Putative biomarkers for evaluating antibiotic treatment: an experimental model of porcine Actinobacillus pleuropneumoniae infection

Biomarkers of infection were screened for their possible role as evaluators of antibiotic treatment in an aerosol infection model of porcine pneumonia caused by Actinobacillus pleuropneumoniae (Ap). Following infection of 12 pigs, clinical signs of pneumonia developed within 20 h, whereafter the animals received a single dose of either danofloxacin (2.5mg/kg) or tiamulin (10 mg/kg). To test the discriminative properties of the biomarkers, the dosage regimens were designed with an expected difference in therapeutic efficacy in favour of danofloxacin. Accordingly, the danofloxacin-treated pigs recovered clinically within 24h after treatment, whereas tiamulin-treated animals remained clinically ill until the end of the study, 48 h after treatment. A similar picture was seen for the biomarkers of infection. During the infection period, plasma C-reactive protein (CRP), interleukin-6 and haptoglobin increased, whereas plasma zinc, ascorbic acid and alpha-tocopherol decreased. In the danofloxacin-treated animals, CRP, interleukin-6, zinc, ascorbic acid and alpha-tocopherol reverted significantly towards normalisation within 24h of treatment. In contrast, signs of normalisation were absent (CRP, zinc and ascorbic acid) or less marked (interleukin-6 and alpha-tocopherol) in the tiamulin-treated animals. Plasma haptoglobin remained elevated throughout the study in both groups. This indicates that CRP, zinc, ascorbic acid and to a lesser extent interleukin-6 and alpha-tocopherol might be used to evaluate antibiotic treatment of acute Ap-infection in pigs. The present model provides a valuable tool in the evaluation of antibiotic treatments, offering the advantage of clinical and pathological examinations combined with the use of biochemical infection markers.     

猪胸膜肺炎放线杆菌的分离和鉴定

某猪场哺乳仔猪及断奶猪临床表现喘气、咳嗽、皮肤发绀以及胸膜肺炎的病理特征，从病猪体内分离出3株菌，根据细菌培养特性、CAMP试验及生化反应等鉴定为猪胸膜肺炎放线杆菌（App)。该菌对小白鼠有一定毒力，回归猪可致死猪只，对常用治疗药有一定的抵抗力，用分离菌制备自家苗可预防本病发生。     

Protective efficacy of conjugate vaccines against experimental challenge with porcine Actinobacillus pleuropneumoniae.

In an attempt to protect pigs against swine pleuropneumonia induced by Actinobacillus pleuropneumoniae (SPAP) by neutralizing the effects of three virulence factors of A. pleuropneumoniae--the capsular polysaccharide (CP), the lipopolysaccharide (LPS), and the hemolysin protein (HP)--two subunit conjugate vaccines were prepared by covalently coupling the CP to the HP and the LPS to the HP. The CP, LPS, and HP were isolated from A. pleuropneumoniae, strain 4074, serotype 1, and the protective efficacy of the conjugate vaccines in swine experimentally infected with A. pleuropneumoniae was evaluated. Following a booster vaccination, a significant (P < 0.05) IgG antibody response to the CP, LPS, and HP was detected in the vaccinated pigs. The pigs vaccinated with the CP-HP and LPS-HP conjugates exhibited significantly less mortality (P < 0.05) and significantly greater weight gain (P < 0.001) than unvaccinated pigs. Vaccinated pigs exhibited significantly fewer and less extensive gross pulmonary lesions (P < 0.001) when compared with unvaccinated pigs. Thus, on the basis of mortality, weight gains, and pulmonary lesion formation, the two conjugate vaccines used in conjunction with one another provide noticeable protective efficacy against SPAP.     

胸膜肺炎放线杆菌引起猪应激时急性期蛋白转录水平变化

为研究胸膜肺炎放线杆菌(APP)感染后猪肝组织急性期蛋白表达变化,选取6头健康三元杂交猪,随机分为应激组和对照组(每组3头,保证饲喂条件一致)。每头猪采血后,应激组滴鼻感染APP,对照组滴鼻等量的无菌生理盐水。感染4 h后对两组猪进行采血、剖杀,分别取肝组织冻存,肺组织进行细菌分离鉴定。采用ELISA试验测定血清中皮质醇质量浓度,荧光定量PCR法测定肝组织急性期蛋白CRP、HP、SAA、Apo-A1、ITIH-4的表达量。结果显示:应激组和对照组猪血清中皮质醇质量浓度在攻毒前无明显差异(P0.05);攻毒4 h后,应激组猪血清中皮质醇含量较对照组显著上调(P0.05);攻毒后,应激组猪肝组织SAA mRNA的表达量较对照组显著上调(P0.05),Apo-A1 mRNA的表达量显著下调(P0.01),CRP、HP和ITIH-4 mRNA表达量无显著变化。以上结果说明APP感染后,机体可通过调节SAA和Apo-A1表达量来参与应激调节。     

