Serotype and apx genotype profiles of Actinobacillus pleuropneumoniae field isolates in Korea.

A total of 100 field isolates of Actinobacillus pleuropneumoniae isolated from lung tissues of pigs with severe pleuropneumonia were serotyped by slide agglutination and precipitation tests. Polymerase chain reactions for apxICA, apxIICA, apxIIICA, apxIBD and apxIIIBD genes were used to determine their genotype prevalence. Serotypes 2 (56 isolates), 5 (28 isolates) and 6 (11 isolates) were the most common; only two isolates belonged to serotype 7, and three were untyped. Among the 97 isolates identified by serotype, 70 had the same apx genes as their respective serotype reference strains, but 27 did not have any of the apx genes present in the corresponding serotype reference strain. Among these 27 isolates, 10 were serotype 2, 12 were serotype 5, three were serotype 6 and two were serotype 7.     

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.     

Detection and localization of ApxI, -II and -III genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization

In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae. In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions. A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7. A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis. Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins. This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A. pleuropneumoniae.     

Adherence of Streptococcus suis to porcine endothelial cells

Streptococcus suis can cause invasive diseases in pigs and humans, such as meningitis or arthritis. Adherence to and invasion of endothelial cells might represent important steps in survival and spread of S. suis within the host. We tested in vitro adherence and invasion of S. suis strains using a porcine brain microvascular and aortal endothelial cell line. Four S. suis strains were tested with and without prior treatment with porcine serum containing anti-S. suis antibodies. Strains included a capsular serotype 2 strain and its non-encapsulated isogenic mutant strain, as well as two non-typeable (NT) strains, which expressed no capsule under our experimental conditions. Strains adhered to both cell lines to different extents depending on encapsulation and pre-treatment with porcine immune serum. The serotype 2 strain showed almost no adherence, whereas the non-encapsulated mutant strain adhered strongly. Similarly, both NT strains adhered substantially better than the serotype 2 strain. Pre-treatment of bacteria with porcine serum increased adherence of the encapsulated serotype 2 strain and decreased adherence of the non-encapsulated strains. None of the strains was able to efficiently invade either of the two cell lines, except for one NT strain, which showed a very low extend of invasion. Our results suggest that S. suis can adhere to but not invade porcine endothelial cells, and that this interaction may involve different bacterial surface structures, such as capsular polysaccharides and/or binding sites for serum components.     

Comparative evaluation of virulence and pathology of Streptococcus suis serotypes 2 and 9 in experimentally infected growers

Streptococcus (S.) suis is an invasive porcine pathogen causing meningitis, septicemia, arthritis and other diseases. Studies on pathogenesis as well as vaccine trials have focused on serotype 2 strains, which are worldwide the most prevalent among invasive isolates. However, in Europe serotype 9 strains also contribute substantially to S. suis-associated invasive diseases of piglets. The objective of this study was to determine the virulence of an MRP* SLY+ serotype 9 S. suis strain in comparison to an MRP+ EF+ SLY+ serotype 2 strain. Experimental intranasal and intravenous infections of 7-8 weeks old SPF piglets were investigated with regard to clinic and pathology. In contrast to the virulent serotype 2 strain, the serotype 9 strain did not cause disease with clinical manifestations after intranasal administration. However, histological screenings of these animals revealed pathological lesions, such as mild focal suppurative meningitis. Clinical manifestations related to meningitis, arthritis and serositis could be induced by intravenous application of this serotype 9 strain. Bacteriological culture and immunohistochemistry of the brain confirmed association with the S. suis challenge strains in all cases with clinical manifestations. Interestingly, expression of MRP within meningitis lesions was demonstrated for both pathotypes via immunohistochemistry. In conclusion, this study demonstrates that MRP* SLY+ serotype 9 strains are less virulent for growers than MRP+ EF+ SLY+ serotype 2 strains. Thus, intravenous application of this serotype 9 strain is required to evaluate heterologous protection in the course of vaccine development based on serotype 2 strains in the future.     

Molecular characterization of Streptococcus suis strains by 16S–23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.     

