Detection of Actinobacillus pleuropneumoniae in the porcine upper respiratory tract as a complement to serological tests.

Attempts were made to isolate Actinobacillus pleuropneumoniae from the nasal cavities and tonsils of 442 healthy pigs from 15 herds. Samples were streaked onto different media formulations. Serum samples were assayed for antibodies to A. pleuropneumoniae by enzyme-linked immunosorbent assay and complement fixation test. Actinobacillus pleuropneumoniae was isolated from the nasal cavities only in 24 pigs, from tonsils only in 90 pigs, and from both the nasal cavities and the tonsils in 11 pigs. A PPLO medium supplemented with lincomycin, bacitracin and crystal violet allowed recovery of A. pleuropneumoniae from more animals than a tryptic soy agar medium from both sites. Incubation of plates in an enriched CO2 atmosphere did not affect the recovery rate. Actinobacillus pleuropneumoniae belonging to serotypes 1, 2, 3, 5a, 5b, 7, 8, 10 and 12 were isolated, and, in several herds, more than one serotype were recovered. Serotypes of A. pleuropneumoniae were isolated from nine herds which were found seronegative to these. The isolation of A. pleuropneumoniae from the upper respiratory tract can be useful for detection of carrier pigs and complements serological screening.     

Serotyping of Haemophilus pleuropneumoniae in the Netherlands: with emphasis on heterogeneity within serotype 1 and (proposed) serotype 9

Four hundred and forty-three Dutch field isolates of Haemophilus pleuropneumoniae were serotyped by rapid slide agglutination (RSA) using specific antisera against serotypes 1 to 5 and against the recently proposed types 6 to 9. The predominant serotypes were 9 (49%) and 2 (32%). Serotypes 1, 3, 5, 7 and 8 were isolated in small numbers: together they accounted for 3% of the total. Five percent of the isolates were not typable either due to autoagglutination or because they were not agglutinated by any of the available antisera. The remaining 49 strains (11%) agglutinated in more than one antiserum and could therefore not be properly classified. Forty-four of these 49 strains agglutinated in both anti type 1 and anti type 9 serum. Antigenic relationships between serotype 1, serotype 9 and isolates reacting with both antisera were studied using immunodiffusion and RSA with adsorbed sera. Serotype 9 strains appeared not to be a homogenous group. Isolates agglutinating exclusively in anti type 9 serum can be divided into two groups: one closely related and another hardly related to serotype 1. Serotype 9 reference strain 13261 belongs to the latter. Type 1 + 9 strains have antigens in common with serotypes 1 and 9, but they also have their own specific antigenic material. Such strains are proposed as a new serotype 10.     

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.     

An Actinobacillus pleuropneumoniae PCR typing system based on the apx and omlA genes--evaluation of isolates from lungs and tonsils of pigs

The genetic variability of a gene coding for an outer membrane lipoprotein (omlA) was used to develop a PCR typing system for Actinobacillus pleuropneumoniae. Sequence differences in the middle region of the gene divided the A. pleuropneumoniae serotypes in five distinct groups. Group I included serotypes 1, 9, 11 and 12 (omlA l), Group II consisted of serotypes 2 and 8 (omlA II), Group III included serotypes 3, 6 and 7 (omlA III), Group IV (omlA IV) consisted of serotype 4 and Group V of serotypes 5a, 5b and 10 (omlA V). The sequence differences were utilized to construct PCR primers specific for each group, except of Group IV, as the amplicon of serotype 4 could be separated from Group III by size. Together with a PCR apx typing system, the omlA PCR typing system could discriminate the majority of A. pleuropneumoniae serotypes of biovar 1 except of serotypes 1, 9 and 11 and serotypes 2 and 8. The PCR typing system was tested on 102 field strains of A. pleuropneumoniae isolated from lungs of diseased pigs. The serotyping results of the investigated field strains were in agreement with the apx and omlA gene patterns found in the reference strains of the bacteria, with the exception of the omlA gene of five strains of serotype 8. To examine the apx and omlA gene pattern of tonsil isolates, the PCR typing system was tested on a total of 280 A. pleuropneumoniae field strains isolated from tonsils of pigs. Agreement between serotyping and DNA typing was found in 96% of the isolates using the apx gene patterns and in 89% of the isolates using the omlA gene. The same serotype specific apx/omlA gene pattern was thus found in the majority of the tonsil isolates and in isolates from diseased lungs. Most of the differences in the omlA gene were found in 18 tonsil isolates of serotype 12. The omlA/apx PCR typing system described in the present study makes it possible to determine the type specificity of the majority of A. pleuropneumoniae isolates by simple PCR technique and enables phenotype independent characterization of isolates non-typable by serotyping.     

