Invivo induced RTX toxin ApxIVA is essential for the full virulence of Actinobacillus pleuropneumoniae

Veterinary Microbiology 137 (2009) 282–289

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In vivo induced RTX toxin ApxIVA is essential for the full virulence of Actinobacillus pleuropneumoniae Jinlin Liu a, Xia Chen c, Chen Tan a, Yi Guo a, Yan Chen a, Shulin Fu a, Weicheng Bei a,b,*, Huanchun Chen a,b a b c

Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China College of Animal Science & Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei 430070, China College of Horticulture & Forestry Science, Huazhong Agricultural University, Wuhan, Hubei 430070, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 October 2008 Received in revised form 2 January 2009 Accepted 5 January 2009

Actinobacillus pleuropneumoniae is a Gram-negative pathogen. It is the aetiological agent of porcine contagious pleuropneumonia (PCP), a severe and highly contagious and severe respiratory disease of swine. Four sets of RTX (repeats in toxin) exotoxins have been described in A. pleuropnuemoniae and three of them have been characterized as important virulence determinants. The aim of this study was to determine the pathogenicity of the in vivo induced RTX toxin ApxIVA during infection of piglets with A. pleuropnuemoniae. An A. pleuropnuemoniae apxIVA mutant was obtained based on an A. pleuropnuemoniae apxIICdeleted mutant strain. An experimental infection assay was performed to evaluate the virulence of ApxIVA in piglets. Clinical signs, lung lesion scores, blood biochemical parameters and histopathologic changes in the piglets were recorded. The results indicated that the pathogenicity of A. pleuropnuemoniae was greater when ApxIVA was present, suggesting that ApxIVA is essential for expression of the full virulence of A. pleuropnuemoniae. ß 2009 Published by Elsevier B.V.

Keywords: Actinobacillus pleuropnuemoniae apxIVA Virulence factor

1. Introduction ‘‘Repeats in toxin’’ (RTX) toxins are produced by a wide range of Gram-negative bacteria. They have been shown to be involved in the virulence of many organisms, including Actinobacillus, Bordetella, Escherichia, Moraxella, Morganella, Pasteurella, Proteus, and Vibrio species (Lally et al., 1999; Schaller et al., 2000; Welch, 2001). The genetic organization of RTX toxins often comprises of four genes, which are designated rtxC, A, B and D in transcriptional order. It has been established that the RtxA gene encodes the structural toxin, RtxC is an acylation activation protein

* Corresponding author at: Division of Animal Infectious Disease, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China. E-mail address: [email protected] (W. Bei). 0378-1135/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.vetmic.2009.01.011

that encodes the post-translational activator of RtxA, and RtxB and D encode proteins that are essential for secretion of the toxin. The structure of the RTX toxins is usually consists of a domain of glycine-rich nonapeptide repeats with the consensus sequence L/I/F-X-G-G-X-G-N/D-D-X (Welch et al., 1995). The number of repeats of this motif varies from 6 to 40 in different toxins. These repeats are essential to the calcium-dependent cytolytic activity of the toxin (Boehm et al., 1990). Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, possesses four sets of RTX toxins, named ApxI, ApxII, ApxIII and ApxIV (Schaller et al., 1999). Published data suggested that Apx toxins play an important role in the pathogenicity of A. pleuropneumoniae (Frey, 1995; Reimer et al., 1995; Boekema et al., 2004). ApxI is strongly haemolytic and strongly cytotoxic; ApxII is weakly haemolytic and moderately cytotoxic; ApxIII is non-haemolytic but stongly cytotoxic (Frey, 1995). ApxIV, first reported by

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The bacterial strains, primers and plasmids used in this study are described in Table 1. Primers were synthesized by Sangon, Shanghai, China. Escherichia coli strains were cultured in Luria–Bertani broth, supplemented with appropriate antibiotics (ampicillin, 50 mg/mL; chloramphenicol, 25 mg/mL). A. pleuropneumoniae strains were cultured in Tryptic Soy Broth (TSB) or Tryptic Soy Agar (TSA) (Becton, Dickinson and Company, U.S.A.) supplemented with NAD (nicotinamide adenine dinucleotide) (10 mg/mL). For the selection of A. pleuropneumoniae transformants, chloramphenicol (1 mg/mL) was added.

