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.     

Serum antibody responses in horses and mice following immunization with Actinobacillus equuli outer membrane proteins and recombinant Aqx toxin

The immune responsiveness of mice (without prior natural exposure) and mares (with naturally acquired antibodies) was determined following vaccination with Actinobacillus equuli outer membrane proteins (OMPs) and/or recombinant A. equuli toxin (rAqx). Mice were vaccinated subcutaneously on days 0 and 21 with one of three doses (5, 25 or 50μg) of A. equuli OMPs, rAqx or both, together with Freund's incomplete adjuvant (FIA). Antibodies against formalin-killed whole bacterial cells (WBCs), OMPs and Aqx were determined on days 0, 21 and 42. Mares were vaccinated subcutaneously on days 0 and 21 with 100μg OMPs, 100μg rAqx or a combination of 50μg of each antigen, together with FIA. Antibodies against WBCs, OMPs and Aqx were determined at 7day intervals for the first 42days, as well as on days 56, 70, 154 and 238. Vaccination of mice stimulated an apparent dose response to OMPs and Aqx. Antibodies against OMPs and Aqx were enhanced following vaccination of mares that had naturally acquired pre-existing antibodies. There was no evidence of interference with antibody responses to the individual antigens when OMPs and rAqx were combined prior to vaccination.     

Serum antibodies in mares and foals to Actinobacillus equuli whole cells, outer membrane proteins, and Aqx toxin

Actinobacillus equuli is carried in the alimentary tract of mares and can cause severe septicemia of neonatal foals. A hemolytic subspecies, A. equuli subsp. haemolyticus, and a non-hemolytic subspecies, A. equuli subsp. equuli, have been identified. Hemolytic strains produce the RTX toxin Aqx. The purpose of this study was to demonstrate sequentially in two sets of mare-foal pairs antibodies to A. equuli whole bacterial cells, outer membrane proteins, and recombinant Aqx and to compare the transfer of antibodies to these antigens between mares and their foals. Two mare/foal sets of sera were evaluated. Cohort A consisted of 18 mare-foal pairs obtained in the spring of 2005. Cohort B consisted of 10 mare-foal pairs obtained in the spring of 2006. For both sets, mare and foal sera were obtained immediately after foaling and prior to nursing (time 0) as well as at 12 and 24h and daily thereafter for 7 days. For Cohort B, sera were also obtained 30 days after birth. At parturition all mares had detectable antibodies to A. equuli whole cells and outer membranes; however, of those mares, two in Cohort A had undetectable antibodies to Aqx and their foals likewise had undetectable anti-Aqx antibodies. Antibodies against whole cells, outer membrane proteins, and Aqx were readily transferred from mares to foals. In most cases, there were significant correlations (p<0.05) between antibodies against whole cells, outer membrane proteins, and Aqx in mares' sera at the time of parturition and foal sera 24 after birth. Antibodies against the three antigen preparations had declined insignificantly (p>0.05) by day 30.     

An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains

Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA. The DNA flanking mbxA was cloned and sequenced to determine if M. bovis contained a classical RTX operon. Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively. Thus, hemolytic M. bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD. In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5'). At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified. Thus, as with the cya RTX operon of Bordetella pertussis, M. bovis appears to have a secretion accessory protein linked to RTX genes. Analysis of genomic DNA isolated from 5 nonhemolytic M. bovis strains by PCR and Southern blotting revealed the absence of mbxCABD. These strains did, however, amplify with primers specific for the 5' region flanking mbxC. M. bovis harbors a classical RTX operon that is absent in nonhemolytic strains.     

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.     

Actinobacillus equuli as a primary pathogen in breeding sows and piglets

The death of over 300 sows in 2 months on a 3000 sow farrow-to-isowean operation in Manitoba was attributed to infection with Actinobacillus equuli. This pathogen commonly infects foals, and is rarely reported in swine. Our report is the second recently published case of this pathogen in North American swine.     