Atypical Actinobacillus pleuropneumoniae isolates that share antigenic determinants with both serotypes 1 and 7.

In the present study, the characterization of 3 atypical isolates of Actinobacillus pleuropneumoniae is presented. Two isolates (1B and 27E) showed positive reactions in coagglutination, immunodiffusion, and indirect hemagglutination tests for serotypes 1 and 7, whereas the third isolate (26B) reacted with antisera to serotypes 1, 4, and 7. These atypical isolates of A. pleuropneumoniae possessed a capsular polysaccharide (CPS) antigenically related to serotype 1 as well as an O-chain lipopolysaccharide antigenically related to serotype 7 or to serotypes 4 and 7, as shown by the use of monoclonal antibodies. Results of toxin profile and virulence assays for mice and pigs showed them to be more related to A. pleuropneumoniae serotype 7 field isolates. All 3 isolates induced antibodies mainly against serotype 7/4 O-long-chain lipopolysaccharide (LC-LPS) and, to a lesser extent, to the CPS of serotype 1, in experimentally infected pigs. Diagnostic laboratories that use a LC-LPS-based enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of A. pleuropneumoniae infection in swine would probably diagnose herds infected with these atypical isolates as being infected by A. pleuropneumoniae serotypes 7 or 4, whereas those that use a CPS-based ELISA would probably consider them as infected by A. pleuropneumoniae serotype 1.     

Serological and pathogenic characterization of Erysipelothrix rhusiopathiae isolates from tonsils of slaughter pigs in Indonesia

Erysipelothrix rhusiopathiae was isolated from tonsils of 245 (35.7%) of 687 apparently healthy slaughter pigs in Indonesia during the period of June 1987 to February 1988. A total of 150 of the 245 E. rhusiopathiae isolated could be serotyped within the 22 recognized serotypes. Serotype 2 was most prevalent with 23.7%, followed by Serotypes 11, 12, 1a, 5 and 6 representing 7.3, 5.3, 4.9, 4.9 and 4.1% of the isolates, respectively. The nine other serotypes (Serotypes 1b, 3, 4, 8, 9, 10, 13, 19 and 22) combined to make up 11.0% of the isolates. Antibiotic-resistant strains were not found. Of 86 selected isolates belonging to various serotypes, 76 (88.4%) were highly virulent for mice (LD50 less than 10(3.0) colony-forming units). In swine, 40 (51.2%) of 78 isolates induced local or generalized urticarial lesions after intradermal inoculation, and the remaining 38 isolates induced no clinical signs. Of 76 isolates used for challenge in the cross-protection study, 29 (38.2%) killed greater than 40% of mice immunized with an erysipelas bacterin marketed in Indonesia. A tendency to be refractory to the bacterin-induced immunity was observed in some isolates of various serotypes, but this characteristic was not consistent.     

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.     

Identification of serotypes of Haemophilus pleuropneumoniae by counterimmunoelectrophoresis

Counterimmunoelectrophoresis, direct immunofluorescence and immunodiffusion procedures were used to serotype 15 strains of Haemophilus pleuropneumoniae isolated from the respiratory tract of pigs in southern Brazil. Antigens were prepared by extracting cultures with a saline solution or by the phenol-water method. Antisera were prepared in rabbits against serotypes 1, 2, 3 and 5. Thirteen of the isolates were type 5 and two were type 3. No differences were observed between the results obtained in serotyping with counter immunoelectrophoresis and direct immunodiffusion, but both procedures were significantly better than immunodiffusion except with the saline extracted antigen. Counterimmunoelectrophoresis was quicker, more sensitive and more easily performed than the other techniques.     