The 5.9 kb PstI-NotI fragment from pMD-apxIV containing the apxIVA gene was ligated into E. coli-APP shuttle vector pJFF224-XN (Frey, 1992), resulted in complementation plasmid pVC3. pVC3 was introduced into QDS01 by electroporation. A chloramphenicol-resistant (Cmr) transformant QA01 was selected. Heredity stability of QA01 was confirmed by series passages and PCR with primers P3 and P4. To confirm the expression of ApxIV in the complementation strain QA01, an experimental infection assay was carried out. Twenty-four 6-week-old BALB/c mice were randomly divided into three experimental groups of eight mice. Group 1 was inoculated intraperitoneally (i.p.) with 1.0  107 CFU of QDS01. Group 2 was inoculated intraperitoneally with 1.0  107 CFU of QA01 in 200 mL TSB. Group 3 was inoculated with 200 mL TSB. Serum samples were collected 3 weeks after injection and were detected by the ApxIVAM-ELISA as describled previously (Liu et al., 2007). Briefly, the 96-well ELISA plates were coated with purified GST-ApxIVAM, 100 mL serial diluted serum samples (from 1:10) were added into each well and incubated at 37 8C for 30 min. After four washes, 100 mL of horseradish peroxidase-conjugated goat anti-mouse IgG was added to each well and the plates were incubated in 37 8C for 30 min, followed by five washes, substrate solution TMB and H2O2 (50 mL) were added to each well, the catalytic reaction was stopped by 50 mL 1% SDS. The optical density was read at 630 nm in an ELISA reader.

2.2. Construction of the complementation strain

2.3. Preparation of infection strains

For the trans-complementation study, the intact apxIV gene was cloned from A. pleuropneumoniae field isolate HB04 with primers P1 and P2, and ligated into the A/T cloning vector pMD18-T, resulted in plasmid pMD-apxIV.

For infection assays, a single colony of each of the four strains of A. pleuropneumoniae was cultured at 37 8C with shaking. The cultures were inoculated into fresh TSB + NAD broth (1:1000 dilution) on the following day, and were

Schaller et al. (1999), has been reported to be expressed only in vivo and is specific to A. pleuropneumoniae. However, whether ApxIV contributes to the pathogenesis of A. pleuropneumoniae infection is still unknown. To assess the pathogenicity of ApxIV and development of a potential marker vaccine that could be used to differentiate vaccinated animals from infected animals, an ApxIVA inactivated mutant has been constructed in our laboratory (Liu et al., 2007). Experimental infection in piglets was carried out to determine the role of the ApxIV toxin in pathogenesis in this study. 2. Materials and methods 2.1. Bacterial strains, primers, plasmids and growth conditions

Table 1 Bacterial strains, plasmids and primers used in this study. Strain, plasmid and primer

Relevant characteristics

Source

A. pleuropneumoniae HB04

A. pleuropneumoniae field isolate, serovar 7.

Isolated from Hubei Province, China Bei et al. (2005) Liu et al. (2007) This work

QP05 QDS01 QA01 Plasmids pMD18-T pMD-apxIV pJFF224-XN pVC3

Primers P1

P2 P3

P4

A. pleuropneumoniae strain HB04 apxIIC-deleted mutant. A. pleuropneumoniae strain HB04 apxIIC/apxIVA double deleted mutant. A. pleuropneumoniae strain QDS01 containing complement plasmid pVC3.

E. coli cloning vector carrying an ampicillin resistance determinant. pMD18-T carrying apxIV gene of A. pleuropneumoniae HB04, for the sequence analysis. E. coli-APP shuttle vector: RSF1010 replicon; mob oriV, Cmr pJFF224-XN carrying the intact apxIVA and orf1 of A. pleuropneumoniae HB04, for the complementation assay.

