Diversity among isolates of Actinobacillus equuli and related organisms as revealed by ribotyping

Objective The objective of this work was to examine the diversity within Australian isolates of Actinobacillus equuli and related organisms by the genotypic method of ribotyping.
Design Ribotyping, performed using the enzyme Hae III, was used to examine the diversity in 12 field isolates of A equuli (five being capable of fermenting L-arabinose), one field isolate of Pasteurella caballi and two unclassifiable field isolates. Isolates were obtained from Australian horses, except for three isolates of A equuli (one L-arabinose positive and two L-arabinose negative) which were obtained from horses and a pig in Africa. In addition, the type strains for A equuli and P caballi and a reference strain for Bisgaard Taxon 9 were included in the study.
Results The ribotype patterns were analysed by computerised cluster analysis, yielding five clusters (A to E). All five of the L-arabinose positive A equuli were assigned to cluster A, with all the other seven A equuli isolates (all L-arabinose negative) and the type strain being assigned to cluster B. One of the two unclassified isolates formed cluster C along with the reference strain for Bisgaard Taxon 9. The remaining unclassified isolate formed cluster D. Cluster E consisted of the field isolate and reference strain of P caballi .
Conclusion The results of this study indicate that A equuli is a diverse species, with L-arabinose positive isolates of A equuli being quite distinct from typical L-arabinose negative isolates. Ribotyping appears to be a useful tool in confirming the identity of A equuli -like organisms from horses.     

Classification of Actinobacillus spp isolates from horses involved in mare reproductive loss syndrome

OBJECTIVE: To identify Actinobacillus spp isolates recovered from fetuses and pericardial fluid from horses affected with mare reproductive loss syndrome (MRLS) and determine whether these bacterial species are the same as those isolated from clinically normal horses. SAMPLE POPULATION: Isolates of actinobacilli recovered from 18 horses with pericarditis and 109 fetuses aborted by mares affected by MRLS. Procedures-Actinobacillus spp isolates were identified to the level of species or subspecies by use of conventional phenotypic tests and biochemical and enzyme test kits. The 16S rRNA gene from selected isolates was amplified, purified, and sequenced. Sequence data were compared with sequence data for actinobacilli in GenBank. RESULTS: Of the 109 isolates obtained from fetuses, 14 were Actinobacillus equuli subsp equuli, 65 were A equuli subsp haemolyticus, 28 were Bisgaard taxon 10-like bacterium, and 2 were Actinobacillus genomospecies 1. Of the 18 isolates from horses with pericarditis, 4 were A equuli subsp equuli, 13 were A equuli subsp haemolyticus, and 1 was Bisgaard taxon 10-like bacterium. Comparisons with published data and GenBank data revealed that the isolates recovered from horses with MRLS were the same as those isolated from the oral cavity or alimentary tract of healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE: Actinobacillus spp isolates recovered from fetuses and pericardial fluid samples of horses affected by MRLS in 2001 to 2003 were identical to Actinobacillus spp found in the oral cavity and alimentary tracts of healthy horses.     

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.     

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.     

Actinobacillus suis-like organisms and evidence of hemolytic strains of Actinobacillus lignieresii in horses

Thirty-seven local isolates of Actinobacillus suis-like organisms from diseased and clinically normal horses and 1 llama were compared with reference strains of A suis, A lignieresii, A equuli, A capsulatus, A hominis, A (Pasteurella) ureae, and equine A suis-like organisms (ASLO) previously described in literature. Comparison was by cultural characteristics, carbohydrate fermentation, enzyme profiles, and whole-cell protein polyacrylamide gel electrophoresis. Carbohydrate fermentation, determined by API-CH gallery, divided 36 equine ASLO isolates into 6 API-CH biotypes. The llama isolate was an additional distinct biotype. The biochemical comparisons between A suis and ASLO did not reveal remarkable and consistent differences. Enzyme analysis revealed 5 API-ZYM biotypes, one of which included the same strains as one of the API-CH biotypes and consisted in both instances of 4 esculin-negative ASLO cultures and the reference strain of A lignieresii. We conclude that the 4 strains were hemolytic variants of A lignieresii. Protein electrophoresis disclosed 15 banding patterns, 10 of which represented equine ASLO strains. The reference strains of A suis shared the pattern predominant among equine ASLO. Four of the remaining reference strains of Actinobacillus species each had a unique profile, whereas the type strain of A capsulatus and the llama isolate had similar profiles. The groupings of cultures resulting from the different testing methods had little relation to each other and to the anatomic source of the strains except the strains comprising API-CH biotype II, which originated in the equine respiratory tract, and the A lignieressi cluster.     