An evaluation of agglutination and coagglutination techniques for serotyping of Haemophilus pleuropneumoniae isolates

A comparative evaluation of rapid slide agglutination, tube agglutination, 2-mercaptoethanol tube agglutination, and coagglutination tests was made for serotyping isolates of Haemophilus pleuropneumoniae. The results indicated that a majority of the isolates could be serotyped by any of these tests. But, it was not uncommon to find isolates which were inagglutinable or poorly agglutinable in homologous sera. Heat treatment of whole-cell suspensions of such isolates was essential to unmask the serotype-specific antigenic determinants; however, in the process of heat treatment, cross-reactive common antigens of minor nature were also exposed. The antibodies involved in such cross-reactions were mainly of immunoglobulin M type, because the cross-reactivities were completely abolished in coagglutination and 2-mercaptoethanol agglutination tests. Thus, both these tests were satisfactory for serotyping inagglutinable mucoid strains. For serotyping strains which were either polyagglutinating or autoagglutinating, agglutination tests could not be used, but the coagglutination test proved to be satisfactory. The coagglutination test was serotype-specific, sensitive, simple, rapid, reproducible, and easier to read and interpret than rapid slide or tube agglutination tests. This test could be used to serotype mucoid, smooth, or rough isolates.     

Serotypes of Erysipelothrix rhusiopathiae in Australian pigs, small ruminants, poultry, and captive wild birds and animals

Serotypes of 93 Australian isolates of Erysipelothrix rhusiopathiae from diseased domestic animals and poultry and a variety of captive wild birds and animals were determined by double diffusion gel precipitation. Two isolates, from the faeces of a swallow were also examined. Serotypes 1a, 1b and 2 were isolated from pigs and serotypes 1a, 1b, 2, 5, 15 and 21 from sheep or goats. Erysipelas in poultry was attributed to serotypes 1b, 5, 15 and 16. In captive wild birds serotypes 1b, 5, 6, 8, 14, 21 and an isolate reactive with antiserum to strain Seehecht were associated with septicaemic deaths. Single isolates from tissues of a bilby (Macrotis lagotis), black rat (Rattus rattus), brown snake (Pseudechis australis) and a bandicoot (Isoodon macrouris) were classified as serotypes 4, 4, 7, and 10 respectively. Six isolates were not able to be typed. Serotype 1b was the most widely distributed and most common (28%), being associated with disease in pigs, sheep, poultry and wild birds. Serotypes 1a or 2 were found in a more restricted range of animals, being commonly associated with erysipelas in pigs, less commonly in sheep and infrequently in other species. From diseased pigs, 26 of 33 isolates (79%) were serotypes 1a and 1b.     

Actinobacillus pleuropneumoniae in Thai pig herds. Prevalence of serum antibodies and relation to performance

The aim of the study was to investigate the prevalence of Actinobacillus pleuropneumoniae infections in market weight pigs in Thailand. ELISA systems employing purified lipopolysaccharide antigens were used to detect antibodies in 549 serum samples collected from pigs of 22 herds. Relevant cut-off values were established from three herds defined seronegative. Serum antibodies were detected to all serotypes except serotype 10. Almost 60% of the samples were seropositive to at least one serotype and 45% of the pigs were seropositive to more than one serotype. Antibodies to the cross-reacting serotypes 1, 9 or 11 were found in 29% of the pigs. Other common serotypes included the cross-reacting serotypes 3, 6 or 8 (26% seropositive pigs) and serotype 5a (also 26%). Antibodies to serotypes 2, 5b and 12 were low in prevalence (<10%). Three herds were regarded to be seronegative and six to have a low pathogen load with respect to the prevalence of seropositive pigs. The remaining 13 herds had a high incidence of pigs with antibodies to A. pleuropneumoniae, dominated by serotypes 1-9-11 and 5a (n = 6), serotypes 3-6-8, and 5a (n = 4) or 1-9-11, 3-6-8, 5a and 4-7 (n = 3). A low pathogen load with respect to A. pleuropneumoniae, as well as small herd size and age-segregated rearing, tended to improve the performance of growers.     