Takara (Dalian, China) This work Frey (1992) This work

50 -GGCGAATTCATGAAAATAAAAAAACGTTAC-30 , upstream primer with EcoRI site (underlined) comprising positions 1–21 of apxIV. This primer was used to clone apxIV gene for the complementation assay. 50 -TAGCGGCCGCTTATAAAGCAGCTGTTAAGC-30 , downstream primer with NotI site (underlined) comprising positions 5892–5911 of apxIV. 50 -GGC TGT CTG TTA GTG GTT CG-30 , upstream primer comprising positions 1446–1465 of apxIVA coding sequence. This primer was used to differentiate A. pleuropneumoniae apxIVA mutant and other strains, since the parent strain will result an 1.2 kbp fragment by PCR, the mutant will have an 0.6 kbp fragment. 50 -CCG TGT GCA GAA ATA CTG CC-30 downstream primer comprising positions 2618–2637 of apxIVA.

This work

Liu et al. (2007)

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cultured for approximately 4 h to an optical density (O.D.) at 600 nm of 0.8. The viable count of A. pleuropneumoniae wild-type (w.t.) strain and mutants at this O.D. was approximately 1.5  109 colony forming units (CFU) per mL. The cultures were placed on ice and diluted in ice-cold TSB to the desired live cell counts. 2.4. Experimental infection in piglets The animal experiments were carried out according to the International Guiding Principles for Biomedical Research Involving Animals (1985) (http://www.cioms.ch/ frame_1985_texts_of_guidelines.htm). Thirty 6-week-old pigs were purchased from an A. pleuropneumoniae-free herd that was confirmed according to previously describled criteria (the absence of clinical signs and no serological reaction in enzyme-linked immunosorbent assays (ELISAs) for ApxII and ApxIVA) (Huang et al., 2005; Liang et al., 2005). The piglets were randomly assigned to five treatment groups each containing six pigs. The virulence of the mutant and w.t. strains was assessed in an intratracheally (i.t.) infection model. For the challenge procedure, pigs were anesthetized by intravenous injection with ketamine (4 mg/ kg of body weight) and xylazine (2 mg/kg). Piglets in the first group were inoculated with 5 mL TSB and used as a negative control; Groups 2–5 were injected with the appropriate dose, suspended in 5 mL TSB, of the double deletion mutant strain QDS01, the single deletion mutant strain QP05, the w.t. strain HB04, or trans-complementation strain QA01. Pigs were monitored clinically for 4 days after inoculation. At 0, 6, 12, 24, 48 and 96 h post-inoculation (hpi) rectal temperatures were measured. Teperature over 41 8C was considered to be in a high fever. To assess the induction and development of disease by the different A. pleuropneumoniae strains in the period after inoculation, blood samples were taken at 0, 24 and 48 hpi. Blood biochemical parameters such as blood glucose (BGlu), glutamic-oxal(o)acetic transaminase (GOT), glutamate-pyruvate transaminase (GPT) were determined using specialized detection kits purchased from Biosino Biotechnology and Science Inc., China, in accordance with the manufacturer’s recommendations. Clinical signs were monitored and scored as described previously (Fuller et al., 2000). Briefly, appetite was scored as 0, did eat; 1, did not eat, total score = number of 12 h periods not eating over 36-h observation period. Dyspnea