Effects of Actinobacillus equuli culture supernatants on equine neutrophil functions and survival

After exposure of equine granulocytes from both foals and adult horses to culture supernatants from clinical isolates of Actinobacillus equuli, phagocytic capacity and respiratory burst was examined by flow-cytometry and a chemiluminescence assay, respectively. One haemolytic isolate of an equine Actinobacillus was also included in the study. An average decrease of 22% in total number of granulocytes, in the flow cytometric assay (P < 0.01), and an average decrease of 26% in light emission, in the chemiluminescence assay (P < 0.001), was seen after exposure to bacterial culture supernatants of A. equuli, indicating that the supernatants contained leukotoxic bacterial products. Supernatants from the haemolytic isolate appeared to contain a higher amount or more potent leukotoxic metabolites when haemolysis was expressed, causing a decrease in total number of granulocytes of 44% (P < 0.01) and a decrease in light emission of 52% (P < 0.01). Evaluation of the stability of the methods used revealed that within-method variation was far less than the observed results. The leukotoxic effects of A. equuli culture supernatants were mainly reflected in the decreased survival of neutrophils and not in neutrophil functions.     

An unusual Actinobacillus equuli strain isolated from a rabbit with Tyzzer's disease

Actinobacillus equuli was isolated in pure culture from the liver and lungs of an adult rabbit with Tyzzer's disease (Clostridium piliforme). Based on the haemolytic features on blood agar plates, a positive reaction in the CAMP-test, hydrolysis of esculin, the inability to ferment l-arabinose, tDNA-PCR and sequencing of the 16S rRNA gene, the isolate was classified as A. equuli subsp. haemolyticus biovar 1. However, the aqxA gene, characteristic for haemolytic A. equuli strains, was not detected by PCR.     

Actinobacillus suis infection of horses

Nineteen isolates of Actinobacillus suis were recovered from horses during the period October 1978-December 1980. Animals varied in age from a full term foetus to 12 years. One isolate was obtained from the nose of an apparently healthy horse, the remainder were obtained from still-born foetuses (2), foals dying within a week of birth (5), older animals with respiratory (6) or genital infections (3) or abscesses in the jaw (1). One isolate was obtained from the lung of a 2-week-old foal which had shown diarrhoea. The bacteriological characteristics of the isolates and the pathological lesions present in eight cases are described. The organism has a wide geographical distribution in New Zealand, and in the northern part of the North Island appears to be more common than A. equuli.     

Characterisation of porcine haemophili isolated from Australian pigs between 1988 and 1992

SUMMARY A total of 362 haemophili, isolated from pigs throughout Australia, were characterised by phenotypic properties. Most were identified as Actinobacillus pleuropneumoniae (296 isolates) or Haemophilus parasuis (52 isolates). The remaining isolates were identified as Haemophilus Taxon ‘minor group’ (12 isolates) and Haemophilus Taxon D (two isolates). All 296 A pleuropneumoniae isolates were serotyped by slide agglutination and/or gel diffusion, using rabbit antisera against all 12 recognised serovars. Of these, only 156 (52.7%) could be assigned to a single serovar as follows: serovar 1–85 isolates, serovar 2–4 isolates, serovar 3–2 isolates, serovar 5–10 isolates, serovar 7–51 isolates, serovar 11–2 isolates and serovar 12–2 isolates. Of the remaining 140 isolates, 91 gave cross-reactions with serovars 3 and 6, one cross-reacted with serovars 9 and 10, one cross-reacted with serovars 9 and 11 whereas 47 gave no reaction with any of the antisera.     