Effectiveness of doxycycline in the prevention of an experimental infection with Actinobacillus pleuropneumoniae in pigs

The effectiveness of medication with doxycycline in feed in the control of pleuropneumonia in pigs was tested using an Actinobacillus pleuropneumoniae serotype 1 aerosol challenge model. Two groups of 10 animals were used for the challenge, a 'medicated group' and an 'unmedicated group'. A third group of four animals was used as a 'control group'. Pigs from the medicated group were provided with feed containing 250 p.p.m. doxycycline (HIPRAMIX/DOXI) for 8 consecutive days and were challenged on the fifth day of treatment. No clinical signs were observed in pigs from the 'control group'. Four animals from the 'unmedicated group' died within the first 48 h after challenge with clinical and lesional evidence of an acute form of pleuropneumonia. Clinical signs of animals surviving the first 48 h were progressively less severe and showed lesions similar to those described for subacute-chronic forms of the disease. However, only one animal from the 'medicated group' showed clinical signs of a chronic form of pleuropneumonia. Reisolation of A. pleuropneumoniae was more evident from lung tissues of animals fed the doxycycline-free feed (70%), coinciding with the presence of both acute and subacute lesions. However, the micro-organism could be reisolated from only one animal which belonged to the 'medicated group'. It is concluded that the treatment of pigs with 250 p.p.m. doxycycline (HIPRAMIX/DOXI) prevents disease caused by A. pleuropneumoniae.     

Survey of Actinobacillus (Haemophilus) pleuropneumoniae infection in swine by different methods

Lung and serum samples from pigs that died or were emergency-slaughtered in a pooled, conventional fattening herd were examined to survey Actinobacillus pleuro-pneumoniae infection and to compare the sensitivity of different testing methods. A total of 110 lungs were used for cultural isolation of the agent and direct immunofluorescence (IF) of impression smears. Boiled lung suspensions were tested by coagglutination (Co-A) and agar gel precipitation (AGP). Eighty-seven sera were tested along with lung samples from the same pigs. The lungs yielded a varied bacterial flora most often containing Pasteurella multocida and less frequently Actinomyces (Corynebacterium) pyogenes, E. coli and Salmonella. A. pleuropneumoniae was isolated from 30 lungs: from 22 lungs it grew out in pure culture, from 7 as mixed culture with P. multocida and from 1 as mixed culture with A. pyogenes. The number of positive samples obtained by the different methods was as follows: coagglutination test (with boiled lung suspensions): 63 (57.3%); immunofluorescence: 43 (39.2%); AGP test (with serum): 31 (35.6%); AFP test (with boiled lung suspension): 25 (22.7%). A total of 23 samples (20.7%) were negative by all serological tests and by cultural isolation. Most samples gave positive results by two or more tests while 26 samples only by one test (most often, on 13 occasions, by the Co-A test). The Co-A test detected antigenic components of serotypes that have not been isolated in Hungary so far. This indicates that it is not enough to test one strain from a given lung sample: several colonies must be cultured and serotyped.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive protection of piglets by monoclonal antibodies against experimental infection with Actinobacillus pleuropneumoniae

Mouse monoclonal antibodies 11C11 (an IgG) and 4A9 (an IgM), which combine with a superficial component of cells belonging, respectively, to serovars 1 or 3 of Actinobacillus pleuropneumoniae, were given intraperitoneally 24 hours before and intranasally one hour before two-week-old, colostrum-deprived piglets were exposed by the intranasal route to 10(9) viable cells of either strain Shope 4074 (serovar 1) or 2/10 (serovar 3). Compared with control piglets given phosphate buffered saline or the heterologous monoclonal antibody, this procedure conferred substantial protection against the development of peracute or acute pleuropneumonia. Protection against the experimental disease was somewhat less in other piglets to which monoclonal antibody 4A9 was given only by the intranasal route one hour before the organism was administered than in those given the antibody intraperitoneally 24 hours beforehand, although its effect was still significantly greater than in piglets given phosphate buffered saline only. These two monoclonal antibodies consequently offer means of investigating at the molecular level the pathogenesis of the disease associated with A pleuropneumoniae and the potential value of anti-idiotypes as immunising agents.     