was scored as: 0, normal; 1, slight; 2, moderate; 3, severe. Lethargy was scored as: 0, normal; 1, slight inactivity; 2, moderate; 3, severe. Observation of pneumonia was determined by histopathological examination, and was charaterized by hyperemia, cellular exudate, consolidation and necrosis. Percentage of pleural surface area exhibiting pleuritis was measured. Seriously diseased pigs, as determined by severe dyspnea and/or lethargy, were euthanized. At day 7 post-challenge, all surviving pigs were euthanized and the lung lesions were recorded as described previously (Hannan et al., 1982). Briefly, a complete lung was divided into seven lobes and each lobe was arbitrarily allotted a maximum possible lesion score of 5. The pneumonic area of each lobe was then assessed and recorded as a fraction of 5 to give the pneumonic score per lobe. For the bacteriological examination, swabs of lung tissue were plated onto the TSA + NAD agar, and incubated at 37 8C for 12 h. Colonies that were morphologically similar to those of A. pleuropneumoniae were picked and verified by PCR using primers P3 and P4. For histological examination, lung samples were fixed in 10% formalin buffer (pH 7.2). Thin sections (5 mm) were stained using hemotoxylin and eosin and examined by light microscopy. 2.5. Statistical analysis All data analysis was performed using the Student’s ttest for comparison of the differences in clinical signs, lung lesion scores and biochemical indicators between different groups. P-values of <0.05 were considered statistically significant; P-values of <0.01 were considered highly statistically significant. 3. Results 3.1. Construction of the complementation strain QA01 An A. pleuropneumoniae complementation strain QA01 was constructed based on the double deleted mutant QDS01 using APP-E. coli shuttle vector pVC03. QA01 was confirmed stabile (Fig. 1). Serum samples of infected mice were detected by ApxIVM-ELISA, results were displayed in Table 2. Mice inoculated with the complementation strain QA01 were positive on the ApxIVAM-ELISA, whereas mice

Fig. 1. Verification of complementation strain QA01 by PCR with specific primers for apxIVA gene and analyzed by electrophoresis in 0.8% agarose gel. Line M: DNA marker 2000, Takara, Dalian, China; Lines 1–16: QA01 of different passages; Line 17: HB04; Line 18: QDS01; Line 19: QP05; Line 20: negative control.

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Table 2 Results of the ApxIVAM-ELISA to measure the response of mice inoculated with QDS01 and QA01. IgG titre (arithmetic mean  S.D.)a

Day 0 Day 21 a b

Group 1

Group 2

Group 3

10  9.0 18.1  4.0

15  7.3 55.0  32.2b

11.9  6.6 9.4  5.7

Antibody titer >28 was assessed positive in ApxIVAM-ELISA. P < 0.01 compared with the QDS01 inoculated group.

inoculated with QDS01 were negative 21 days after injection, suggesting that QA01 possesses a functioning ApxIV. 3.2. Virulence in piglets The wild-type strain was used as a virulent control in the experimental infection of piglets. The clinical signs and lung lesion scores of pigs infected intratracheally with A. pleuropneumoniae serovar 7 parent strain HB04 or the mutants are summarized in Table 3. The values of blood biochemical parameters in the various groups are shown in Fig. 2. Lung sections are shown in Fig. 3. All pigs inoculated with HB04 showed severe clinical signs, and exhibited labored breathing, coughing, depression, and little or no appetite. The rectal temperature was up to 41 8C 6 hpi and remained elevated for about 2 days. One pig was euthanized at about 48 hpi and extensive lung lesions and severe pleuritis were found on postmortem examination. The A. pleuropneumoniae challenge strain was isolated from all pigs inoculated with the w.t. strain. There was a significant reduction in BGlu in the pigs of this group compared to the negative control group 24 and 48 hpi (P < 0.01). Compared with the negative control group, the GOT and GPT were transiently increased (P < 0.01) in the infected piglets. The lung sections were examined by microscopy (Fig. 3D), which showed that the lung pleura and parenchyma were edematous, with massive proliferation of fibroblasts and formation of connective tissue. The bronchioles were filled with cellular exudate, comprised of neutrophils and alveolar epithelial cells. Portions of the parenchyma were completely necrotic or collapsed. These data suggested HB04 was highly virulent in piglet.