Serotype specificity of immunological assays for the capsular polymer of Actinobacillus pleuropneumoniae serotypes 1 and 9.

The cross-reactivity of the purified polysaccharides of Actinobacillus pleuropneumoniae serotypes 1 and 9 were examined using a variety of highly sensitive assays, such as radioimmunoassay, latex agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. In addition, conventional immunodiffusion was included for comparison. Latex agglutination, utilizing affinity-purified IgG to capsule, was also used to serotype whole cells. Agglutination or precipitation tests (radioimmunoassay, latex agglutination, and immunodiffusion) indicated no cross-reactivity between the capsules of serotypes 1 and 9, and no cross-reactivity between whole cells by latex agglutination. Assays that required binding of the capsule to a solid support (ELISA and immunoblotting) did demonstrate cross-reactions between serotypes 1 and 9 capsules, although reactions with the heterologous serotype were weaker than with the homologous serotype. The cross-reactivity could not be attributed solely to nonspecific factors because similar cross-reactivity did not occur with serotype 5 or 7 capsules by any assay. Reactivity of antisera with homologous or heterologous capsule was reduced, but not completely eliminated, by adsorption with washed, live bacteria of the heterologous serotype. Thus, the assay, as well as the antigen or specificity of the antibody reagent used, may influence the results of A. pleuropneumoniae serotyping or serological tests.     

Biochemical and serological identification of strains of Haemophilus pleuropneumoniae

Eighteen field isolates of Haemophilus pleuropneumoniae were studied biochemically and serotyped using the complement fixation test (CFT), agglutination test and the immunodiffusion test. Three biochemical tests (V-dependency, CAMP-reaction and urease activity) were found to be very useful for the biochemical characterization of the H. pleuropneumoniae. Haemolysis on blood agar plates, although present, was not sufficiently pronounced in all cases to warrant absolute dependence on this characteristic. Serological typing revealed the isolates belong to Serotypes 1 and 5. The immunodiffusion test proved to be the most serotype specific, while a marked cross-reaction was observed with the CFT.     

Actinobacillus (Haemophilus) pleuropneumoniae: use of coagglutination and complement fixation to determine the relationship between presence of organism and antibody titer in slaughterhouse pigs

The conventional culture method was compared to coagglutination for detection of Actinobacillus (Haemophilus) pleuropneumoniae in 425 sets of pig lungs. Sera from the same animals were evaluated for antibodies to A. pleuropneumoniae by the complement fixation (CF) test. All samples were collected at 2 packing plants in Iowa. In 2 nonvaccinated herds with no history of respiratory disease, the difference between standard culture results and coagglutination was highly significant (P less than 0.001). None of the 57 pigs in this group were positive for A. pleuropneumoniae by conventional culture, but 7 were positive by the coagglutination test. There were 15 animals with CF titers between 1:8 and 1:32. Animals from 6 herds vaccinated for A. pleuropneumoniae and without recent respiratory problems were evaluated. One out of 118 animals tested was positive for A. pleuropneumoniae by standard culture as compared to 9 positive by coagglutination. The difference in positive results between culture and coagglutination was highly significant (P less than 0.001). Twenty-eight animals had CF titers to A. pleuropneumoniae (1:4 to greater than or equal to 1:128). Two hundred fifty lungs and sera samples were collected from 7 herds which had recently experienced varying degrees of respiratory disease. Thirty-nine lungs were positive for A. pleuropneumoniae by culture and 182 were positive by coagglutination. The number of positives detected by coagglutination was significantly different (P less than 0.001) from the number positive by culture. There were 172 animals with antibody titers ranging from suspect to greater than or equal to 1:128. There were significantly fewer positive animals detected by standard culture than with the CF test (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Serological cross-reactivity between a porcine Actinobacillus strain and Haemophilus pleuropneumoniae.

