Antimicrobial susceptibility of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis recovered from Taiwanese swine.

Minimum inhibition concentrations (MICs) were determined for ampicillin, ceftiofur, cephalothin, chloramphenicol, enrofloxacin, gentamicin, lincomycin, lincospectin (lincomycin/spectinomycin), neomycin, premafloxacin, spectinomycin, sulfamethoxazole/trimethoprim, and tetracycline against a total of 180 isolates of Actinobacillus pleuropneumoniae, Escherichia coli, and Salmonella choleraesuis (60 each) clinically isolated from pigs on farms in Taiwan from 1994 to 1996. No more than 3 isolates per farm were used. Ceftiofur had the highest activity in vitro against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, with MIC90 values of 0.03, 2, and 1 microg/ml, respectively. Premafloxacin was highly active against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, with MIC90 values of 2, 8, and 0.5 microg/ml, respectively, which were lower than those with enrofloxacin (MIC90 8, 32, and 2 microg/ml, respectively). Neomycin was moderately active against A. pleuropneumoniae and E. coli, with MIC90 values of 8 and 64 microg/ml, respectively, but was inactive with S. choleraesuis. Gentamicin showed high activity against A. pleuropneumoniae (MIC90 of 2 microg/ml) but was only moderately active with E. coli and S. choleraesuis (MIC90 of 64 and 32 microg/ml). Cephalothin was highly active against isolates of A. pleuropneumoniae (MIC90 of 1 microg/ml) but was inactive with E. coli (MIC90 of 128 microg/ml). Lincomycin had moderate activity (MIC90 of 32 microg/ml) against A. pleuropneumoniae. Chloramphenicol, lincomycin, and tetracycline were inactive with E. coli and S. choleraesuis (MIC90 > 128 microg/ml). In conclusion, ceftiofur and premafloxacin were highly active against isolates of A. pleuropneumoniae, E. coli, and S. choleraesuis, enrofloxacin and gentamicin were highly to moderately active; cephalothin was highly active against A. pleuropneumoniae and moderately active against S. cholearesuis; chloramphenicol, lincomycin, and tetracycline were active only with A. pleuropneumoniae; neomycin was moderately active against A. pleuropneumoniae and E. coli. The other antimicrobials tested were inactive.     

Antimicrobial resistance of Actinobacillus pleuropneumoniae isolated from swine

The aim of this retrospective study was to evaluate the antimicrobial resistance rates and the trend in resistance of Actinobacillus pleuropneumoniae isolated from pigs in Italy from 1994 to 2009. A total of 992 A. pleuropneumoniae isolates were tested for their susceptibility to a panel of antimicrobial agents in a disk diffusion method. Resistance to 7 drugs (amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, cotrimoxazole, penicillin G and tilmicosin) showed a significant increasing trend over the time, while for 2 drugs (gentamycin and marbofloxacin) a significant decrease was observed. Resistance to the remaining 14 antimicrobial agents tested did not change significantly over the study period. Most of the isolates retained high susceptibility to antimicrobials usually effective against A. pleuropneumoniae such as amphenicols, fluoroquinolones and ceftiofur. However, high rates of resistance were observed for potentiated sulfa drugs, tetracyclines and penicillins which are currently recommended antimicrobials for pig pleuropneumonia therapy. Our results suggest the importance of continued monitoring of A. pleuropneumoniae clinical isolates in order to choose the most appropriate treatment of infections and to control the increase of resistance to currently used antimicrobials.     

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.     

Serologic studies of Actinobacillus (Haemophilus) pleuropneumoniae strains of serotype-3 and their antigenic relationships with other A pleuropneumoniae serotypes in swine

Serotype-3 strains of Actinobacillus (Haemophilus) pleuropneumoniae possess epitopes shared with almost all other Actinobacillus serotypes. Common epitopes detected in particulate antigens were heat-labile and heat-stable and were of minor nature. Additional cross-reactive epitopes were detected in soluble and particulate antigens prepared from strains of serotypes 3, 6, and 8. Cross-reactions occurring between serotype-3 antigens and those of other serotypes were of 2 types: one associated mainly with 2-mercaptoethanol (2-ME)-sensitive IgM antibodies and the other associated mainly with 2-ME-resistant IgG antibodies. The cross-reactivity between serotypes 3, 6, and 8 was associated mainly with IgG antibodies, as shown by the results of 2-ME tube agglutination, 2-ME-indirect hemagglutination, and coagglutination tests. None of the tests was entirely satisfactory for serotyping serotype-3 isolates, mainly as a result of overlapping of type-specific antigenic determinants of serotypes 3, 6, and 8 in different combinations in the same strain. A combination of tests, using particulate and soluble antigens, may be necessary for typing of serotype-3 isolates.     

Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates and their antigenic relationships with other A pleuropneumoniae serotypes

Antigenic relationship of Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates with other serotypes was studied, using tube agglutination, with and without 2-mercaptoethanol, indirect hemagglutination with and without 2-mercaptoethanol, ring precipitation, coagglutination, and immunodiffusion tests. Serotype-8 isolates possessed serotype-specific, group-specific common antigens cross-reactive with serotypes 3 and 6 and species-specific common antigens cross-reactive with other serotypes. Absorption studies were done to study the antigenic relationship of serotype 8 with serotypes 3 and 6. Rabbit antisera against whole-cell (WC) suspensions of reference strains of serotypes 3, 6, and 8 were used for absorption studies with WC and boiled WC suspensions of homologous and heterologous serotypes. Unabsorbed and absorbed sera were tested for antibodies against WC and boiled WC antigen preparations of serotype 8, using various serotests. Absorption studies revealed that serotype-8 strains possessed 2 main types of epitopes, one of which was serotype-specific and did not have cross-reactivity with other serotypes. The second type of epitopes was group specific and was cross-reactive with serotypes 3 and 6.     

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)     

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.     

Seroprevalence of Actinobacillus (Haemophilus) pleuropneumoniae serotype-1 infection in swine herds in Quebec

Seroprevalence of Actinobacillus (Haemophilus) pleuropneumoniae serotype-1 infection was evaluated in pigs on 7 farms in Quebec. Commercial cross-bred herds A to G, ranging from 110 to 235 sows and infected with A pleuropneumoniae serotype-1 were selected. Five pigs/litter were selected at random and were identified (group 1). Blood samples were obtained from group-1 pigs at 2 to 4, 14, 28, 42, and 56 days of age. Blood also was obtained from group-1 pigs remaining in the postweaning unit at 70 days of age, and from 20 to 40 sows 1 to 3 times. To determine prevalence of seropositive pigs in all age groups for the entire study period in herds C to G, blood samples were obtained from 20 pigs/age group (group 2) selected at random at 28, 42, and 56 days of age at each visit. Group-1 pigs were included when they reached 28, 42, and 56 days of age. Pigs were serologically monitored in herds A and B for 3 months and in herds C to G for 5 to 6 months. Serologic status of pigs at 2 to 4 days of age was not statistically associated with status at 42 days (P = 0.6293) and at 56 days (P = 0.3098) of age for the same pigs. Therefore, seronegative pigs 2 to 4 days old did not seroconvert earlier than did those with detectable maternal antibodies at 2 to 4 days old. Only about 50% of the 70-day-old pigs were seropositive at 56 days. Seemingly, pigs seroconverted late in the postweaning period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial susceptibility of 51 strains of Haemophilus pleuropneumoniae.

Fifty-one strains of Haemophilus pleuropneumoniae were tested for susceptibility to 27 antimicrobial agents using agar disc diffusion, broth-tube dilution and microdilution methods. There was generally good agreement between the interpretation of the disc diffusion inhibition zones and the actual minimal inhibitory concentrations obtained with the dilution methods. The agreement between the results obtained with the broth-tube dilution method and the microdilution method was very good. Three strains were resistant to penicillin, ampicillin, carbenicillin, methicillin and tetracycline. One of those was also resistant to chloramphenicol. Forty strains were resistant to streptomycin, 23 strains were resistant to novobiocin and seven were resistant to triple sulfa. It is thus necessary to consider resistance development against antimicrobial agents chosen for the treatment of pleuro-pneumonia in pigs caused by Haemophilus pleuropneumoniae.     

Characterization and classification of Actinobacillus (Haemophilus) pleuropneumoniae plasmids.

Actinobacillus (Haemophilus) pleuropneumoniae plasmids were characterized and classified. They were isolated from A pleuropneumoniae strains different in serotype, year isolated, or location from which isolated. Six of 8 plasmids encoded streptomycin (Sm) and sulfonamide (Su) resistance (SmSu). One of the other plasmids, pVM105, encoded ampicillin (Ap) resistance and another, pHM0, encoded no drug resistance. All SmSu plasmids were transferred to Escherichia coli strains by transformation. Among them, pABO and pMS260 were 8.1 kb and incompatible with each other; they were stable in E coli. The other SmSu plasmids, pHM1, pVM104, pVM106, and pKD25, were 4.3 kb and did not replicate stably in E coli. The former SmSu plasmids were mobilized in E coli strains by a plasmid RP4, which belonged to incompatibility (Inc) group P, but the latter plasmids were not. Further, each 8.1-kb SmSu plasmid and each 4.3-kb plasmid had the same respective restriction pattern. These results indicated that there were at least 2 types of SmSu plasmids in A pleuropneumoniae. The 2 types were classified in 2 groups: H1(pMS260 and pABO) and H2(pHM1, pVM104, pVM106, and pKD25). The H1 and H2 plasmids belonged to different Inc groups, and H2 plasmids belonged to a different Inc group from that of pHMO and pVM105.     