Prevalence of cpb2, encoding beta2 toxin,in Clostridium perfringens field isolates: correlation of genotype with phenotype

Beta2 toxin, encoded by the cpb2 gene, has been implicated in the pathogenesis of porcine, equine and bovine enteritis by type A Clostridium perfringens. By incorporating primers to cpb2 into a multiplex genotyping PCR, we screened 3270 field isolates of C. perfringens. Of these, 37.2% were PCR positive for the cpb2 gene. The majority of isolates from cases of porcine enteritis were positive for cpb2 (>85%), and this was even more true for C. perfringens isolated from cases of porcine neonatal enteritis (91.8%). In contrast, isolates from normal pigs only contained cpb2 in 11.1% of cases. The correlation between enteritis in other animal species and the presence of cpb2 was not so strong. cpb2 was found in 21.4% of C. perfringens isolates from cattle with enteritis, and in 47.3% of isolates from calves with enteritis or abomastitis. The prevalence of cpb2 varied with genotype, with type A isolates being positive for this gene in 35.1% of cases. Furthermore, enterotoxigenic type D or type E strains almost always carried cpb2. We cloned a 6xHIS-tagged beta2 (HIS-beta2) and used this protein to raise antiserum against beta2. Culture supernatants from 68 cpb2-positive and 13 cpb2-negative strains were tested for the presence of beta2 by Western blotting. In cpb2-positive isolates of porcine origin, beta2 was almost always detected (96.9%). However, in cpb2-positive isolates from other animal species, only 50.0% expressed beta2 protein. The high rate of cpb2-positivity among strains from neonatal pigs with enteritis and the high correlation of genotype with phenotype, supports the contention that beta2 toxin plays a role in the pathogenesis of these infections. However, it may be important to consider the use of an additional method for the detection of beta2 toxin in non-porcine cpb2-positive isolates when making claims about the role of beta2 in enteritis in non-porcine species.     

广西某猪场猪链球菌分离鉴定及其生物学特性研究

2019年12月广西玉林某猪场猪连续发生多起保育猪突然死亡病例,为确诊该猪场猪发病死亡原因并调查分离菌株的致病性与耐药情况,对发病猪场进行采样和细菌分离培养,并对分离菌株进行形态学观察、生化鉴定、gdh基因PCR扩增、血清型和7个毒力基因的鉴定、耐药性检测以及耐药基因的鉴定。结果显示,从两头病死猪中各分离出1株细菌,在TSB固体培养基表面培养为灰白色、表面光滑、边缘整齐且大小一致的圆形菌落,初步鉴定为革兰阳性链球菌,分别命名为GXYL-F1、GXYL-N2。经猪链球菌特异性基因gdh及血清型引物鉴定,两分离株均为9型猪链球菌。生化鉴定结果显示,两分离菌株均可发酵水杨素、麦芽糖、M.R.和七叶苷。PCR检测菌株的毒力基因结果显示,gdh、fbps、sao、sbp2'和orf2均为阳性,mrp、epf、sly基因均为阴性。药敏试验结果显示,两分离株均对新霉素、链霉素、替米考星、泰乐菌素、红霉素、四环素、左氧氟沙星、青霉素和磺胺嘧啶这9种药物呈耐药性,此外分离菌株GXYL-F1还对恩诺沙星耐药。两分离菌株均扩增出耐药基因aadA1、qnrB、qnrS。表明该猪场保育猪突然死亡是由9型猪链球菌引起。上述研究结果为该猪场制定9型猪链球菌防治措施提供参考依据,并为了解猪链球菌流行血清型多样性与防控技术研究提供了新的数据。     

我国部分地区猪繁殖与呼吸综合征病毒分离株ORF5基因的遗传变异分析

参考Genbank发表的猪繁殖与呼吸综合征病毒（PKRSV）ATCC VR-2332的ORF5基因序列，设计并合成了一对引物．对来自福建、浙江、山东等地的PRRSV分离毒株进行RT-PCR扩增．获得约748bp的DNA片断，将其分别克隆入pMD18-T载体中，并进行测序。应用DNAStar软件分析所测序列，并与ATCCVR-2332、CH-1a、MLV、Lv等毒株的ORF5序列进行比较，结果表明：SHDl与F114、MLV、ATCCVR-2332同源性高达98．5％，与CH-1a同源性为91．0％，与其它毒株同源性为86．6％~88．4％；F114与MLV、ATCCVR-2332同源性为99．3％～99．7％．其余分离毒株在遗传关系上和CH-1a又分为明显的两个群．显示近年来各地PRRSV分离毒株与cH-1a株的遗传差异越来越大。     

Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15.

