Prevalence of "Actinobacillus porcitonsillarum" in porcine tonsils and development of a diagnosis duplex PCR differentiating between "Actinobacillus porcitonsillarum" and Actinobacillus pleuropneumoniae

"Actinobacillus porcitonsillarum" is a newly suggested commensal species colonizing porcine tonsils. In the diagnostic laboratory the sole difference to the porcine lung pathogen Actinobacillus pleuropneumoniae is a negative mannitol reaction. In order to substantiate and improve this important differentiation a PCR test was developed using the relevant reference strains including Actinobacillus minor. The practicability of the test was confirmed on 20 clinical isolates of Actinobacillus spp. cultured from 100 tonsil samples originating from 18 farms in Thailand. Applying the newly developed PCR test 10 isolates were identified as A. pleuropneumoniae, and 10 as "A. porcitonsillarum" with one of them being mannitol-positive in biochemical testing. Subsequent 16S rRNA sequencing confirmed classification of all 10 strains as "A. porcitonsillarum"/A. minor. These results emphasize that suspected A. pleuropneumoniae isolates, particularly from porcine tonsils, should be confirmed by PCR in order to prevent false positive diagnoses.     

Phylogenetic relationship of equine Actinobacillus species and distribution of RTX toxin genes among clusters

Equine Actinobacillus species were analysed phylogenetically by 16S rRNA gene (rrs) sequencing focusing on the species Actinobacillus equuli, which has recently been subdivided into the non-haemolytic A. equuli subsp. equuli and the haemolytic A. equuli subsp. haemolyticus. In parallel we determined the profile for RTX toxin genes of the sample of strains by PCR testing for the presence of the A. equuli haemolysin gene aqx, and the toxin genes apxI, apxII, apxIII and apxIV, which are known in porcine pathogens such as Actinobacillus pleuropneumoniae and Actinobacillus suis. The rrs-based phylogenetic analysis revealed two distinct subclusters containing both A. equuli subsp. equuli and A. equuli subsp. haemolyticus distributed through both subclusters with no correlation to taxonomic classification. Within one of the rrs-based subclusters containing the A. equuli subsp. equuli type strain, clustered as well the porcine Actinobacillus suis strains. This latter is known to be also phenotypically closely related to A. equuli. The toxin gene analysis revealed that all A. equuli subsp. haemolyticus strains from both rrs subclusters specifically contained the aqx gene while the A. suis strains harboured the genes apxI and apxII. The aqx gene was found to be specific for A. equuli subsp. haemolyticus, since A. equuli subsp. equuli contained no aqx nor any of the other RTX genes tested. The specificity of aqx for the haemolytic equine A. equuli and ApxI and ApxII for the porcine A. suis indicates a role of these RTX toxins in host species predilection of the two closely related species of bacterial pathogens and allows PCR based diagnostic differentiation of the two.     

Antimicrobial resistance profile of Actinobacillus pleuropneumoniae and Actinobacillus porcitonsillarum

A total of 83 Actinobacillus pleuropneumoniae and 58 Actinobacillus porcitonsillarum strains collected from slaughtered pigs in Switzerland were screened for susceptibility to 20 antimicrobial agents by MIC determinations. Resistance to sulfamethoxazole, the combination sulfamethoxazole-trimethoprim, tiamulin, tilmicosin, tetracycline, penicillin and ampicillin were found. A few A. porcitonsillarum isolates displayed decreased susceptibility to enrofloxacin. PCR analysis revealed the presence of the sul2 gene in approximately one-fifth of the sulfonamide-resistant A. pleuropneumoniae and A. porcitonsillarum isolates. The tetracycline-resistant A. pleuropneumoniae harbored tet(B) and tet(H), whereas the tetracycline-resistant A. porcitonsillarum isolates harbored the tet(B) gene. The penicillin and ampicillin-resistant A. pleuropneumoniae and A. porcitonsillarum harbored the bla(ROB-1) gene.     

