The Journal of Veterinary Medical Science

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1 Advance Publication The Journal of Veterinary Medical Science Accepted Date: Sep 0 J-STAGE Advance Published Date: Oct 0

2 FULL PAPER Bacteriology SEROTYPES, ANTIMICROBIAL SUSCEPTIBILITY, AND MINIMAL INHIBITORY CONCENTRATIONS OF ACTINOBACILLUS PLEUROPNEUMONIAE ISOLATED FROM SLAUGHTER PIGS IN TAIWAN (00 00) Cheng-Yao YANG 1,), Chao-Nan LIN ), Chuen-Fu LIN ), Tsung-Chou CHANG ) and Ming-Tang CHIOU )* ) Division of Animal Medicine, Animal Technology Institute Taiwan, Chunan, Miaoli 0, Taiwan, ROC; ) Department of Biological Science and Technology, China Medical University, Taichung 0, Taiwan, ROC; ) Department of Veterinary Medicine, National Pingtung University of Science and Technology, Neipu, Pingtung 1, Taiwan, ROC; ) Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Takun, Taichung 0, Taiwan, ROC 1 1 Running head: IN VITRO ACTIVITIES OF A. PLEUROPNEUMONIAE * Corresponding author: Ming-Tang Chiou Department of Veterinary Medicine, National Pingtung University of Science and Technology No. 1, Hsueh-Fu Road, Nei-pu, Pingtung 1, Taiwan, ROC Tel: +--00; Fax: +--00; mtchiou@mail.npust.edu.tw 1

3 1 1 ABSTRACT: In total, isolates of A. pleuropneumoniae were collected from pigs with hemorrhagic pneumonia at slaughterhouses during Serotypes, antimicrobial susceptibility and minimum inhibitory concentration (MIC) values were determined for each isolate of A. pleuropneumoniae to antimicrobial agents. Serovar 1 of A. pleuropneumoniae was predominant in Taiwan in 1 of the isolates, followed by serovars and. More than 0% of collected isolates were sensitive to ceftiofur, cephalothin, and chloramphenical. However, lincospectin and gentamicin were relatively less susceptible with sensitivities of only.% and.%, respectively. Additionally, ceftiofur had the highest in vitro activity with an MIC 0 of. µg/ml, followed by cephalothin (. µg/ml) and chloramphenicol (. µg/ml). Lincospectin had the least activity with MIC 0 and MIC 0 values of. µg/ml and. µg/ml, respectively. The data indicate that ceftiofur and cephalothin were extremely active against A. pleuropneumoniae and with minimum MIC values. These drugs are suitable for controlling and treating hemorrhagic pleuropneumonia outbreaks in swine Keywords: Actinobacillus pleuropneumoniae; Antimicrobial susceptibility; Minimum inhibitory concentration; Pig; Serotype

4 INTRODUCTION Porcine necrotizing hemorrhagic pleuropneumonia caused by Actinobacillus pleuropneumoniae is one of the most costly respiratory diseases in swine industries. The acute type of this disease is highly contagious and has a high mortality rate, it is economically important for pig producers [1]. Fifteen serovars of A. pleuropneumoniae have been recognized []. The distributions of serovars differ among countries [1,, ]. Serovars 1,,,,, and of A. pleuropneumoniae have been isolated and confirmed in Taiwan [,,, ]; serovar 1 was the predominate isolate []. Administering antimicrobial agents is the most common and effective strategy employed for controlling and treating this lethal disease in the field. Several antimicrobial agents have been employed clinically. Since antimicrobial susceptibility can change over time in the same area [], an effective treatment using antimicrobial agents against A. pleuropneumoniae in the field requires detailed information about the antimicrobial activities of antimicrobial agents. This study investigates the serotypes of A. pleuropneumoniae isolates and in vitro activity of antimicrobial agents against A. pleuropneumoniae isolates collected from slaughterhouse pigs in Taiwan MATERIALS AND METHODS A. pleuropneumoniae isolation and serotyping: In total, lung specimens with typical hemorrhagic necrotizing pneumonia obtained from pig slaughterhouse were collected during All isolates were cultured on tryptic soy blood agar plates (Difco Lab., MI, USA) with 1% nicotinamide adenine dinucleotide (Sigma-Aldrich, MO, USA). Plates were incubated for h at C under % CO. Isolates were purified and then characterized by standard biochemical and morphological tests to confirm their identities. Each isolate was