We report on the re-examination of nine Australian isolates of Actinobacillus pleuropneumoniae that have been previously assigned to serovar 12. In the ring precipitation test, none of the nine isolates reacted with antisera to serovars 1-14 of A. pleuropneumoniae. Antiserum prepared against one of the Australian isolates gave no reaction with any of the 14 recognised serovar reference strains, except serovar 7. This reaction of the HS143 antiserum with serovar 7 antigen could be removed by adsorption with serovar 7 antigen. The adsorbed antiserum remained reactive with HS143 and the other eight Australian isolates. The nine Australian isolates were all shown to express ApxII and ApxIII, found in serovars 2, 4, 6 and 8, as well as the 42kDa outer membrane protein found in all serovars of A. pleuropneumoniae. The nine Australian isolates were found to possess the following toxin associated genes apxIBD, apxIICA, apxIIICA, apxIIIBD and apxIVA. The toxin gene profile of the Australian isolates is typical of A. pleuropneumoniae serovars 2, 4, 6 and 8. On the basis of the serological characterisation results and the toxin gene profiles, we propose that these isolates represent a new serovar of A. pleuropneumoniae--serovar 15--with HS143 being the reference strain for the new serovar.     

Experimental infections of mice and pigs with Streptococcus suis type 2.

Five inbred strains of mice were tested for their susceptibility to Streptococcus suis type 2 including the type strain, two isolates from meningitis in pigs and two isolates from tonsils of clinically healthy pigs. C57BL/6, ICR and ddY strain mice showed lower susceptibility to all strains of S. suis type 2 than BALB/c and SS strain mice. The type strain and the isolates from diseased pigs produced septicaemia and meningitis in BALB/c and SS mice inoculated with 10(8) colony forming unit of the bacteria and 60 to 100% of these infected mice died. On the other hand, mice inoculated with the isolates from healthy pigs showed mild clinical signs but none of them died. In BALB/c mice which died or developed nervous signs, the purulent meningo-encephalitis, myocarditis, ophthalmitis, labyrinthitis and otitis media were observed. S. suis type 2 antigen was demonstrated in these lesions by immunoperoxidase staining using rabbit S. suis type 2 antiserum. These results were similar to those in the experimentally infected pigs with these virulent and avirulent strains against mice. These results indicate that BALB/c and SS strains of mice are useful as an experimental model of S. suis type 2 infections in pigs, and that there are virulent and avirulent strains against mice and pigs among the strains of S. suis type 2.     

猪链球菌PCR检测技术研究进展

猪链球菌是猪的一种重要病原菌,并且也会引起人的链球菌病。有35个荚膜血清型(1/21、~34),通常自发病或死亡猪体分离获得1,2,7,9型和14型菌株,其中2型是毒力最强的血清型。根据已知猪链球菌16 SrRNA及溶血素(sly)、谷氨酸脱氢酶(gdh)、荚膜多糖(cps)、胞壁蛋白或溶菌酶释放相关蛋白(mrp)、胞外因子(epf)编码基因序列设计特异性引物,建立猪链球菌群和1(14),2(1/2),7型和9型特异性PCR或多重PCR,建立2型致病性菌株和1型高致病性菌株毒力鉴定PCR或多重PCR,用于检测和鉴别临床病料和细菌分离物中的猪链球菌,具有高敏感性和高特异性,与其他致病菌及其他血清的猪链球菌型无交叉反应,为疫病诊断及流行病学的研究提供了快速、简便和有用的工具。     

Colonization of suckling pigs by Streptococcus suis with particular reference to pathogenic serotype 2 strains.