Apx toxins in Pasteurellaceae species from animals

Pasteurellaceae species particularly of porcine origin which are closely related to Actinobacillus pleuropneumoniae were analyzed for the presence of analogues to the major A. pleuropneumoniae RTX toxin genes, apxICABD, apxIICA and apxIIICABD and for their expression. Actinobacillus suis contains both apxICABD(var.suis) and apxIICA(var. suis) operons and was shown to produce ApxI and ApxII toxin. Actinobacillus rossii contained the operons apxIICA(var.rossii) and apxIIICABD(var.rossii). However, only the toxin ApxII and not ApxIII could be detected in cultures of A. rossii. The Apx toxins found in A. suis and A. rossi may play a role in virulence of these pathogens. Actinobacillus lignieresii, which was included since it is phylogenetically very closely related to A. pleuropneumoniae, was found to contain a full apxICABD(var.lign.) operon which however lacks the -35 and -10 boxes in the promoter sequences. As expected from these results, no expression of ApxI was detected in A. lignieresii grown under standard culture conditions. Actinobacillus seminis, Actinobacillus equuli, Pasteurella aerogenes, Pasteurella multocida, Haemophilus parasuis, and also Mannheimia (Pasteurella) haemolytica, which is known to secrete leukotoxin, were all shown to be devoid of any of the apx toxin genes and did not produce ApxI, ApxII or ApxIII toxin proteins. However, proteins of slightly lower molecular mass than ApxI, ApxII and ApxIII which showed limited cross-reactions with monospecific, polyclonal anti-ApxI, anti-ApxII and anti-ApxIII were detected on immunoblot analysis of A. equuli, A. seminis and P. aerogenes. The presence of Apx toxins and proteins that imunologically cross react with Apx toxins in porcine Actinobacillus species other than A. pleuropneumoniae can be expected to interfere with serodiagnosis of porcine pleuropneumonia.     

Acquisition of haemoglobin-bound iron by strains of the Actinobacillus minor/"porcitonsillarum" complex

Members of the Actinobacillus minor/"porcitonsillarum" complex are common inhabitants of the swine respiratory tract. Although avirulent or of low virulence for pigs, these organisms, like pathogens, do grow in vivo and must, therefore, be able to acquire iron within the host. Here, we investigated the abilities of six members of the A. minor/"porcitonsillarum" complex to acquire iron from transferrin and various haemoglobins. Using growth assays, all six strains were shown to acquire iron from porcine, bovine and human haemoglobins but not from porcine transferrin. Analyses of whole genome sequences revealed that A. minor strains NM305(T) and 202, unlike the swine-pathogenic actinobacilli, A. pleuropneumoniae and A. suis, lack not only the transferrin-binding protein genes, tbpA and tbpB, but also the haemoglobin-binding protein gene, hgbA. Strains NM305(T) and 202, however, were found to possess other putative haemin/haemoglobin-binding protein genes that were predicted to encode mature proteins of ～ 72 and ～ 75 kDa, respectively. An affinity procedure based on haemin-agarose allowed the isolation of ～ 65 and ～ 67 kDa iron-repressible outer membrane polypeptides from membranes derived from strains NM305(T) and 202, respectively, and mass spectrometry revealed that these polypeptides were the products of the putative haemin/haemoglobin-binding protein genes. PCR approaches allowed the amplification and sequencing of homologues of both haemin/haemoglobin-binding protein genes from each of the other four strains, strains 33PN and 7ATS of the A. minor/"porcitonsillarum" complex and "A. porcitonsillarum" strains 9953L55 and 0347, suggesting that such proteins are involved in the utilization of haemoglobin-bound iron, presumably as surface receptors, by all six strains investigated.     

Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections, development and prevalidation of the ApxIV ELISA

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, which causes worldwide severe losses in pig farming. The virulence of the 15 serotypes of A. pleuropneumoniae is mainly determined by the three major RTX toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX toxin, ApxIV, is produced by all 15 serotypes only during infection of pigs, but not under in vitro conditions. Pigs infected with A. pleuropneumoniae show specific antibodies directed against ApxIV. In contrast, antibodies against the other three toxins ApxI, ApxII and ApxIII are also found in pigs free of A. pleuropneumoniae. The antibodies to the three latter might result from other, less pathogenic Actinobacillus species such as A. rossii and A. suis. We used a recombinant protein based on the N'-terminal part of ApxIV to serologically detect A. pleuropneumoniae infections in pigs by immunoblot analysis. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected A. pleuropneumoniae infections in the second to third week post infection. We developed an indirect ELISA based on the purified recombinant N'-terminal moiety of ApxIV. The analysis of sera from pigs that were experimentally or naturally infected by A. pleuropneumoniae, and of sera of pigs that were free of A. pleuropneumoniae, revealed that the ELISA had a specificity of 100% and a sensitivity of 93.8%. The pre-validation study of the ApxIV-ELISA revealed that the latter was able to detect A. pleuropneumoniae-positive herds, even when clinical and pathological signs of porcine pleuropneumonia were not evident. Pigs vaccinated with a subunit vaccine Porcilis App were serologically negative in the ApxIV-ELISA.     

Non-pathogenic Actinobacillus isolates antigenically and biochemically similar to Actinobacillus pleuropneumoniae: a novel species?

Two unusual Actinobacillus isolates were recovered from pigs with no clinical signs, no lesions and no history of swine pleuropneumonia. Two representative strains (9953L55 and 0347) analyzed in this study were initially biochemically and antigenically identified as A. pleuropneumoniae serotypes 1 and 9, respectively, by traditional identification methods. Both strains presented, however, negative results with three A. pleuropneumoniae-specific PCR tests and revealed in particular the absence of the apxIV toxin genes. However, both strains produced and secreted ApxII toxin although they only harbored the toxin genes apxIICA, which is an uncommon feature for any of the known A. pleuropneumoniae serotypes. Upon experimental inoculation of pigs, these strains proved to be totally non-pathogenic. Animals infected with one of the strains produced antibodies that cross-react with A. pleuropneumoniae serotypes 1-9-11-specific LC-LPS ELISA. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that these strains form a separate phylogenetic group that is distinct from other Actinobacillus species and is particularly different from A. pleuropneumoniae.     

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.     

Distribution of RTX toxin genes in strains of [Actinobacillus] rossii and [Pasteurella] mairii

Strains of [Actinobacillus] rossii, [Pasteurella] mairii and [Pasteurella] aerogenes can be isolated from abortion in swine. The RTX toxin Pax has previously been found only in those [P.] aerogenes strains isolated from abortion. Nothing is known about RTX toxins in field isolates of the other two species. To gain insight into the distribution of selected RTX toxin genes and their association with abortion, PCR screening for the pax, apxII and apxIII operons on 21 [A.] rossii and seven [P.] mairii isolates was done. Since species can be phenotypically misidentified, the study was backed up by a phylogenetic analysis of all strains based on 16S rRNA, rpoB and infB genes. The pax gene was detected in all [P.] mairii but not in [A.] rossii strains. No apx genes were found in [P.] mairii but different gene combinations for apx were detected in [A.] rossii strains. Most of these strains were positive for apxIII, either alone or in combination with apxII. Whereas pax was found to be associated to strains from abortion no such indication could be found with apx in [A.] rossii strains. Phylogenetically [A.] rossii strains formed a heterogeneous cluster separated from Actinobacillus sensu stricto. [P.] mairii strains clustered with [P.] aerogenes but forming a separate branch. The fact that [P.] aerogenes, [P.] mairii and [A.] rossii can phylogenetically clearly be identified and might contain distinct RTX toxin genes allows their proper diagnosis and will further help to investigate their role as pathogens.     