During serological screening of a closed SPF-herd free of pleuropneumonia, more than half of the pigs were positive for complement-fixing antibodies to Haemophilus pleuropneumoniae. Actinobacillus bacteria closely related to A. suis were isolated from tonsillar tissue of 14 out of 20 slaughtered pigs submitted for pathological and bacteriological evaluation. None of the pigs had evidence of respiratory disease. Two pigs inoculated endobronchially with a selected Actinobacillus strain developed mild focal pneumonia and complement-fixing antibodies cross-reacting with H. pleuropneumoniae. Five pigs exposed and vaccinated with the Actinobacillus strain and five pigs spontaneously infected with the strain also developed complement-fixing antibodies against H. pleuropneumoniae and appeared to be less susceptible to experimental Haemophilus pleuropneumonia than pigs not exposed to the Actinobacillus infection. The agglutination test applied on serum treated with 2-mercaptoethanol detected antibodies against H. pleuropneumoniae serotype 5 but not against serotype 1 in pigs exposed to the Actinobacillus strain. Antibodies reactive with the Actinobacillus strain were also found in pigs hyperimmunized against H. pleuropneumoniae serotypes 1-5 in 2-mercaptoethanol tube agglutination test and rabbits hyperimmunized against serotypes 1,2 and 7, and strain 73567 in the immunodiffusion test. Conversely rabbits immunized against the Actinobacillus strain had antibodies against H. pleuropneumoniae serotypes 1, 3, 4, 5 and 6. It is concluded that pigs infected with Actinobacillus organisms may become false positive reactors against H. pleuropneumoniae.     

猪胸膜肺炎放线杆菌的分离与鉴定

从山东省泰安市采集的38份具有严重呼吸道症状的病死猪的肺脏、气管和扁桃体进行胸膜肺炎放线杆菌(APP)的分离鉴定,其中14株菌株表现出形态多样性、革兰氏染色阴性等特点,小鼠试验显示有较强的毒力,PCR电泳结果得到了650bp的预期目的片段。系统的生物学鉴定表明,这14株分离菌为胸膜肺炎放线杆菌(Actinobacillus Pleuropneaumoniae,APP)。对确诊的14株APP采用凝集试验进行血清型检测。结果从山东省泰安市分离到14株2个血清型的胸膜肺炎放线杆菌,其中,血清7型8株、血清5型6株,血清7型、5型为绝对优势血清型。     

Isolation of Streptococcus suis from diseased pigs in Canada

A total of 260 isolates of streptococci collected over a 9-year period from diseased pigs submitted for necropsy were studied. Seventy-seven percent of isolates were identified as S. suis and 32% of S. suis isolates were retrieved in pure culture. S. suis was found more frequently in lungs and was often isolated in conjunction with Actinobacillus pleuropneumoniae, Pasteurella multocida, Escherichia coli and other microorganisms. A total of 151 (76%) of S. suis isolates could be serotyped within the 9 recognized serotypes. Serotype 2 was the most prevalent with 33%, followed by serotypes 3, 5 and 7. All isolates were sensitive to ampicillin, penicillin, cephradine, chloramphenicol and trimethoprim-sulfamethoxazole. Resistance to streptomycin, neomycin and tetracycline appeared to be very high.     

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.     

Detection of strains of Haemophilus pleuropneumoniae using the coagglutination test

Three tests were used for the serological identification of the strains of Haemophilus pleuropneumoniae (Actinobacillus pleuropneumoniae) isolated from pigs and coming from 66 sites where pleuropneumonia, caused by Haemophilus, occurred in pigs. The coagglutination test was found to be the best for the identification of the causal agent; the ring precipitation test was somewhat less sensitive, and worse results were obtained when rapid slide agglutination was used. Of all the field isolates of H. pleuropneumoniae, serovar 2 occurred most frequently (56%), followed by serovar 1 (39%); one strain was identified as serovar 7. Two strains have remained unidentified. The serological identification of the strains was performed on the basis of their comparison with eight type serovars of H. pleuropneumoniae.     

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.