Direct detection of Actinobacillus (Haemophilus) pleuropneumoniae in the lungs of swine

By means of cultural examination, coagglutination test (CT) and indirect fluorescent-antibody-technique (IFAT) a total of 199 lung specimens from necropsy pigs from Northwestern Germany with symptoms of pleuropneumonia was examined for Actinobacillus (Haemophilus) pleuropneumoniae (AP). The CT was used to detect type specific antigens in lung extracts and the IFAT was performed on tissue sections. Both tests were found to be specific. Detection and identification of AP by either test were successful in 68 of 199 lung specimens. AP was isolated out of 40 lungs, antigen detection by CT was successful in 40 and by IFAT in 65 lung samples. In 26.5% of the positive samples AP was demonstrated only by IFAT. In 4.4% of the positive specimens AP was demonstrated only by cultural examination, but the detected serovars were not accounted in IFAT and CT. In 44.1% of the positive specimens AP was isolated or detected by all three techniques. The predominating serovar was serovar 9 followed by 2 and 7. One field isolate could be identified as serovar 3 and another one as serovar 10. Furthermore one isolate was untypable. IFAT and CT were limited for detection of serovars 2, 7 and 9. Detection of multiple serovars in few lung samples was successful only by IFAT. Indirect fluorescent-antibody-technique was found to be more sensitive than coagglutination test and cultural examination. On the other hand CT was found to be less time consuming and easier to evaluate than other tests. By this, coagglutination test seems to be preferable in examining large numbers of lung samples.     

猪胸膜肺炎放线杆菌菌影的制备与免疫试验

通过鲎素抗菌肽和超高静水压联合作用,制备出一种胸膜肺炎放线杆菌菌影。利用胸膜肺炎放线杆菌血清5型（CVCC263）制备菌影并检测其灭活率。CVCC263菌影疫苗接种免疫仔猪,二免14d后攻毒,每天测量体温,并观察精神状态,呼吸,食欲等。攻毒第8天对存活猪进行剖杀,测量肺部病变面积,进行病理检测。结果显示,免疫组抗体效价及血清中的IgG、IgM、IgA、IL-2、IL-4含量均较对照组显著增加。免疫组临床症状和肺部病变面积均小于对照组。CVCC263菌影疫苗免疫仔猪抗APP感染的保护效果是明显的,并且APP5型的菌影疫苗可对APP7型感染有交叉保护。     

Functional genome analysis investigating resistance to respiratory tract disease in a porcine Actinobacillus pleuropneumoniae infection model

Here we present the work of the multidisciplinary consortium IRAS (Development of Genetic Markers for Immune Defence and Resistance in the Porcine Respiratory Tract) which includes different commercial and research institutions and was formed as a response to the call "Functional Genome Analysis in the Animal Organism (FUGATO)" by the German Ministry of Education and Research. IRAS started work in the fall of 2005 and--using the experimental infection of pigs with Actinobacillus pleuropneumoniae as model pathogen--aims at i) characterizing the course of infection by clinical as well as advanced laboratory tools (phenotypic-genetic approach) and ii) defining the diversity and distribution of allels known to be associated with immune defence in mouse and man (homolog-genetic approach). The intention is to identify genetic markers for increased resistance to infection thereby providing additional tools for the estimation of breeding values to the pig industry.     

Differential proteomic analysis reveals increased cathelicidin expression in porcine bronchoalveolar lavage fluid after an Actinobacillus pleuropneumoniae infection

Accurate definition of respiratory health in pigs is an important problem for swine producers and veterinarians. In an approach to identify potential biomarkers, two-dimensional gel electrophoresis and mass spectrometry on bronchoalveolar lavage fluid (BALF)-derived proteins from pigs experimentally infected with Actinobacillus pleuropneumoniae were performed at different time points post infection. Mock-infected pigs were used as a control. It was shown that the antimicrobial peptides, prophenin-2 and PR-39, and the calcium-binding protein calgranulin C were reproducibly upregulated in BALF of pigs chronically infected with A. pleuropneumoniae. Concentrations of PR-39 were significantly (p<0.05) increased in BALF (median of 4.8 nM) but not in serum (median of 2.5 nM) on day 21 after infection. A Receiver Operating Characteristics (ROC) plot showed that PR-39 in BALF is an accurate and easily accessible marker to detect clinically healthy pigs convalescent from an experimental A. pleuropneumoniae infection. These results imply that PR-39 might have a potential as a general biomarker to determine porcine respiratory health.     