Fig. 2. Blood biochemical parameters BGlu (A), GOT (B) and GPT (C) in pigs inoculated with different A. pleuropneumoniae strains at different timesa. a Blood biochemical parameters blood glucose (BGlu), glutamicoxal(o)acetic transaminase (GOT) and glutamate-pyruvate transaminase (GPT) were evaluated using detection kits produced by Biosino Biotechnology and Science Inc., Beijing, China. bP < 0.01 compared with the QP05 challenged group. c P < 0.01 compared with the QA01 challenged group. dSignificant difference between the negative control group and the HB04 challenged group (P < 0.01).

Pigs inoculated with QP05 showed moderate to severe clinical signs. Pigs showed increased respiratory rate and high rectal temperatures 6 hpi, which lasted for about 20 h. They also showed decreased appetite and depression. In contrast, only two pigs displayed moderate clinical signs in

Table 3 Virulence of serovar 7 w.t. and isogenic mutant strains following intratracheally inoculation. Group

Challenge dose (CFU)

Appetite

Negative control QDS01 QP05 HB04 QA01

0

0

a b c d

9

2.2  10 2.0  109 9.0  108 2.2  109

0.5  0.5 1.5  0.5 2.5  0.5 1.5  0.8

Dyspnea

Lethargy

0 a,b

0.3  0.5 1.5  0.5 2.2  0.8 1.7  0.5

0 c,d

0.3  0.5 1.3  0.5 2.5  0.5 1.5  0.8

Lung lesion score

0 c,b

0.9  0.4 4.1  0.9 14.3  3.6 4.6  3.1

No. of animals with pneumonia/ total no. 0/6

c,b

1/6 5/6 6/6 6/6

Pleuritis

0 0 13.3  5.1 35  15.5 13.3  6.8

P < 0.05 compared with the QP05 challenged group. P < 0.05 compared with the QA01 challenged group. Significant difference between the QDS01 challenged group and the QP05 challenged group (P < 0.01). Significant difference between the QDS01 challenged group and the QA01 challenged group (P < 0.01).

No. of animals with reisolation of the challenge strain

Observations >41 8C/ total observations post-infection

0/6

0/36

0/6 0/6 6/6 1/6

2/36 9/36 17/35 10/36

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the group inoculated with QDS01. A significant difference was observed in appetite and the incidence of dyspnea between the groups inoculated with the QP05 mutant strain and that inoculated with the QDS01 mutant strain (P < 0.05). Pigs challenged with QP05 were exhibited

moderate adhesive pleuritis and focal lung lesions at postmortem examination. However, only mild pathological changes were observed in the group challenged with QDS01. The results of histological examination were coincident with those of the postmortem examination.

Fig. 3. Lung sections of pigs in different treatment groups, negative control (A), QDS01 (B), QP05 (C), HB04 (D) and QA01 (E). (A1) Profile of normally inflated alveoli, H&E 10; (A2) normal thickness of alveolar wall and absence of exudates in alveoli, H&E 100. (B1) Mild edema of pleura and lung parenchyma, with some serous exudate, H&E 10; (B2) fibroblast proliferation in pleura, H&E 100. (C1) Lung section showing bronchiectasia, H&E 10; (C2) significant thickening of pleura, H&E 10; (C3) encapsulated lung lesion filled with extensive connective tissue, H&E 50; (C4) alveoli filled with cellular exudate composed of desquamated epithelial cells, erythrocytes and neutrophils, H&E 100. (D1) Thickening of pleura and lung parenchyma, H&E 10; (D2) encapsulation formed to prevent the extension of suppurative change, H&E 50; (D3) lung section showing cellular exudate filling the bronchioles, H&E 50; (D4) proliferation of epithelial cells, neutrophils and erythrocytes in alveolar cavity, H&E 100. (E1) Lung section showing thickening of pleura, H&E 10; (E2) alveoli and bronchioles filled with cellular epithelial cells, erythrocytes and neutrophils, H&E 25; (E3) destructed pulmonary lobule filled with epithelial cells, erythrocytes and neutrophils, H&E 25; (E4) micrangium hyperemia or hemorrhage, H&E 100.

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Fig. 3. (Continued ).