Serological characterisation of Actinobacillus pleuropneumoniae isolated from pigs in 1993 to 1996

OBJECTIVES: To clarify the serological identity of the prototype strain of a group of Actinobacillus pleuropneumoniae isolates that could not be serotyped in previous studies and to establish the serovar of 378 isolates of A pleuropneumoniae obtained from pigs in Australia over the period 1993 to 1996. DESIGN: After initial validation, QGD and IHA tests were used to characterise the prototype isolate (HS143) selected to represent the cross-reacting isolates that were found in a previous study. Next, 378 recent field isolates of A pleuropneumoniae were characterised using the existing gel diffusion serotyping technique and/or the IHA or QGD tests. RESULTS: The indirect haemagglutination test was shown to be capable of correctly recognising the reference strain for all serovars except serovar 11. While the quantitative gel diffusion test was not as effective as indirect haemagglutination, it could recognise serovar 11. When the two tests were used to examine the prototype strain (HS143) of the cross-reactive isolates, the results indicated that HS143 is serologically distinct from all 12 of the recognised serovars of A pleuropneumoniae. However, as HS143 was subsequently identified as serovar 12 by one of the leading international reference laboratories, the antiserum to isolate HS143 was used as the serovar 12 antiserum. A total of 346 of the 378 A pleuropneumoniae field isolates examined could be confidently serotyped with almost 90% of the isolates being either serovar 1 (104 isolates); serovar 7 (83 isolates) or serovar 12 (142 isolates). A range of other serovars and some cross-reactive isolates made up the remainder of the isolates. CONCLUSION: The serovar 12 antiserum produced against the international reference strain (1096) does not recognise Australian serovar 12 isolates. The antiserum raised against isolate HS143 does recognise the Australian serovar 12 isolates. The dominant serovars of A pleuropneumoniae infecting Australian pigs are (in decreasing order) serovars 12, 1 and 7.     

Specific immune response of mares and their newborn foals to Actinobacillus spp. present in the oral cavity

Oral swab samples, serum and colostrum was taken from 15 mares and 14 of their foals, within 24 h of birth. The presence of antibody against Actinobacillus spp. isolated from the oral cavity was investigated using agar gel immunodiffusion. Antibodies against 48 out of the 77 Actinobacillus isolates from all horses in the study were present in the respective sera of 13 mares and 9 foals. In 11 mother-foal pairs, the antibody content of the foal serum was similar to that of the mare, and in 9 cases this was reflected in the antibody content of colostrum from the mare. The results indicate that an immune response to Actinobacillus spp. colonising the oral cavity is present in many adult horses and that this immune response can be transferred from mother to foal via colostrum.     

Biochemical fingerprinting and ribotyping of isolates of Actinobacillus equuli from healthy and diseased horses

A total of 112 isolates of Actinobacillus equuli, including both clinical isolates and isolates from the oral cavity of healthy horses, were included in this study. All isolates were ribotyped and 92 of the isolates were also typed biochemically, with the commercially available Pheneplate (PhP) system, which includes 48 different substrates. As expected, ribotyping was more sensitive than biochemical fingerprinting in detecting differences between the isolates. The correlation between the two methods used was poor. It was not possible to distinguish clinical isolates from normal flora isolates by either of the two methods used.     

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.     

Characterisation of a novel Mannheimia sp from Australian feedlot cattle

OBJECTIVE: To characterise eight isolates of a Gram-negative organism obtained from the upper respiratory tract of cattle showing evidence of mild upper respiratory tract disease. DESIGN: The isolates were compared with the five recognised species within the genus Mannheimia - M haemolytica, M glucosida, M granulomatis, M ruminalis and M varigena--using a range of phenotypic and genotypic methods. RESULTS: Phenotypic characterisation indicated that the isolates belonged to the trehalose-negative [Pasteurella] haemolytica complex. This complex has recently been reorganised into five species within the new genus Mannheimia. Ribotyping performed using HindIII and a computerised analysis system indicated that the eight Australian isolates formed a distinct cluster that was related to, but different from, the five recognised species of Mannheimia. The 16S rRNA sequence of one isolate (BNO311) was determined and a phylogenetic analysis performed. Isolate BNO311 was distinct from the five named Mannheimia spp but did join a larger cluster consisting of rRNA cluster IV (M varigena) and the unnamed rRNA cluster V of Mannheimia. DNA:DNA hybridisation between isolate BNO311 and M haemolytica NCTC 9380T, M granulomatis P411 and Actinobacillus ligniersii NCTC 4189T all suggested similarities of approximately 30%. CONCLUSIONS: These phenotypic and genotypic characterisation studies suggest that the eight Australian isolates represent a new species of Mannheimia. Until further characterisation studies are performed, we are unwilling to propose a name for this taxon, preferring to refer to this possible new species as Bisgaard taxon 39 of cluster V of Mannheimia.     