Three swine commercial farms with high mortality rates in nursery pigs due to Streptococcus suis serotype 2 were studied. Brain samples from diseased animals were collected for a period of 6 to 10 mo and used to isolate the strain that was responsible for the mortality (virulent strain) in each farm. Tonsil swabs from piglets at 5, 10 and 15 d were taken to assess both total colonization and colonization by the virulent strain. The effect of sow vaccination against S. suis on colonization was evaluated in 1 of the farms. All suspect tonsil isolates were identified biochemically and then tested against serotype 2. The genomic patterns of serotype 2 isolates were compared to that of the virulent strain using Rep-PCR. Results showed that total colonization by S. suis occurred very early in the pigs' life, with most animals being colonized by weaning age. Prevalence of colonization by serotype 2 strains was much lower than total colonization. After comparing serotype 2 isolates with the virulent strains, only 1 tonsillar isolate had the same genomic pattern as the virulent strain and it belonged to a 4-week-old weaned pig. The genomic pattern of the virulent strain was not found in any tonsillar isolate from 15-day-old or younger pigs. Although limited by sample size, sow vaccination against S. suis increased total colonization at the same time significantly decreasing colonization by serotype 2 strains. Even though most pigs are colonized early in age by S. suis, colonization by the virulent strain is of low prevalence and delayed in time. This could constitute a risk factor for developing the disease later in time, because animals would be colonized when maternal immunity is no longer present, allowing the organism to become systemic.     

Comparison of pig, rabbit and mouse IgG response to Streptococcus suis serotype 2 proteins and active immunization of mice against the infection.

The aim of this study was to compare the IgG response of different animal species to Streptococcus suis serotype 2 proteins and to evaluate the immunogenic potential of these proteins in the mouse experimental model of infection. The protein profiles of ten different S. suis capsular type 2 isolates were compared by Western blotting using antisera produced in mice, rabbits and pigs against the reference strain. Strains were grown overnight in Todd-Hewitt broth, harvested by centrifugation, processed in a French press cell and digested with lysozyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then performed and proteins transferred to nitrocellulose. The rabbit antiserum recognized seventeen common immunoreactive proteins, of which, proteins of 33, 44, 96, 122 kDa were present in all strains. Two, 128 and 136 kDa proteins were recognized by swine serum in many strains. An additional protein of 30 kDa was recognized by the mouse antiserum. These seven proteins, originating from the reference strain, were excised directly from polyacrylamide gels, mixed with incomplete Freund's adjuvant and given to groups of five mice on days 0 and 10. Immunoglobulin G response to each protein was monitored on day 20 using Western blots. Mice were then experimentally infected on day 21. Results indicated that vaccination with proteins of 33, 44, 128 and 136 kDa resulted in an IgG response and protection against the challenge with the reference strain, but gave only a partial protection against another virulent S. suis serotype 2 strain.     

Detection and molecular typing of Streptococcus suis in tonsils from live pigs in France

Streptococcus suis is an important pathogen of swine, causing meningitis, arthritis, polyserositis, septicemia, and sudden death in weaning piglets as well as fattening pigs. Recently, 3 molecular tests have been developed in our laboratory: a multiplex polymerase chain reaction (m-PCR) assay for the detection of S. suis species and serotypes 2 and 1/2, and 2 molecular typing methods, pulsed-field gel electrophoresis and an approach based on PCR amplification of a fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S rDNA intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis (ISR-RFLP). In the present study, we used these tests to analyze tonsil samples from clinically healthy pigs and to identify individual isolates of S. suis during epidemiologic investigations of 8 related herds with a history of septicemia caused by S. suis serotype 2. Capsular typing showed that 58% of the strains were nontypable. Of the 17 serotypes present, serotype 22 was the most prevalent. In the 7 farms without clinical signs on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, in less than 5% of the pigs by m-PCR or by bacteriologic culture. In the 8th farm, on which 2 pigs had clinical signs of septicemia on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, by m-PCR in the tonsils of 40% of fattening pigs (21 wk old) that lacked symptoms. Molecular typing of the serotype 2 strains showed a common origin of contamination in these herds, given that 1 pattern (C1) was detected in the isolates from 6 of the 8 herds. However, up to 4 patterns were associated with septicemia and sudden death. Several patterns of S. suis serotype 2 can be responsible for disease in the same herd. These molecular tools may be useful for confident studies of the transmission of S. suis, thereby contributing to the control of S. suis infection.     