Prevalence of Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella multocida, and Streptococcus suis in representative Ontario swine herds

Tonsillar and nasal swabs were collected from weanling pigs in 50 representative Ontario swine herds and tested for the presence of 5 important bacterial upper respiratory tract pathogens. All but 1 herd (2%) tested positive for Streptococcus suis by polymerase chain reaction (PCR); 48% of herds were S. suis serovar 2, 1/2 positive. In all but 2 herds there was evidence of Haemophilus parasuis infection. In contrast, toxigenic strains of Pasteurella multocida were detected by a P. multocida--enzyme-linked immunosorbant assay (PMT-ELISA) in only one herd. Seventy-eight percent of the herds were diagnosed positive for Actinobacillus pleuropneumoniae by apxIV PCR. Sera from finishing pigs on the same farms were also collected and tested by ELISA for the presence of A. pleuropneumoniae antibodies. Seventy percent of the herds tested had evidence of antibodies to A. pleuropneumoniae including serovars 1-9-11 (2%), 2 (4%), 3-6-8-15 (15%), 5 (6%), 4-7 (26%), and 12 (17%). This likely represents a shift from previous years when infection with A. pleuropneumoniae serovars 1, 5, and 7 predominated. At least 16% and possibly as many as 94% of the herds tested were Actinobacillus suis positive; only 3 of the 50 herds were both A. pleuropneumoniae and A. suis negative as judged by the absence of a positive PCR test for apxII. Taken together, these data suggest that over the past 10 years, there has been a shift in the presence of pathogenic bacteria carried by healthy Ontario swine with the virtual elimination of toxigenic strains of P. multocida and a move to less virulent A. pleuropneumoniae serovars. As well, there appears to be an increase in prevalence of S. suis serovar 2, 1/2, but this may be a reflection of the use of a more sensitive detection method.     

ISApl1, a novel insertion element of Actinobacillus pleuropneumoniae, prevents ApxIV-based serological detection of serotype 7 strain AP76

Actinobacillus pleuropneumoniae, a gram-negative rod of the Pasteurellaceae family, causes pleuropneumonia in pigs. Establishing A. pleuropneumoniae free herds is difficult due to the occurrence of persistently infected animals. The ApxIV toxin is expressed by A. pleuropneumoniae in vivo and an ELISA based on the toxin is used to detect infection and to differentiate between infected and vaccinated animals. In this study, we have identified a 1070bp insertion element of the IS30 family, designated ISApl1, in the A. pleuropneumoniae serotype 7 strain AP76. ISApl1 contains a 924bp ORF encoding a transposase, which is flanked by 27bp inverted repeats showing six mismatches. We investigated the occurrence of ISApl1 in other A. pleuropneumoniae strains, and its possible interference with virulence associated factors. Four insertion sites were identified in AP76: within the apxIVA toxin ORF, within a putative autotransporter adhesin ORF, upstream of a capsular polysaccharide biosynthesis gene cluster, and downstream of a beta-lactamase gene. ISApl1 is also present in some serotype 7 field isolates, but not in reference or field strains of other serotypes. In A. pleuropneumoniae AP76, the transposase gene is transcribed in vitro. The insertion in the apxIVA toxin gene remains stable after animal passage. Since this insertion should disrupt toxin expression, we tested 7 pigs infected with AP76 at day 21 post-infection. All were negative in the ApxIV ELISA but four out of seven were positive in an ApxII toxin ELISA. These results show that insertion elements can affect the detection of A. pleuropneumoniae infected animals.     

Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia

ABSTRACT: Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia.     

Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.     

