Comparison and partial characterization of the protein profiles and outer membrane antigens of Actinobacillus species isolated from ram lambs with epididymitis

Three monoclonal antibodies (LG17, LG30, LG33) were used in the indirect fluorescent antibody test, the ELISA, and the immunoelectrotransfer blot technique to identify group-specific and strain-specific epitopes on the outer membranes of Actinobacillus seminis, A actinomycetemcomitans, and 17 field isolates of Actinobacillus spp. The field isolates had been obtained by bacteriologic culture of specimens from ram lambs with epididymitis. Only antibody LG33 consistently had specificity for an outer membrane epitope shared by most of the bacterial isolates tested. Staining of polyacrylamide gels with periodic acid-Schiff reagent, Sudan black B, and Coomassie brilliant blue R250 indicated that target antigens for antibodies LG17 and LG33 contained carbohydrate and lipoprotein components, respectively. The chemical composition of the LG30 target antigen was not determined because of its instability after exposure to sodium dodecyl sulfate. Discontinuous-gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate and spectrophotometric scans of the gels were used to analyze n-octyl-beta-D-glucopyranoside protein extracts from A seminis, A actinomycetemcomitans, and 13 representative field isolates of Actinobacillus spp. Bacterial isolates could be grouped according to their protein profiles. The first group consisted of A seminis, A actinomycetemcomitans, and 7 field isolates of Actinobacillus spp, all of which shared common protein bands with molecular masses of approximately 94 kilodaltons (kD), 64 kD, 60 kD, 52 kD, 44 kD, and 26 kD. The second group was composed of 6 field isolates, each with unique protein profiles; isolates had relatively few protein bands in common. These data suggested that members of the genus Actinobacillus cultured from ram lambs with epididymitis probably include a number of various strains.     

An evaluation of the API ZYM system as a means of identifying Haemophilus somnus and related taxa.

The commercially available API ZYM microbiological identification system was evaluated for the rapid identification of Haemophilus somnus. Eighty-seven isolates of the organism had API ZYM profiles which were characteristic. The API ZYM profiles demonstrate clear differences between H. somnus and other genera but suggest a close association to three related organisms. Enzyme activity of H. somnus isolates were similar to organisms identified as Histophilus ovis, Haemophilus agni and strains UQV of Actinobacillus actinoides and Actinobacillus seminis but was clearly different from isolates of Pasteurella haemolytica, Pasteurella multocida, Bordetella bronchiseptica and group EF4. The API ZYM system allowed more rapid identification of H. somnus than conventional biochemical tests and may be a useful adjunct to conventional methods used for identification of H. somnus isolates. The test did not reveal obvious differences between isolates from various anatomic locations.     

Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis.     

Association of sexual experience with isolation of various bacteria in cases of ovine epididymitis

Testicles from rams in flocks experiencing ram epididymitis in Idaho and eastern Oregon were cultured. Twenty-six breeding rams from 6 flocks were cultured and only Brucella ovis was isolated. Virgin rams (65) harbored numerous species of small fastidious gram-negative rods, including Actinobacillus actinomycetemcomitans, Haemophilus spp, Moraxella spp, and Pasteurella spp. Thus, there appeared to be 2 separate disease entities, dependent on sexual experience of the animal.     

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.     

Use of monoclonal antibodies to identify outer membrane antigens of Actinobacillus species

Three monoclonal antibodies (LG17, LG30, and LG33) were used to identify outer membrane antigens of Actinobacillus sp (As8C isolate) cultured from the epididymides of an infected ram lamb. Specificity of the 3 antibodies to As8C antigens was determined by use of bacterial agglutination, the enzyme-linked immunosorbent assay, and the indirect fluorescent antibody test. Results of immunoelectron microscopy confirmed that each antibody was specific for epitopes on As8C outer membrane antigens. Evaluation by use of enzyme-linked immunoelectrotransfer blot indicated that target antigens for LG17 and LG33 antibodies had molecular weights of 10 kilodaltons and 43 kilodaltons, respectively. Multiple-band staining was observed with the LG33 antibody. The target antigen for the LG30 antibody could not be discerned by use of enzyme-linked immunoelectrotransfer blot. For each of the 3 monoclonal antibodies, enzyme-linked immunosorbent assay titers were obtained for Actinobacillus seminis, A actinomycetemcomitans, and 10 field isolates of Actinobacillus spp. Target antigens for LG17 and LG30 antibodies occurred infrequently or were absent on these bacteria. However, the target antigen for the LG33 antibody was shared by Actinobacillus seminis, A actinomycetemcomitans, and the 10 field isolates of Actinobacillus spp, indicating some diversity of outer membrane antigens between isolates.     