5 serotyped via a rapid slide agglutination test as described previously [1]. Antisera were prepared in rabbit against each serovar 1 to 1 of A. pleuropneumoniae with reference strains. Antimicrobial agents: The following ten antimicrobial agents, commonly recommended and administered to treat hemorrhagic pneumonia, were tested: amoxicillin, ampicillin, ceftiofur, cephalothin, chloramphenicol, doxycycline, gentamicin, lincospectin, nitrofurantoin, and oxytetracycline. All antimicrobial agents, except for ceftiofur and lincospectin (Pharmacia & Upjohn, PA, USA), were purchased from a single source (Sigma-Aldrich, MO, USA) Antimicrobial susceptibility test: The agar disk diffusion method was employed in antimicrobial susceptibility tests as described previously [1]. The American Type Culture Collection (ATCC) quality control strain (A. pleuropneumoniae ATCC 00), recommended by the National Committee for Clinical Laboratory Standards (NCCLSs), was used as a reference strain for quality control. The antimicrobial agent concentration of disks (Becton-Dickinson, Heidelberg, Germany) used in the study were following the NCCLSs recommendation [1]. Isolates were categorized as susceptible, intermediate, poor, or resistant using zone diameter standards according to the manufacturer s instructions Minimal inhibitory concentration (MIC) determinations: Minimal inhibitory concentration (MIC) determinations were performed in microtiter wells according to NCCLSs guidelines [1]. These antimicrobial agents were prepared by dissolving appropriate amounts of each agent in suitable solvents [] according to the manufacturer s instructions. Stock solutions were prepared to obtain 0 µg of each antimicrobial agent per milliliter. Serial two-fold dilutions at µg/ml were then prepared. Test plates were incubated for 1 h at C in atmosphere containing % CO. The MIC values was defined as the lowest concentration of

6 antimicrobial agent at which no bacterial growth existed. MIC 0 and MIC 0 values were defined as the concentration of antimicrobial agent inhibited at least 0% and 0% of isolates, respectively, and calculated according the previously described method [1] RESULTS In total, isolates of A. pleuropneumoniae were collected from pigs with hemorrhagic pneumonia at slaughterhouses during Among the serotypes of the isolates, 1 (.%), (.1%), and 1 (0.%) belonged to serovars 1,, and, respectively. Serovar 1 was the predominant isolate. No other serotypes were identified (Table 1). Table shows the agar disk diffusion susceptibility results. Of all tested antimicrobial agents, ceftiofur was most effective with no isolates showing resistance. Fewer than isolates were resistant to cephalothin and chloramphenicol. Amoxicillin, ampicillin, doxycycline, nitrofurantoin, and oxytetracycline had sensitivities of..%. Conversely, fewer than % of the isolates were sensitive to lincospectin and gentamicin. Table lists the MIC values of antimicrobial agents against the isolates. Ceftiofur had the highest in vitro activity against isolates of A. pleuropneumoniae with an MIC 0 value of. µg/ml, followed by cephalothin (1. µg/ml), nitrofurantoin (. µg/ml), and chloramphenicol (. µg/ml). Lincospectin had the lowest activity an MIC 0 value of. µg/ml. Table shows the effective dilution ranges and distribution of MICs obtained from the isolates. The MICs of all ten tested antimicrobial agents were distributed widely. Ceftiofur had the highest activity with MIC values in the range of 0.1 µg/ml, followed by cephalothin (0. 0 µg/ml) and nitofurantoin (0. 0 µg/ml), whereas the distribution of MICs values of amoxicillin, ampicillin, chloramphenicol, doxycycline, gentamicin, lincospectin, and oxytetracycline were same range (0. >0 µg/ml).