Experimental Actinobacillus pleuropneumoniae infection in piglets with different types and levels of specific protection: immunophenotypic analysis of lymphocyte subsets in the circulation and respiratory mucosal lymphoid tissue

Actinobacillus pleuropneumoniae (APP) infection in piglets results in severe and fatal fibrinous hemorrhagic necrotizing pneumoniae. The aim of our study was to analyze changes in lymphocyte subset distribution in peripheral blood, bronchoalveolar lavage fluid (BALF) and tracheobronchal lymph nodes (TLN) in non-immune piglets upon a challenge with a high dose of APP and to compare the quality of such changes in unprotected piglets with counterparts exhibiting specific immunity mediated by high titers of colostrum-derived APP-specific antibodies and/or a low dose APP infection in the early postnatal period. Challenge with APP resulted in a massive increase in CD8-negative gammadelta T-cells in parallel with a reduction in numbers of CD3-CD8low cells in BALF independent of the type and level of immunity and this seems to be a general phenomenon associated with experimental infection. An increase in B-lymphocyte numbers in TLN was another characteristic feature accompanying APP infection in all experimental groups. In piglets with colostrum-derived APP-specific antibodies, this was associated with higher relative numbers of IgM+CD2+ lymphocytes in TLN, while B-cells with the CD2- surface phenotype apparently expanded in the absence of passive humoral immunity.     

Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelial cells and frozen lung sections

The ability of 23 different Actinobacillus pleuropneumoniae isolates to adhere in vitro to porcine tracheal epithelial cells and to porcine frozen lung sections was examined. It was found that A. pleuropneumoniae adhered poorly to isolated tracheal epithelial cells. On the other hand, A. pleuropneumoniae adhered to frozen lung sections and marked variations were observed between and within serotypes. Adherence to lung sections did not seem related to the hemagglutinating activity of the isolate. Two noncapsulated variants adhered to lung sections in greater numbers than their capsulated parent strains. Adherence to lung sections was not inhibited by the extracellular matrix components tested namely, laminin, fibronectin, and collagen, but was inhibited by homologous serotype-specific antiserum. The data indicated that the A. pleuropneumoniae isolates tested possess the ability to adhere to porcine lung tissue, a property which did not seem to be related to the serotype and did not seem to involve the capsular material or the hemagglutinins of the isolates.     

Complement resistance in Actinobacillus (Haemophilus) pleuropneumoniae infection of swine

The possible role of the complement-mediated bactericidal system in protection of swine against contagious pleuropneumonia was investigated. Strains of Actinobacillus (Haemophilus) pleuropneumoniae representing serotypes 2, 3 and 5 were found to be fully resistant to the bactericidal action of porcine serum from precolostral, clinically normal adult, and chronically infected pigs. All strains were also resistant to hyperimmune rabbit serum, but 3 of 4 strains were sensitive to normal human serum. This bactericidal effect was lost when human serum was previously absorbed with the homologous bacteria, indicating that antibody was necessary for killing. Addition of human serum to porcine serum or to absorbed human serum did not restore the bactericidal system. Pretreatment of the bacteria with undiluted heat-treated human serum also failed to sensitize the bacteria to the absorbed serum, indicating that a heat-labile, absorbable factor may have been required for killing of A pleuropneumoniae. None of the strains was sensitized to porcine serum by sublethal treatment with polymyxin B, a treatment that is known to disrupt the integrity of the outer membrane and induce serum sensitivity in gram-negative bacteria. The ability of A pleuropneumoniae to resist complement killing in vitro may reflect a virulence mechanism in vivo that assists bacteria in avoiding the pulmonary defenses of swine and promotes bacterial invasion of the lungs.     

Studies on the interactions of two different serotypes of Actinobacillus pleuropneumoniae

In vitro and in vivo interactions of various field strains of Actinobacillus pleuropneumoniae of serotypes 1, 2, 5 and 7 were studied. There was no influence of one serotype over the other when strains belonging to two serotypes were cultivated together in vitro. In vivo interactions showed predominance of serotype 1 over other serotypes when a strain of serotype 1 was inoculated together with a strain of serotype 2 or 5 in mice. Serotype 1 strain remained predominant irrespective of whether it was inoculated before or after the inoculation of serotype 2 strain. The mortality caused by the inoculation of two strains was higher than the mortality caused by a single strain. Early mortality was observed on inoculation of strains of serotype 2, 5 or 7 along with a strain of serotype 1. Both serotypes could be detected in the blood on cultural examination of mice infected with mixed serotypes.     