Characterization of the type I secretion system of the RTX toxin ApxII in "Actinobacillus porcitonsillarum"

Strains of Actinobacillus porcitonsillarum are regularly isolated from the tonsils of healthy pigs. A. porcitonsillarum is non pathogenic but phenotypically it strongly resembles the pathogenic species Actinobacillus pleuropneumoniae, thereby interfering with the diagnosis of the latter. A. porcitonsillarum is hemolytic but unlike A. pleuropneumoniae, it contains only apxII genes and not apxI or apxIII genes. In contrast to the truncated apxII operon of A. pleuropneumoniae, which lacks the type I secretion genes BD, characterization of the apxII operon in A. porcitonsillarum revealed that it contains an intact and complete apxII operon. This shows a typical RTX operon structure with the gene arrangement apxIICABD. The region upstream of the apxII operon is also different from that in A. pleuropneumoniae and contains an additional gene, aspC, encoding a putative aspartate aminotransferase. Trans-complementation experiments in Escherichia coli and A. pleuropneumoniae indicated that the entire apxII operon of A. porcitonsillarum is sufficient to express and secrete the ApxIIA toxin and that the ApxIIA toxin of A. pleuropneumoniae can be secreted by the type I secretion system encoded by apxIIBD. These findings suggest that the complete apxII operon found in A. porcitonsillarum might be an ancestor of the truncated homologue found in A. pleuropneumoniae. The genetic context of the apxII locus in A. porcitonsillarum and A. pleuropneumoniae suggests that in the latter, the contemporary truncated operon is the result of a recombination event within the species, rather than a horizontal transfer of an incomplete operon.     

Characterisation of Australian isolates of Actinobacillus capsulatus, Actinobacillus equuli, Pasteurella caballi and Bisgaard Taxa 9 and 11

Objective The objective of this work was to perform a comprehensive phenotypic characterisation of 16 isolates of bacteria previously identified as Actinobacillus equuli.
Design The 16 isolates that had been obtained from Australian animals – 15 from horses and one from a rabbit – were compared with reference strains of A equuli, A capsulatus, Pasteurella caballi and Bisgaard Taxa 9 and 11.
Results The characterisation study demonstrated that only nine of the isolates were A equuli . The other isolates were identified as A capsulatus (the isolate from rabbit), P caballi (one isolate), Bisgaard Taxon 11 (two isolates) and Bisgaard Taxon 9 (one isolate). The final two isolates could not be assigned to any recognised species or taxa.
Conclusion This study has highlighted the importance of a complete characterisation of Actinobacillus -like organisms isolated from horses and rabbits. The study represents the first time that A capsulatus, P caballi and Bisgaard Taxa 9 and 11 have been recognised as being present in Australia.     

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.     

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.     

猪传染性胸膜肺炎放线杆菌毒素Ⅰ抗原模拟表位的筛选与鉴定

将猪传染性胸膜肺炎放线杆菌毒素Ⅰ(APP ApxⅠ)抗血清用饱和硫酸铵分级粗提后,再经DEAE-sepha-dex-A50低压层析系统纯化得到IgG,用此IgG作为配基对噬菌体随机十二肽库进行4轮不同条件的富集筛选。通过改变筛选条件,噬菌体的回收率从1.44×10-6增加到了8.9×10-5,P/N值逐步提高,阳性噬菌体得到了富集。ELISA结果表明,随机挑取的10个噬菌体克隆中,有5个与纯化的IgG有较强的特异性结合能力。对筛选到的5个阳性克隆提取ssDNA进行测序,并对其递呈的氨基酸序列进行分析。结果有3个克隆的4个连续的氨基酸序列(RVDV)相同,并与APP ApxⅠ蛋白的原始序列具有较高的同源性。     

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.     

A haemolytic variant of Actinobacillus equuli causing an acute septicaemia in a foal

Abstract

Extract

This paper describes the isolation of a haemolytic variant of Actinobacillus equuli from a foal which died from acute septicaemia. Foal deaths due to non-haemolytic A. Equuli (also known as Shigella equirulus) in New Zealand have been described previously by Hutchinson (1956 Hutchinson, Sally C. 1956. Shigella equirulus infection in a foal. N.Z. vet. J., 4: 119–120. [Taylor &; Francis Online] , [Google Scholar]) and Bloomberg (1963 Bloomberg, J. H. 1963. Atypical symptoms in a case of sleepy foal disease (Actinobacillus equuli infection). N.Z. vet. J., 11: 125–126. [Taylor &; Francis Online] , [Google Scholar]).     

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.     

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.