7 DISCUSSION Hemorrhagic pleuropneumonia is a lethal swine disease worldwide. This study examined the serotype and in vitro activities of antimicrobial agents against A. pleuropneumoniae isolates collected from swine slaughterhouses. The data provide important and useful trace-back information compared with those from other studies. The prevalence of different serovars of A. pleuropneumoniae varies among countries. Serovars 1 and are common in Japan [1]. Serovar is predominant in Korea [], In Spain, serovars and are predominant []. Based on our study results, serovar 1 is predominant in Taiwan, followed by serovar ; serovar was seldom isolated. These data are consistent with those in a previous study of 0 isolates collected in Taiwan during 1 1 []. Ceftiofur had the highest susceptibility rates against A. pleuropneumoniae isolates in vitro in this study, followed by cephalothin. These experimental results are similar with those in a previous study []. Notably, the MIC 0 values of ceftiofur and cephalothin were. µg/ml and 1. µg/ml, respectively, markedly higher than those in other studies from Taiwan (0.0 µg/ml for ceftiofur and 1 µg/ml for cephalothin), Spain ( µg/ml for cephalothin), and Korea (1 µg/ml for ceftiofur) [,, ]. Although these agents are recommended for treatment and control of hemorrhagic pleuropnemonia in the field, their efficacy against A. pleuropneumoniae must be determined periodically. A previous in vitro study showed that chloramphenicol had a susceptible rate of % against A. pleuropneumoniae isolates collected in Taiwan during 1 1 []. Interestingly, in this study, chloramphenicol had the high sensitivity (. %) against A. pleuropneumoniae. Chloramphenicol was widely used for controlling hemorrhagic pneumonia in the field until it was banned for use in food animals in Taiwan in 00. The high sensitivity of chloramphenicol against A. pleuropneumoniae isolates collected during may

8 1 1 result in the banning of this antimicrobial agent for use in the field within a few years. Of the tested isolates, 1 were resistant to oxytetracycline. This antimicrobial agent had a limited effect against A. pleuropneumoniae. A large proportion of A. pleuropneumoniae strains were resistant to oxytetracycline, as reported in another in vitro study []. Since oxytetracycline is a commonly used antimicrobial agent added to feed for disease prevention in herds in Taiwan, this may partially account for the high percentage of resistant strains in this study. Gentamicin, another popular antimicrobial agent, had a lower susceptibility rate and a higher MIC 0 value (. µg/ml) than those in other studies as,, and 1 µg/ml obtained from Taiwan, Spain, and Korea, respectively (,, ). Ampicillin, amoxicillin, and lincospectin, also widely used in Taiwan for treating respiratory disease, had weak activity against A. pleuropneumoniae isolates. These antimicrobial agents should not be employed to treat hemorrhagic pleuropneumonia ACKNOWLEDGMENTS The authors would like to thank the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture of the Republic of China, Taiwan, for financially supporting this research under contract numbers AS-.1.-BQ-B REFERENCES 1. Asawa, T., Kobayashi, H., Mitani, K., Ito, N. and Morozumi, T. 1. Serotypes and antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolated from piglets with pleuropneumonia. J. Vet. Med. Sci. : -.. Blackall, P.J., Klaasen, H.L.B.M., Van den Bosch, H., Kuhnert, P. and Frey, J. 00. Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 1. Vet. Microbiol.