Peritonitis associated with Actinobacillus equuli in horses: 51 cases

OBJECTIVE: To review the clinical findings, diagnosis and treatment of 51 horses with peritonitis attributed to Actinobacillus equuli. DESIGN: Retrospective study of clinical cases. METHODS: Breed, age and gender of horse, history, physical examination findings, treatment and outcome were determined from the hospital records of 51 horses in which a diagnosis of peritonitis attributed to A. equuli was made between January 1993 and June 1999. Results of abdominal fluid cytology and bacteriology, antimicrobial sensitivity patterns, haematology and faecal egg counts, when performed, were also retrieved. RESULTS: There was a variety of breeds of horses affected. There were 35 male and 17 female horses, aged from 9 months to 22 years, presented. Lethargy, signs of depression with mild to moderate signs of abdominal pain and inappetence were the most common reasons for presentation. Most horses had elevated heart and respiratory rates, an elevated rectal temperature and reduced intestinal borborygmi heard on auscultation of the abdomen. Abnormal colour with an elevated protein were features of an abdominal fluid sample in 98% of horses and a marked elevation in nucleated cell count was present in all samples. Pleomorphic gram-negative rods were seen on cytology in 53% of samples and a positive culture of A. equuli was returned in 72% of samples. Other laboratory findings in some horses included mild haemoconcentration, hypoproteinaemia, an elevated circulating nucleated cell count with a left shift, an elevation in fibrinogen concentration and an elevated faecal egg count. All horses demonstrated a rapid response to treatment with procaine penicillin alone, or a combination of procaine penicillin and gentamicin sulphate. Where antimicrobial sensitivity tests were performed, all but two isolates were sensitive to procaine penicillin. All horses responded to antimicrobial and supportive therapy and were discharged from hospital. CONCLUSION: Horses with A. equuli peritonitis present with similar clinical signs as horses with other causes of abdominal pain. However, these signs, when evaluated in conjunction with the results of abdominal fluid analysis and response to treatment, are characteristic of A. equuli peritonitis. Pleomorphic gram-negative bacteria may be seen on a cytological preparation of the abdominal fluid sample, and a positive bacterial culture may be obtained in some, but not all, cases. Most isolates are sensitive to procaine penicillin, so treatment with procaine penicillin and gentamicin sulphate is recommended until antimicrobial sensitivity is known.     

Study of agglutinins to Brucella abortus, B canis and Actinobacillus equuli in horses

Horses at a veterinary teaching hospital and a slaughterhouse were surveyed for antibodies to Brucella abortus, B canis and Actinobacillus equuli. Four of the 141 hospitalised horses and none of the 73 slaughtered horses had titres of 1:100 or greater to B abortus. Six horses of both populations reacted to the card test. One was culture positive. A card test using B canis antigen was positive in 38 per cent of the sera from hospitalised horses and all of the slaughtered horses. Twenty (27.4 per cent) of the latter group had high tires in a tube agglutination test. High titres could not be reduced by 2-mercaptoethanol serum treatment. The titres appeared to be associated with advanced age but not to sex. Adsorption of sera with B canis did not affect titres to A equuli but the reverse was true.     

Septicemia and peritonitis due to Actinobacillus equuli infection in an adult horse

Actinobacillus equuli is a rare cause of peritonitis in adult horses. Septicemia and peritonitis due to A. equuli were diagnosed at necropsy in an 8-year-old Saddlebred mare. The origin of the infection was not known; however, small necrotic colonic mucosal lesions presumed to have been caused by phenylbutazone treatment may have allowed bacterial invasion. A good response to antimicrobial treatment has been documented in the small numbers of previously reported acute cases of peritonitis. Because it is potentially treatable, it is important for pathologists and clinicians to identify horses with A. equuli peritonitis.     

Antibodies to Aqx toxin of Actinobacillus equuli in horses and foals

Actinobacillus equuli is found in the normal oral flora of horses, but has been associated with several diseases, and particularly with the usually fatal septicaemia in neonatal foals which is thought to be associated with a failure of the passive transfer of immunoglobulins via the colostrum. The Aqx protein of A equuli, belonging to the RTX family of pore-forming toxins, is also cytotoxic to horse lymphocytes. The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx.     

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.