The usefulness of the API 20 E classification system in the identification of Actinobacillus actinomycetem comitans, Actinobacillus seminis and Pasteurella haemolytica

The prepuces of lambs aged 6--8 months and semen of 2 adult rams were found to be infected with gram negative, non-motile, non-haemolytic, pleomorphic bacilli. These organisms were compared with those of known strains of actinobacillus actinomycetem comitans. Actinobacillus seminis and Pasteurella haemolytica, using the API 20 E classification system. Applying the principles of numerical taxonomy, the majority of suspected strains of A. seminis could be classified as A. actinomycetem comitans and 3 examples as Histophilus ovis. Although some of the suspected strains of A. seminis could be classified as P. haemolytica, obvious differences between the genera Actinobacillus and Pasteurella were evident.     

Bacteriological characteristics of Histophilus ovis and its relationship to similar bacteria

Seventeen strains of Histophilus ovis were obtained from a variety of pathological conditions and geographical areas and were examined to determine the bacteriological characteristics of the organism. All were Gram-negative pleomorphic rods which only grew under micro-aerophilic conditions in media enriched with blood, serum or meat particles. All strains were catalase negative, indole positive and nitrate positive while most produced acid from glucose, fructose, galactose, mannose, xylose, mannitol and sorbitol. Eight strains were examined serologically and compared with two strains of Actinobacillus seminis. In agglutination and precipitation tests, all strains of H ovis appeared antigenically identical and showed antigenic relationships to A seminis. Results of complement fixation tests confirmed the close, if not identical, antigenic relationship of H ovis and A seminis. It is suggested that H ovis and A seminis are biochemical variants of the same organism.     

SEROLOGICAL COMPARISON BETWEEN HISTOPHILUS OVIS, ACTINOBACILLUS SEMINIS AND BRUCELLA OVIS

SUMMARY: The serological relationships between 4 strains of Histophilus ovis , the neotype strain of Actinobacillus seminis and Brucella ovis were examined using a cross-absorption complement-fixation technique.
It was found that the 4 strains of H. ovis were serologically homologous and that an incomplete relationship existed between these organisms and A. seminis. Anteriserums prepared against one strain of H. ovis and the strain of A. seminis gave a weak, apparently nonspecific cross-reaction with Br. ovis antigen.
The practical significance of these results is discussed.     

A Comparison of Various Haemophilus somnus Strains

Sixty-eight Haemophilus somnus strains isolated from the bovine in Canada and the U.S.A. were compared. In media enriched with 5% ovine serum, 5% bovine serum and 10% yeast extract, H. somnus fermented glucose, levulose, maltose, mannitol, mannose, sorbitol, trehalose and xylose, but failed to ferment arabinose, dulcitol, galactose, inositol, lactose, raffinose, rhamnose, salicin and sucrose. The organisms acidified litmus milk, produced cytochrome oxidase, indole and hydrogen sulfide (H₂S) and reduced nitrates to nitrites. The motility, methyl-red, acetylmethyl-carbinol urease catalase, citrate, malonate, lysine, ornithine and arginine tests were negative. Haemophilus somnus was resistant to lincomycin, neomycin and triple sulfa, but susceptible to ampicillin, chloramphenicol, streptomycin, penicillin and tetracycline. No antigenic differences were noted between strains when tested against rabbit antisera of eight strains using agglutination, complement-fixation, immunodiffusion and counterimmunoelectrophoresis tests. Low titre cross-reactions were found in the agglutination tests with some of the anti-H. somnus rabbit sera with Actinobacillus lignieresi and Moraxella bovis. No distinct antigenic similarities to nine other species of pathogenic bacteria of animal origin were found. No difference was observed between H. somnus isolates from Ontario and those from western Canada and the U.S.A.     