Host cell specific activity of RTX toxins from haemolytic Actinobacillus equuli and Actinobacillus suis

We assessed and compared host cell specificity of the haemolytic and cytotoxic activity of the RTX toxins from Actinobacillus equuli, an equine pathogen, and Actinobacillus suis, which is pathogenic for pigs. The two bacterial species are closely related, phenotypically as well as phylogenetically, sharing the same 16S rRNA gene sequence. Both species contain specific protein toxins from the family of pore-forming RTX toxins, however, the two species differ in their RTX toxin profiles. Haemolytic A. equuli contains the operon for the Aqx toxin, whereas A. suis harbours genes for ApxI and ApxII. We tested the toxic activity of the corresponding proteins on erythrocytes as well as on lymphocytes isolated from horse and pig blood. The strength of the haemolytic activity for each of the toxins was independent of the origin of erythrocytes. When testing cytotoxic activity, the Aqx protein showed a higher toxic effect for horse lymphocytes than for porcine lymphocytes. On the other hand, ApxI and ApxII showed a strong cytotoxic effect on porcine lymphocytes and a reduced toxicity for horse lymphocytes; the toxicity of ApxII was generally much lower than ApxI. Our results indicate a host species specificity of the toxic activity of RTX toxins Aqx of A. equuli and ApxI and ApxII of A. suis.     

Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model

Actinobacillus pleuropneumoniae is an infective agent that leads to porcine pleuropneumonia, a disease that causes severe economic losses in the swine industry. Based on the fact that the respiratory tract is the primary site for bacterial infection, it has been suggested that bacterial exclusion in the respiratory tract through mucosal immune induction is the most effective disease prevention strategy. ApxIIA is a vaccine candidate against A. pleuropneumoniae infection, and fragment #5 (aa. 439–801) of ApxIIA contains the major epitopes for effective vaccination. In this study, we used mice to verify the efficacy of intranasal immunization with fragment #5 in the induction of protective immunity against nasal challenge with A. pleuropneumoniae and compared its efficacy with that of subcutaneous immunization. Intranasal immunization of the fragment induced significantly higher systemic and mucosal immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. Intranasal immunization not only efficiently inhibited the bacterial colonization in respiratory organs, but also prevented alveolar tissue damage in infectious condition similar to that of a contaminated pig. Moreover, intranasal immunization with fragment #5 provided acquired protective immunity against intranasal challenge with A. pleuropneumoniae serotype 2. In addition, it conferred cross-protection against serotype 5, a heterologous pathogen that causes severe disease by ApxI and ApxII secretion. Collectively, intranasal immunization with fragment #5 of ApxIIA can be considered an efficient protective immunization procedure against A. pleuropneumoniae infection.     

Isolation of Actinobacillus pleuropneumoniae serovar 15-like strain from a field case of porcine pleuropneumonia in Japan

An Actinobacillus pleuropneumoniae strain isolated from a field case of porcine pleuropneumonia in Japan, was closely related to a reference strain of serovar 15, which is a newly proposed serovar according to an analysis of field isolates originating from Australia. The isolate had biological and biochemical properties consistent with A. pleuropneumoniae biovar 1, and reacted strongly to a rabbit antiserum raised against a reference strain of serovar 15 in an agar gel precipitation test. The nucleotide sequence of a hyper variable region in the 16S RNA gene of the isolate was identical to that of the reference strain of serovar 15. The isolate possessed A. pleuropneumoniae-RTX toxin (Apx) II, III, and IV genes, consistent with serovar 15. Its virulence in mice was lower than that of ApxI-bearing strains but higher than that of other ApxIII-bearing strains. This is the first report describing the isolation of A. pleuropneumoniae serovar 15-like strain from a country or region other than Australia.     

大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体构建

合成胸膜肺炎放线杆菌质粒pKMA2425上长度为126bp及2214bp的2个DNA片段,克隆到大肠埃希氏菌质粒pGSI中,获得重组质粒pGSIA。PCR扩增pTKRED上的大观霉素抗性基因及线性化的pGSIA（126bpDNA片段-pGSI-2214bpDNA片段）,连接,电转化胸膜肺炎放线杆菌,在大观霉素的选择压力下筛选鉴定到连接正确的质粒,命名为pGSIAS。pGSIAS测序结果符合预期。在大观霉素的选择压力下,含有pGSIAS的胸膜肺炎放线杆菌血清1-10型菌株均生长良好;在氨苄青霉素的选择压力下,含有pGSIAS的大肠埃希氏菌生长良好。结果表明本研究构建的大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体pGSIAS序列正确,在胸膜肺炎放线杆菌及大肠埃希氏菌中均能复制并表达质粒上的相关基因。     