We have previously reported that the apxIIC mutant showed decreased virulence in pigs, and no severe clinical signs could be observed even when pigs were inoculated with a high dose (Liu et al., 2007). Therefore, an extremely high dose, up to 2.0  109 CFU, was used in the experimental infection in the current study. There were no deaths in the group infected with the apxIIC mutant QP05, but the pigs developed moderate to severe signs of pneumonia. Lung sections showed that all pigs had pneumonic changes, including pleura, cellular exudates

and neutrophil infiltration (Fig. 3C). Compared with pigs inoculated with the QP05, however, pigs infected with the double mutant strain QDS01 were affected more mildly (Fig. 3B). It was also notable that the level of BGlu was significantly different 24 hpi in the group infected with the QP05 when compared with the group infected with QDS01 (P < 0.01). To assess whether virulence could be restored in QDS01 by complementation, the trans-complementation strain QA01 was constructed, and tested in the pig infection

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assay. The virulence of QA01 was more powerful than QDS01. Pigs were showed moderate to severe clinical signs. At postmortem examination, one pig showed a large lung lesion, others had spread small lung lesions. Lung sections (Fig. 3E) indicated severe pulmonitis. Therefore, the data suggest that the virulence of A. pleuropneumoniae decreased after deletion of apxIVA, and ApxIVA is therefore a critical virulence factor in A. pleuropneumoniae infection of pigs. 4. Discussion Actinobacillus pleuropneumoniae contains four sets of RTX toxins, three of them have been considered as important virulence factors. ApxI is strongly haemolytic and strongly cytotoxic and appeared to be primarily responsible for virulence, whereas ApxII is relatively mild, is weakly haemolytic and moderately cytotoxic (Jansen et al., 1995; Reimer et al., 1995). Deletion of ApxI resulted in attenuation of 100-fold and deletion of ApxII caused a 10-fold attenuation in murine toxicity experiments (Lin et al., 2007). ApxIII is strongly cytotoxic to porcine neutrophils and pulmonary alveolar macrophages, endobronchial inoculation with rApxIII can trigger the development of clinical symptoms and lung lesions typical of porcine pleuropneumonia (Kamp et al., 1997; Bosse´ et al., 2002). The contribution of the fourth RTX toxin ApxIV in the induction of clinical symptoms and pneumonic lesions was still unclear. ApxIVA possesses features of RTX proteins, including N-terminal hydrophobic domains, potential acylation sites and glycine-rich nonapeptides repeats in the C-terminal. Recombinant ApxIVA has haemolytic and co-haemolytic activities (Schaller et al., 1999). We hypothesized that ApxIV would be a virulence determinant of A. pleuropnuemoniae. This hypothesis was approved by our present work. Previous study suggested that recombinant ApxIV had weak haemolytic activity in vitro (Schaller et al., 1999). It presumed that ApxIV might less virulent than ApxII toxin, and it suggested that the apxIIC/apxIVA double deleted mutant would be less virulent than the apxIV single deleted mutant in animals. To avoid the interference of toxic ApxII on the observation of ApxIV induced pathogenesis, therefore, the double mutant was used in our study. Complementation strain was used to complement apxIV gene mutation, results stated here strengthened our hypothesis. Post-translational activation is essential for expression of the full virulence for the RTX toxins. Deletion of the apxIC or apxIIC affected the virulence of A. pleuropnuemonia (Lin et al., 2007). ORF1, which is located immediately upstream of apxIVA, may act as an acyl-carrier, and might be involved in the activation of ApxIV and is required for the observation of haemolytic and cohaemolytic phenotypes (Schaller et al., 1999). We hypothesize that this is at least part of the reason why deletion of the apxIVA gene causes the attenuation of the virulence of A. pleuropneumoniae. Osicka et al. (2004) confirmed that ApxIVA had a similar properties to the Neisseria meningitidis RTX protein FrpC,