Serological studies of Actinobacillus (Haemophilus) pleuropneumoniae strains of serotype 6 and their antigenic relationship with other serotypes

Serological tests such as agglutination, coagglutination, precipitation and indirect haemagglutination were used to study the antigenic relationship of reference and field strains of Actinobacillus (Haemophilus) pleuropneumoniae of serotype 6 with reference strains of other serotypes. Both cell-associated particulate and cell-free soluble antigens prepared from unheated and heat-treated bacterial suspensions of reference and field strains of serotype 6 were used in the studies. Species-specific, common antigenic determinants associated mainly with heat-treated particulate antigens of serotype 6 were cross-reactive in tube agglutination tests with almost all the serotypes. The species-specific antigens were of a minor nature because the cross-reactivities were abolished in both 2-mercaptoethanol agglutination and coagglutination tests. Cell-free saline extracts of both unheated and heat-treated suspensions of serotype 6 strains possessed epitopes specific for serotypes 3, 5 and 8 in addition to their own specific determinants. The epitopes were dominant because the reactions of strains of serotype 6 with antisera against serotypes 3, 5 and 8 persisted in almost all the serological tests used. Serotype 6 strains were antigenically closer to serotype 8 than to serotypes 3 or 5. A combination of serological tests such as coagglutination followed by 2-mercaptoethanol tube agglutination and, or, immunodiffusion tests differentiated serotype 6 strains from those of other cross-reacting serotypes.     

Improved protection of swine from pleuropneumonia by vaccination with proteinase K-treated outer membrane of Actinobacillus (Haemophilus) pleuropneumoniae

The immunogenic and protective potentials of an outer membrane-enriched fraction (OM) from a serotype 5 strain of Actinobacillus (Haemophilus) pleuropneumoniae (APP) and the same OM degraded with proteinase K or periodate were evaluated in swine. Groups of pigs were vaccinated with two doses of OM, proteinase K-treated OM (P-OM), periodate-treated OM (PI-OM), or placebo vaccine and challenged intranasally with the homologous strain of APP. Results from triplicate experiments indicated that proteinase K treatment of OM resulted in an improved efficacy. This improved efficacy of P-OM vaccine over untreated OM vaccine was evidenced not only by less severe lung lesions in P-OM vaccinated pigs but also by significant reduction (P less than 0.05) in the number of P-OM vaccinated pigs which developed lung lesions upon challenge with APP. Assessment of sera from vaccinated animals by immunoblotting, complement fixation test, or ELISA indicated that the immunogenicity of some but not all protein or carbohydrate components were reduced (or eliminated) by proteinase K and periodate treatments respectively.     

Effects of Mycoplasma hyopneumoniae and Actinobacillus (Haemophilus) pleuropneumoniae infections on alveolar macrophage functions in swine

Alveolar macrophages were collected at necropsy from pigs inoculated with Mycoplasma hyopneumoniae or Actinobacillus pleuropneumoniae or both and were tested for phagocytic capabilities, using in vitro techniques. Macrophages from noninoculated littermates were used as controls. Alveolar macrophages from pigs inoculated with either M hyopneumoniae or A pleuropneumoniae had significantly (P less than 0.05 to P less than 0.0025) higher phagocytic capacity than that of noninoculated controls. Macrophages from A pleuropneumoniae-inoculated pigs were comparatively more stimulated than were those from M hyopneumoniae-inoculated pigs. Pigs inoculated with M hyopneumoniae and then challenge-exposed with A pleuropneumoniae 2 and 4 weeks later had greatly reduced phagocytosis. Infection with M hyopneumoniae or A pleuropneumoniae caused stimulation of alveolar macrophage functions, and M hyopneumoniae infections may have suppressed phagocytic responses when pigs were challenge-exposed with a secondary pathogen (A pleuropneumoniae). This potential suppression may represent a prediposition of the host by M hyopneumoniae to secondary bacterial infections.     

Antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolates from clinical outbreaks of porcine respiratory diseases

Limited data regarding the susceptibility of Actinobacillus pleuropneumoniae to antimicrobials has been published during recent years. Accordingly, the aim of the present study was to investigate the distribution of MICs for the isolates of A. pleuropneumoniae from diseased pigs in the Czech Republic between 2007 and 2009. A total of 242 isolates were tested for susceptibility to 16 antimicrobial agents by a broth microdilution method. A low degree of resistance was observed for florfenicol (0.8%), amoxicillin and clavulanic acid (0.8%), tilmicosin (1.2%), tiamulin (1.7%) and ampicillin (3.3%), whereas resistance to tetracycline was detected more frequently, 23.9% of isolates. Interestingly, resistance to florfenicol has not yet been reported in any study investigating antimicrobial resistance of A. pleuropneumoniae. By PCR the presence of the floR gene was confirmed in all florfenicol resistant isolates.