Systemic and local antibody responses after experimental infection with Actinobacillus pleuropneumoniae in piglets with passive or active immunity

The objectives of the present study was to describe different dynamics of humoral immune responses to experimental infection in piglets of different stages of infection and immunity. Two groups of piglets originating from non-immune (group 1) and immune (group 2) sows at the age of 3 weeks were subdivided as follows: a half of each group of piglets was exposed to a low-dose infection with Actinobacillus pleuropneumoniae (APP) strain 9. At the age of 8 weeks, all four groups of piglets were challenged with a high infection dose of APP of the same strain. Isotype characterization of the specific antibodies in sera and in bronchoalveolar lavage fluids (BALF) to a lipopolysaccharide was carried out, besides monitoring clinical signs and post-mortem examinations. A typical primary immune response was observed in specific antibody-free piglets infected with a challenge infection. Colostrum-derived immunoglobulin-G (IgG) antibodies persisted in sera and BALF of piglets up to the age of 8 weeks. However, they did not prevent induction of specific-primary antibody response, either in 8 or 4 weeks of age, when levels of specific colostrum-derived antibodies were still high. It was demonstrated by the increase of specific IgM antibodies in sera. The infection induced an increase in the levels of IgA antibodies in BALF regardless the severity of infection and presence of specific colostrum-derived antibodies. The specific antibodies of IgG isotype increased only in BALF from piglets without colostrum-derived antibodies.     

痤疮丙酸杆菌对小鼠抗胸膜肺炎放线杆菌感染的研究

为研究痤疮丙酸杆菌(PA)对小鼠抗胸膜肺炎放线杆菌感染的影响,从人的面部皮肤分离得到革兰氏阳性短杆菌,通过培养性状观察、生化试验、PCR鉴定,证实7株菌为PA.经ELISA检测发现PA与猪传染性胸膜肺炎放线杆菌(APP)存在免疫交叉反应.以PA免疫小鼠15 d后用10倍LD50的APP攻毒,观察1周,最高保护率为60%;将制备的抗PA高免血清注射小鼠,分别用10倍LD50的APP血清1型、APP血清5型感染小鼠,保护率均为100%.结果表明,PA对APP血清1型,APP血清5型感染具有良好的保护作用.     

猪圆环病毒与胸膜肺炎放线杆菌混合感染的诊治

2019年3月,大庆某规模化猪场个别猪出现发育不良、咳喘、消瘦、皮肤苍白和呼吸困难等症状,随后出现死亡。为明确发病原因,降低损失,对送检病死猪进行临床症状观察、剖检及实验室检测,并对病原菌进行药物敏感性试验。检测结果为致病性胸膜肺炎放线杆菌和猪圆环病毒混合感染所致,分离的胸膜肺炎放线杆菌对阿莫西林和头孢唑高度敏感。本研究为养殖场饲养猪的死亡病因分析和防治提供了参考。     

猪胸膜肺炎放线杆菌PCR检测方法的建立及临床应用

为建立直接检测可疑病料中胸膜肺炎放线杆菌（APP）的PCR诊断方法，对用以扩增apxⅣA毒力基因3’端长约442bp的核苷酸片段的引物、模板、Taq DNA聚合酶、dNTPs的最适剂量及退火温度进行了优化筛选，并进行了特异性及重复性试验；以筛选出的优化条件，对临床可疑病料进行了PCR检测。结果，在25μL体系中，以5pmol／μL引物、0．25μL Taq DNA聚合酶、2mmol／L dNTPs、56℃退火温度对APP标准菌株apxⅣA毒力基因的扩增效果最好，重复性和稳定性高；而对类症菌株的扩增结果则为阴性，表明其特异性强。对分离到APP的病料PCR阳性率为100％（5／5）；未分离到APP的纤维渗出性病料中，新鲜与陈旧病料PCR阳性率分别为70．59％（12／17）、50．00％（4／8），而非纤维渗出性病料的PCR结果均为阴性（0／45、0／9）。结果表明，建立的PCR方法特异、快速、稳定、敏感，优于细菌学方法，它可作为APP可疑病料的理想诊断方法。