Lack of influence of the anaerobic [NiFe] hydrogenase and L-1,2 propanediol oxidoreductase on the outcome of Actinobacillus pleuropneumoniae serotype 7 infection

The genes for the large subunit of [NiFe] hydrogenase 2, (hybB) and for L-1,2 propanediol oxidoreductase (fucO), were identified in an Actinobacillus (A.) pleuropneumoniae serotype 7 strain. Based on the hypothesis that adaptation to anaerobic conditions in damaged lung tissue may play a role in A. pleuropneumoniae persistence in host tissues, deletion mutants with a deletion in the hybB or the fucO gene were constructed and examined in an aerosol infection model. Deletion of the hybB or fucO genes appeared to have no significant effect on A. pleuropneumoniae virulence.     

Development of a multiplex PCR test for identification of Actinobacillus pleuropneumoniae serovars 1, 7, and 12

A PCR assay for simultaneous species identification and separation of Actinobacillus pleuropneumoniae serovars 1, 7 and 12 was developed. Primers specific for genes involved in biosynthesis of the capsular polysaccharides (cps genes) of serovars 1, 7, and 12 were combined with a species-specific PCR test based on the omlA gene. The PCR test was evaluated with the serovar reference strains of A. pleuropneumoniae as well as 183 Danish field isolates. For all typable strains, a complete correspondence was found between results obtained with the multiplex PCR test and results from the traditional serotyping methods. Among eight serologically cross-reacting strains designated K1:O7, seven isolates produced amplicons of similar sizes as serovar 1 and one isolate produced amplicons of similar sizes as serovar 7. The species specificity of the assay was evaluated using a collection of 126 strains representing 25 different species within the family Pasteurellaceae including 45 field strains of the phylogenetically affiliated species Actinobacillus lignieresii. All these isolates tested negative for the cps genes by the multiplex PCR test except for 6 isolates of A. lignieresii. Five of these isolates produced an amplicon identical to the cps gene of serovar 7, whereas one isolate produced an amplicon identical to the cps gene of serovar 1. In addition, four isolates of Actinobacillus genomospecies 1 tested positive for the omlA gene but negative for the cps genes. The test represents a convenient and specific method for serotyping A. pleuropneumoniae in diagnostic laboratories.     

Revised definition of Actinobacillus sensu stricto isolated from animals. A review with special emphasis on diagnosis

The taxonomy of the members of the genus Actinobacillus associated with animals has been reviewed with focus on classification and identification including molecular based characterization, typing and identification. Out of the 22 species or species like taxa reported as Actinobacillus, 19 are associated with animals. When classified on the basis of 16S rRNA sequence based phylogenetic analysis, DNA-DNA hybridizations and phenotypic analysis, Actinobacillus sensu stricto is restricted to include A. lignieresii, A. pleuropneumoniae, A. equuli subsp. equuli, A. equuli subsp. haemolyticus (taxon 11 of Bisgaard), A. hominis, A. suis, A. ureae, A. arthritidis (taxon 9 of Bisgaard), Actinobacillus genomospecies 1 and 2 and the taxa 8 and 26 of Bisgaard. The remaining 11 species of Actinobacillus are unrelated to A. sensu stricto and should consequently be grouped with other genera or be renamed as new genera depending on new data. Identification of members of Actinobacillus at species level is possible through phenotypic characterization combined with information on host of isolation. PCR tests are available for specific detection of A. pleuropneumoniae. Only A. pleuropneumoniae is presently considered as a primary pathogen. Based on different types of RTX genes it is possible to PCR type A. pleuropneumoniae to serotype level. PCR might also be used for the specific detection of A. equuli subsp. haemolyticus. Epidemiological investigations and surveillance have so far included serotyping, multilocus enzyme electrophoresis (MLEE), ribotyping and restriction fragment length profiling.