9 : -.. Chang, C.F. and Chang, Y.D. 1. Distribution of Actinobacillus pleuropneumoniae in healthy pigs in Taiwan. J. Chin. Vet. Sci. 0: Chang, C.F., Chang, L.C., Chang, Y.F., Chen, M. and Chiang, T.S. 00. Antimicrobial susceptibility of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis recovered from Taiwanese swine. J. Vet. Diagn Invest. 1: Chang, C.F., Yeh, T.M., Chou, C.C., Chang, Y.F. and Chiang, T.S. 00. Antimicrobial susceptibility and plasmid analysis of Actinobacillus pleuropneumoniae isolated in Taiwan. Vet. Microbiol. : Chang, C.N., Shen, Y.M. and Yu, P.H.. Epidemiology of swine Haemophilus pleuropneumonia. Bull. Inst. Taiwan Sugar Co., pp Gutiérrez, C.B., Píriz, S., Vadillo, S. and Rodríguez Ferri, E.F. 1. In vitro susceptibility of Actinobacillus pleuropneumoniae strains to antimicrobial agents. Am. J. Vet. Res. : -0.. Gutiérrez-Martin, C.B., Blanco, N.G.., Blanco, M., Navas, J. and Rodríguez-Ferri, E.F. 00. Changes in antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolates from pigs in Spain during the last decade. Vet. Microbiol. : 1-.. Hong, S.S., Huang, S.T., Chen, W.L. and Tsai, M.C.. Serotypes and antimicrobial susceptibility of Haemophilus pleuropneumoniae from pigs. J. Taiwan Vet. Med. Anim. Husb. :.. Hsu, S.R., Yei, C.M., Shen, Y.M. and Chou, N.Y. 1. Epidemiology, serovars and antimicrobial susceptibility of swine hemophilus pleuropneumonia. Bull. Inst. Taiwan Sugar Co., pp Kim, B., Min, K., Choi, C., Cho, W.S., Cheon, D.S., Kwon, D., Kim, J. and Chae, C Antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolated from pigs in

10 Korea using new standardized procedures. J. Vet. Med. Sci. : National Committee for Clinical Laboratory Standards (NCCLS): 1, Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animal, approved stand. NCCLS document M1-A. Wayne, PA, USA. 1. Nielsen, R. 1. Serological characterization of Actinobacillus pleurpneumoniae strains and proposal of a new serotype 1. Acta Vet. Scand. : Reed, L.J. and Muench, H.A. 1. A simple method of estimating fifty percent endpoints. Am. J. Hygiene. : Sebunya, T.N.K.and Saunders, J.R. 1. Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 1: -1.

11 Table 1. Relationship between resistance and serotype of isolates of A. pleuropneumoniae. Antimicrobial agents Serotype I (1) a) II () V (1) No. of resistant isolates Amoxicillin 1 0 Ampicillin 1 Ceftiofur Cephalothin 0 0 Chloramphenicol 0 0 Doxycycline 0 Gentamicin Lincospectin Nitrofurantoin 0 0 Oxytetracycline a) Number of isolates used for analysis. 1

12 Table. Susceptibility of isolates of A. pleuropneumoniae to antimicrobial agents. Antimicrobial agents Disc Number of isolates Content (µg) R a) P I S Sensitivity(%) b) Amoxicillin Ampicillin. Ceftiofur Cephalothin Chloramphenicol Doxycycline 1 0. Gentamicin Lincospectin Nitrofurantoin 0 1. Oxytetracycline a) R: resistant; P: poor; I: intermediate; S: susceptible. b) Susceptible and intermediate isolates were combined to calculate sensitivity to A. pleuropneumoniae. 1

13 Table. MIC 0 and MIC 0 for various antimicrobial agents against A. pleuropneumoniae isolates. Antimicrobial agents MIC (µg/ml) 0% 0% Amoxicillin.. Ampicillin 0.. Ceftiofur.. Cephalothin. 1. Chloramphenicol.. Doxycycline..1 Gentamicin.. Lincospectin.. Nitrofurantoin.. Oxytetracycline 1. 1

14 Table. MIC distributions of antimicrobial agents against A. pleuropneumoniae isolates. Antimicrobial agents MIC (µg/ml) Range >0 Amoxicillin 0.~> Ampicillin 0.~>0 1 Ceftiofur 0.1~ 1 1 Cephalothin 0.~0 1 1 Chloramphenicol 0.~> Doxycycline 0.~> Gentamicin 0.~> Lincospectin 0.~> Nitrofurantoin 0.~0 1 1 Oxytetracycline 0.~>