天津地区2型猪链球菌的分离鉴定及耐药性分析

为了解天津地区2型猪链球菌的流行及耐药情况,本研究收集该地区部分规模化养猪场疑似猪链球菌病病料317份,通过病原分离培养、染色特性观察、生化试验、PCR扩增等方法进行鉴定,并对分离菌进行致病性及药敏试验。结果从样品中分离鉴定出11株2型猪链球菌。对小鼠的致病力试验结果显示,应用0.5×10⁸ CFU/mL的细菌攻击小鼠,11株2型猪链球菌分离株中有6株具有致死性,其中1株致死率较强,5株致死率较弱。药敏试验结果表明,11株分离菌对9种临床常见药物均表现出很强的耐药性,其中对阿米卡星、四环素和强力霉素耐药性最强,耐药率达到100.00%;且所有分离株均呈多重耐药,其中4个分离株为9重耐药,占36.36%（4/11）。本试验结果表明,天津地区存在2型猪链球菌感染情况,且对多种抗菌药均产生了耐药性,应引起养殖户的高度重视,在防制猪链球菌病时,应有针对性地合理、科学、规范用药和交替用药。     

9型猪链球菌广东株的分离鉴定和基因序列分析

为了解9型猪链球菌广东分离株毒力因子基因型、基因变异和进化情况，本试验采集发病猪脑脊液、关节液、脾脏、肝脏、淋巴结进行细菌分离培养和生化鉴定，采用PCR方法鉴定其血清型及部分毒力基因，对cps9D、gdh、gapdh与orf2基因进行序列测定和分析，并构建系统发育树。结果显示，分离菌镜检为革兰氏阳性链状球菌，在鲜血琼脂平板中呈β溶血，可发酵大多数糖类，能成功扩增cps9D基因，含重要的保守基因gdh及毒力基因gapdh和orf2，表明分离菌株为9型猪链球菌。分离菌株cps9D、gdh、gapdh、orf2基因核苷酸序列与GenBank上国内外参考菌株的同源性分别为95.9%~100.0%、98.6%~99.8%、98.7%~99.6%和95.5%~99.9%，说明分离菌株与国内外其他9型猪链球菌毒株核苷酸同源性都较高，且与国内外的流行毒株基本一致，未发生太大的变异。系统发育树表明，分离株与国内外来源不同的猪链球菌分离株同源性高，亲缘关系密切。     

Serotyping and antigenic comparison of some animal rotaviruses isolated in China.

Eight strains of rotaviruses isolated from diarrheal animals (4 from calves and 4 from piglets) in China were compared by serotyping with reference animal rotavirus strains (bovine NCDV, porcine OSU and simian SA-11 and human rotavirus Wa strain). Two-way cross neutralization test showed no antigenic difference between all 4 local strains of bovine rotavirus (BRV007, BRV014, HN-7 and BRV6555) and reference NCDV, so they belonged to rotavirus serotype 6 (bovine rotavirus serotype 1 or NCDV-serotype). Meanwhile, the four strains of Chinese porcine rotavirus could be determined into 2 different serotypes. One (Li99) was neutralised to a high titer with the antiserum against reference OSU virus and probably related to OSU (serotype 5 or porcine serotype 1). The other three strains (Lin71, Nan86 and Jiang150) were antigenically obviously different from Li99 and did not react with the antiserum against OSU. They were tentatively considered as porcine rotavirus serotype 2. All the strains of bovine and porcine rotavirus did not cross-neutralise with simian SA-11 and human Wa strain. There was also no antigenic relationship between bovine rotaviruses and porcine rotaviruses.     

辽宁省部分地区猪繁殖与呼吸综合征病毒流行株ORF5-7基因遗传变异分析

根据GenBank发表的猪繁殖与呼吸综合征病毒(PRRSV)ATCC VR-2332全基因序列,设计并合成2对引物,采用RT-PCR技术,对辽宁省站送检的病料进行分离扩增,分别获得相应的目的片段,将其分别克隆入pMD18-T载体中,并送上海生工测序。应用DNAStar软件分析所测序列,并与ATCC VR-2332、CH-1a、BJ-4、LV-M96262及疫苗株的ORF567基因进行序列比较,通过核苷酸序列分析表明这5株流行株与VR-2332、CH-1a、BJ-4等代表株的同源性为89.5%～95%,与LV-M96262的同源性为54%～58.3%.氨基酸序列分析表明这5株流行株与VR-2332、CH-1a、BJ-4等代表株的同源性为82.8%～94.7%.     

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.