programme including routine checks of all cattle carcasses at the abattoir and subsequent epidemiological investigation of suspected cases are recommended.

牛支原体和丝状支原体丝状亚种双重TaqMan荧光定量PCR检测方法的建立与应用

牛传染性胸膜肺炎和牛支原体病均为重要的牛传染病,两者均可致牛出现呼吸系统症状,临床上难以区分。为建立鉴别诊断牛传染性胸膜肺炎和牛支原体病的双重Taq Man荧光定量PCR检测方法,本研究根据这两种病的病原丝状支原体丝状亚种和牛支原体的基因组保守区域(丝状支原体丝状亚种nt826-nt1742,牛支原体nt189-nt618)分别设计引物与探针,通过优化反应体系与反应条件,建立同时检测这两种病原的Taq Man荧光定量PCR检测方法。特异性试验结果显示,该方法仅对丝状支原体丝状亚种模拟样品和牛支原体检测结果为阳性,而巴氏杆菌、牛传染性鼻气管炎病毒、无乳支原体、山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、关节炎支原体Leachii株检测结果均为阴性,特异性较强。分别以1.0×10~7拷贝/μL~1.0×10~1拷贝/μL的p EASY-Mmm和p EASY-Mb质粒标准品为模板,进行敏感性试验,结果显示,该方法对丝状支原体丝状亚种和牛支原体重组质粒标准品的检测限均为1.0×10~1拷贝/μL,敏感性较高。对不同浓度质粒标准品混合物的重复性试验结果显示,组内与组间重复性试验变异系数均小于2.5%,重复性较好。利用该方法对112份临床样品(104份鼻拭子、8份肺组织样品)检测,结果显示,丝状支原体丝状亚种检测结果和已发表PCR方法检测结果一致,均为阴性,而本实验建立的方法检测出4份牛支原体阳性样品,且经测序证实检出样品为真实阳性样品,而已发表的多重PCR方法检测该病原结果均为阴性。本研究建立了能够同时检测丝状支原体丝状亚种和牛支原体的双重Taq Man荧光定量PCR方法,其特异性强、敏感性高、重复性好,可用于各种临床样品的检测,为这两种病原的快速检测和流行病学调查提供了技术手段。

A Serological Survey of Contagious Caprine Pleuropneumonia in Taizhou City,Jiangsu Province

[ Objective] This study aimed to investigate the prevalence of contagious caprine pleuropneumonia in Taizhou City, Jiangsu Province. [ Method] By using indirect hemagglutination assay kits for detection of antibodies against Mycoplasma capricolum subsp, capripneumoniae （ Mecp）, Mycoplasma mycoides subsp. capriclonum （Mmc） and Mycopalsam ovipneumonia （ Movi）, 1 157 goat sera from Taizhou City of Jiangsu Province were detected. [ Result ] The average positive detection rate of Mccp and Mmc in infected goats was 51.85% and 39.81%, respectively; the average positive detection rate of Mccp and Mmc in non-infected goats was 10.52% and 5.84%, respectively, and the total positive detection rate of Mycoplasma infections was 16.37%. [ Conclusion ] Mycoplasma infections oc- cur commonly in Taizhou City. The prevention and control of Mycoplasma infections should be strengthened.

Mycoplasmas and Mycoplasmosis in Sheep and Goats

The known mycoplasmas isolated from goats and sheep were listed. The pathogenicity of these mycoplasmas and their effects during occurrence of mycoplasmosis were summarized.

胸膜肺炎放线杆菌ApxⅠ蛋白单克隆抗体制备和抗原表位初步鉴定

为了制备胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)Ⅰ型ApxⅠ毒素(ApxⅠ)AI2蛋白特异性单克隆抗体,进而建立用于评估亚单位疫苗免疫效果的阻断ELISA方法,以实验室前期筛选得到的ApxⅠ抗原优势决定簇AI2重组蛋白为免疫原,免疫6周龄BALB/c雌鼠。利用常规细胞融合和间接ELISA筛选阳性杂交瘤细胞,获得4株单克隆抗体,命名为2C2、4E4、5E7和6F2,上清液效价分别为1∶6400、1∶6400、1∶12800和1∶6400。ELISA与Western blot结果表明,制备的4株单克隆抗体均与重组AI2蛋白有良好的反应性。其中5E7可与APP天然ApxⅠ毒素蛋白特异性结合,同时4株单克隆抗体均不与多杀性巴氏杆菌、支气管败血波氏菌、猪丹毒丝菌、副猪格拉菌和猪链球菌2型反应。亚类分型结果表明,2C2、4E4、5E7属于IgG1亚类,6F2属IgG2a亚类,4株单抗轻链均为κ链。通过构建AI2重组蛋白截短体,初步确定线性表位在74~93 aa之间。本研究成功制备了4株针对AI2蛋白单克隆抗体,为APP亚单位疫苗免疫效果评估方法的建立奠定了基础。