which possesses self-processing ‘‘clip-and-link’’ activity. This is a unique calcium-dependent autocatalytic processing at an Asp-Pro peptide bond, and which is accompanied by formation of high molecular weight oligomeric species of FrpC that contain subunits covalently cross-linked through a new type of isopeptide bond. This process may be an important factor for both the colonization and pathogenicity of meningococci (Osicka et al., 2004). It may also be a mechanism for the ApxIVA-mediated pathogenicity of A. pleuropneumoniae. The investigation of the blood biochemical parameters in the current study revealed that infection of A. pleuropneumoniae resulted in the fluctuation of BGlu and GOT/GPT, they might be useful markers for the evaluation of the virulence of A. pleuropneumoniae. The BGlu was significantly decreased 24 hpi after infection with the virulent A. pleuropneumoniae w.t. strain or the apxIIC mutant, possibly because of the loss of appetite or a disorder of metabolism caused by the infection. This is similar to the severe hypoglycemia induced by infection with virulent Salmonella strains (Itoh et al., 1996; Santos et al., 2002). The temporary alteration in GOT/GPT may have been caused by the hepatic damage (Ohba et al., 2008), although A. pleuropneumoniae infection is mainly associated with pulmonary pathology. In conclusion, the results described here indicated that deletion of apxIVA led to the attenuation of the virulence of A. pleuropneumoniae. It was confirmed that ApxIVA was essential for the expression of the full virulence of the bacterium. In addition, QDS01 has the potential to be used as a safe, efficient live vaccine. This vaccine could be used to differentiate infected animals from vaccinated animals, as ApxIVA was expressed in vivo and was specific to A. pleuropneumoniae. Acknowledgements The authors thank Dr. Joachim Frey (Institute of Veterinary Bacteriology, University of Berne, Switzerland) for the kindly gift of the shuttle vector pJFF224-XN. This study is supported by grants from National Nature Science Foundation of China (Nos. 30600025 and 30530590), National Key Technology R&D Program (No. 2006BAD06A04), and Innovation Teams of Ministry of Education (No. IRT0726). References Bei, W., He, Q., Yan, L., Fang, L., Tan, Y., Xiao, S., Zhou, R., Jin, M., Guo, A., Lv, J., Huang, H., Chen, H., 2005. Construction and characterization of a live, attenuated apxIICA inactivation mutant of Actinobacillus pleuropneumoniae lacking a drug resistance marker. FEMS Microbiol. Lett. 243, 21–27. Boehm, D.R., Welch, A., Snyder, I.S., 1990. Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infect. Immun. 58, 1959–1964. Boekema, B.K., Kamp, E.M., Smits, M.A., Stockhofe-Zurwieden, N., 2004. Both ApxI and ApxII of Actinobacillus pleuropneumoniae serotype 1 are necessary for full virulence. Vet. Microbiol. 100, 17–23. Bosse´, J.T., Janson, H., Sheehan, B.J., Beddek, A.J., Rycroft, A.N., Kroll, J.S., Langford, P.R., 2002. Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect. 4, 225–235. Frey, J., 1992. Construction of a broad host range shuttle vector for gene cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae. Res. Microbiol. 143, 263–269.