A study for the differentiation of Actinobacillus seminis, A. actinomycetem-comitans, Histophilus ovis and Pasteurella haemolytica

By using well-defined techniques under optimum conditions it is possible adequately to define the biochemical characteristics of typical A. seminis strains. A. seminis can be distinguished from Histophilus ovis on the latter's distinctive colony morphology, but it cannot be distinguished from Actinobacillus actinomycetem-comitans. These organisms, however, can be differentiated from Pasteurella haemolytica on serological grounds and the latter's greater pathogenicity for mice. It is appreciated, however, that intermediate forms occur which cannot as yet be satisfactorily allocated to any of the above-mentioned genera.     

Genetic homogeneity of Actinobacillus seminis isolates

Isolates of Actinobacillus seminis from clinical cases and reference sources had markedly similar Bam H1 restriction endonuclease profiles but were readily distinguishable from the Bam H1 profiles of the Histophilus-Haemophilus group as well as from A lignieresii. For epidemiological purposes this lack of interstrain variation in Bam H1 profiles makes restriction endonuclease analysis of isolates of A seminis unsuitable.     

猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立

目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。     

Leukotoxins of gram-negative bacteria.

Leukotoxins are a group of exotoxins that produce their primary toxic effects against leukocytes, especially polymorphonuclear cells (PMNs). Leukotoxins include a variety of chemicals ranging from 9,10-epoxy 12-octadecenoate, a fatty acid derivative secreted by leukocytes themselves, to proteins such as RTX (repeats in toxin). This review focuses on leukotoxins of three species of gram-negative bacteria, Mannheimia (Pasteurella) haemolytica, Actinobacillus actinomycetemcomitans, and Fusobacterium necrophorum.     

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.     

Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae

Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.     

Comparison of DNA:DNA homology and enzymatic activity between Pasteurella haemolytica and related species

A commercially available microbiological identification system and DNA:DNA hybridization were used to determine relationships between and within serovars 1-13 of Pasteurella haemolytica, and between P haemolytica and P multocida and 4 species of Actinobacillus. All serovars of P haemolytica that belonged to biovar A were related with mean DNA homology of 78%, whereas all serovars of P haemolytica that belonged to biovar T were related to each other with mean DNA homology of 90%. The DNA:DNA hybridization between strains of biovars A and T ranged from 3 to 13%, indicating little or no genetic relationship between the 2 biovars of P haemolytica. The DNA homology between all serovars of P haemolytica and other species of non-P haemolytica bacteria tested (P multocida and actinobacilli) was less than 14%, suggestive of essentially no genetic relationship of P haemolytica with the ATCC reference strains of the genus Pasteurella or the genus Actinobacillus. Enzymatic differences were observed between P haemolytica and the other non-P haemolytica bacteria tested; however, the microbiological identification system that uses enzymatic reactions could not distinguish among biovars of P haemolytica. Results of this research support other data that suggest that biovars A and T of P haemolytica should be classified as separate species, but do not support the inclusion of either biovar A or T within the genus Actinobacillus.     

Isolation of Actinobacillus seminis from rams in the United Kingdom.

Actinobacillus seminis was isolated from the semen of five rams on four farms. Four of the rams had abnormal semen and three were also infertile. The isolates of A seminis showed similar phenotypic profiles and electrophoretic protein patterns to the type strain of A seminis but were distinct from Histophilus ovis previously isolated from rams with epididymitis in Scotland. The infection appeared to be subclinical but two of the five rams had palpable abnormalities of their testes. Three rams were treated with antibiotics but the infection persisted. No gross lesions were found in the genitalia of two of three rams examined post mortem but one had necrotic abscesses in the testes and epididymis. A seminis was isolated from the seminal vesicles and epididymis of one ram without gross lesions but not from the genitalia of the other two. On one farm the infection in a recently purchased ram led to the detection of another case as a result of the bacteriological screening of 11 stock rams not in contact with the initial case. These five subclinical cases, which included a supposedly healthy stock ram, suggest that A seminis infection may be widespread and should be considered in cases of infertility.     

Multiplex PCR for the detection of Brucella ovis, Actinobacillus seminis and Histophilus somni in ram semen

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.