J. Liu et al. / Veterinary Microbiology 137 (2009) 282–289 Frey, J., 1995. Virulence in Actinobacillus pleuropneumoniae and RTX toxins. Trends Microbiol. 3, 257–261. Fuller, T.E., Thacker, B.J., Duran, C.O., Mulks, M.H., 2000. A geneticallydefined riboflavin auxotroph of Actinobacillus pleuropneumoniae as a live attenuated vaccine. Vaccine 18, 2867–2877. Hannan, P.C., Bhogal, B.S., Fish, J.P., 1982. Tylosin tartrate and tiamutilin effects on experimental piglet pneumonia induced with pneumonic pig lung homogenate containing mycoplasmas, bacteria and virus. Res. Vet. Sci. 33, 76–88. Huang, H., Zhou, R., Chen, M., Liu, J., Xu, X., Chen, H., 2005. Cloning and expression of the apxIVA gene of Actionbacillus pleuroneumoniae and development of an indirect ApxIVA-ELISA. Chin. J. Biotechnol. 21, 294–299 (in Chinese). Itoh, N., Kikuchi, N., Hiramune, T., 1996. Biochemical changes in fowl serum during infection with Salmonella typhimurium. J. Vet. Med. Sci. 58, 1021–1023. Jansen, R., Briaire, J., Smith, H.E., Dom, P., Haesebrouck, F., Kamp, E.M., Gielkens, A.L., Smits, M.A., 1995. Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils. Infect. Immun. 63, 27– 37. Kamp, E.M., Stockhofe-Zurwieden, N., van Leengoed, L.A., Smits, M.A., 1997. Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia in pigs. Infect. Immun. 65, 4350–4354. Lally, E.T., Hill, R.B., Kieba, I.R., Korostoff, J., 1999. The interaction between RTX toxins and target cells. Trends Microbiol. 7, 356–361. Liang, W., He, Q., Liu, Z., Chen, H., Wu, B., Xu, X., Li, T., 2005. Cloning and expression of ApxII and development an indirect ELISA for detection of antibody against A. pleuropneumoniae. Chin. J. Vet. Sci. 25, 145–147 (in Chinese). Lin, L., Bei, W., Sha, Y., Liu, J., Guo, Y., Liu, W., Tu, S., He, Q., Chen, H., 2007. Construction and immunogenecity of a DeltaapxIC/DeltaapxIIC dou-

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ble mutant of Actinobacillus pleuropneumoniae serovar 1. FEMS Microbiol. Lett. 274, 55–62. Liu, J., Chen, X., Lin, L., Tan, C., Chen, Y., Guo, Y., Jin, M., Guo, A., Bei, W., Chen, H., 2007. Potential use an Actinobacillus pleuropneumoniae double mutant strain DeltaapxIICDeltaapxIVA as live vaccine that allows serological differentiation between vaccinated and infected animals. Vaccine 25, 7696–7705. Ohba, T., Shibahara, T., Kobayashi, H., Takashima, A., Nagoshi, M., Osanai, R., Kubo, M., 2008. Multifocal granulomatous hepatitis caused by Actinobacillus pleuropneumoniae serotype 2 in slaughter pigs. J. Comp. Pathol. 139, 61–66. Osicka, R., Procha´zkova´, K., Sulc, M., Linhartova´, I., Havlı´cek, V., Sebo, P., 2004. A novel ‘‘Clip-and-link’’ activity of repeat in toxin (RTX) proteins from Gram-negative pathogens. J. Biol. Chem. 279, 24944–24956. Reimer, D., Frey, J., Jansen, R., Veit, H.P., Inzana, T.J., 1995. Molecular investigation of the role of ApxI and ApxII in the virulence of Actinobacillus pleuropneumoniae serotype 5. Microb. Pathog. 18, 197–209. Santos, R.L., Tsolis, R.M., Baumler, A.J., Adams, L.G., 2002. Hematologic and serum biochemical changes in Salmonella ser Typhimurium-infected calves. Am. J. Vet. Res. 63, 1145–1150. Schaller, A., Kuhn, R., Kuhnert, P., Nicolet, J., Anderson, T.J., MacInnes, J.I., Segers, R.P., Frey, J., 1999. Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae. Microbiology 145, 2105–2116. Schaller, A., Kuhnert, P., de la Puente-Redondo, V.A., Nicolet, J., Frey, J., 2000. Apx toxins in Pasteurellaceae species from animals. Vet. Microbiol. 74, 365–376. Welch, R.A., 2001. RTX toxin structure and function: a story of numerous anomalies and few analogies in toxin biology. Curr. Top. Microbiol. Immunol. 257, 85–111. Welch, R.A., Bauer, M.E., Kent, A.D., Leeds, J.A., Moayeri, M., Regassa, L.B., Swenson, D.L., 1995. Battling against host phagocytes: the wherefore of the RTX family of toxins? Infect. Agents Dis. 4, 254–272.