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.     

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.     

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.     

Microbiologic and pathologic findings in an epidemic of equine pericarditis.

During the spring and summer of 2001 and in association with the mare reproductive loss syndrome, 22 terminal and 12 clinical cases of equine pericarditis were diagnosed in central Kentucky. Actinobacillus species were the principal isolates from 8 of 10 nontreated, terminally affected and 3 of 10 clinically affected horses. Enterococcus faecalis and Streptococcus zooepidemicus were cultured from the remaining 2 nontreated terminal cases. No viruses were isolated in tissue culture. Nucleic acid of equine herpesvirus-2 was detected in pericardial and tracheal wash fluids of 3 and 1 individuals, respectively. Microscopic alterations in sections of heart and parietal pericardium were consistent with chronic fibrinous bacterial pericarditis. This report confirms a significant role of Actinobacillus species in equine pericarditis and describes an epidemic of this infrequently observed syndrome in the horse.     

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.     

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.     

REVIEW paper: mare reproductive loss syndrome

An epidemic of early fetal loss (EFL), late fetal loss (LFL), fibrinous pericarditis, and unilateral uveitis which occurred during the spring of 2001, are together now known as the mare reproductive loss syndrome (MRLS). A similar epidemic with less intensity was reported during the same period of time from southern Ohio, West Virginia, and Tennessee. The same syndrome with lesser intensity recurred in 2002. The estimated economic loss from the syndrome in 2001 and 2002 together was approximately $500 million. Both EFL and LFL were characterized by the absence of specific clinical signs in aborting mares. Nonhemolytic Streptococcus spp. and Actinobacillus spp. accounted for 65% of the organisms isolated from fetuses submitted for a postmortem during the MRLS period in 2001 and 2002. The pathologic findings in fetoplacental units of LFL included bronchopneumonia and funisitis, and there were no findings in EFL. Epidemiologic studies conducted in 2001 suggested an association between the presences of eastern tent caterpillars (ETC) in pastures with MRLS. Experimental studies in pregnant mares by exposure to ETC, or administration by stomach tube or with feed material, reproduced EFL and LFL. Similar experimental studies in mouse, rats, and goats with ETC were unsuccessful. Currently, 2 hypotheses are proposed for MRLS. One hypothesis proposes that an ETC-related toxin with secondary opportunistic bacterial invasion of the fetus leads to MRLS. The second hypothesis suggests that a breach of gastrointestinal mucosal integrity by hairs of ETC leads to a bacteremia and results in MRLS. In 2004, a similar equine abortion storm was reported from Australia and caterpillar exposure was identified as a risk factor for the abortion. In 2006, the syndrome was observed in Florida and New Jersey.     

PERITONITIS IN HORSES ASSOCIATED WITH ACTINOBACILLUS EQUULI

Actinobacillus equuli was the cause of peritonitis in 5 horses. In 3 the onset was sudden with intestinal stasis and acute abdominal pain as predominant findings. Two others presented with chronic disease and weight loss. Characteristically the peritoneal fluid had a high nucleated cell count with non-degenerate neutrophils as the predominant cell type. Four horses were treated and recovered.     

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.     

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.     

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.     

Postoperative infection with Actinobacillus spp in horses: 10 cases (1995-2000)

OBJECTIVE: To determine features of postoperative wound infection caused by Actinobacillus spp in horses undergoing clean, elective surgery and to evaluate bacterial susceptibility profiles of bacteria isolated. DESIGN: Retrospective study. ANIMALS: 10 horses. PROCEDURE: Data were retrieved from medical records and the microbiology laboratory database. RESULTS: 1,604 horses underwent clean, elective surgical procedures during the study period. Of these, 23 (1.43%) had postoperative wound infections, and Actinobacillus spp was isolated from 10 of these 23 (43%). Surgical procedures in these 10 horses included laryngoplasty with ventriculocordectomy (n = 3), arthroscopy (3), desmotomy of the accessory ligament of the superficial digital flexor tendon (2), removal of laryngoplasty prostheses (1), and hygroma resection (1). Seven horses survived, and 3 were euthanatized. All 10 Actinobacillus isolates were resistant to penicillin, and 6 were resistant to trimethoprim-sulfamethoxazole. All isolates were susceptible to ceftiofur and gentamicin. During the 5-year period of the study, Actinobacillus organisms were isolated from 35 of 513 (6.8%) samples from the general hospital population submitted for bacterial culture and antimicrobial susceptibility testing. CONCLUSIONS AND CLINICAL RELEVANCE: During the study period, Actinobacillus spp was isolated from a higher than expected percentage of horses that developed postoperative wound infections after clean, elective surgery. Susceptibility profiles for these isolates were different from typical susceptibility profiles for Actinobacillus isolates, suggesting that a pattern of resistance may be emerging.     

Bacteria in enteric lesions of horses

Thirty-three species of bacteria were isolated from the gastrointestinal mucosa of 23 adult horses and two foals. The bacteria isolated could be related to gross and microscopical lesions in some cases. Clostridium perfringens type A, Actinobacillus equuli, Salmonella typhimurium and Campylobacter coli biotype 1 could all be associated with gastrointestinal lesions. C jejuni biotype 1 and Aeromonas hydrophila were both recovered in this study and have been identified as causes of enteritis in horses or in other species. The case of C coli enteritis appears to be the first such report. The difficulties in examining adult horses with enteritis and relating the lesions seen to the bacteria isolated are discussed.     

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.     

Theory of ammonia toxicity as the mechanism of abortion in the mare reproductive loss syndrome

In late April/early May of 2001, for the equine industry in Kentucky, a previously unknown factor led to the loss of up to 40% of the new pregnancies (20-60 days) for that year, with a following significant number of late abortions and ill new born foals. In addition, encephalopathy, ocular lesions, pericarditis, laminitis, and other diseases were suspected to be associated with the syndrome. This occurrence was known as the mare reproductive loss syndrome (MRLS). On examination of the weather data, this period was seen to have an unusual pattern of severe late spring frost. Acute changes in the pasture diet affected the horses gut microflora, allowing the overgrowth of opportunistic pathogens and resulting in the excessive absorption of nitrogen and ammonia from the large bowel. Clinical blood chemistries from the horses on pasture at this time showed abnormally high levels of blood urea nitrogen (BUN). Elevated blood ammonia levels were found in mares and newborn foals affected with MRLS. Ammonia is metabolically one of the most toxic substances to the body. It is present naturally as a breakdown product of nitrogen turnover. If the body nitrogen balance is disturbed for any reason, with nitrogen accumulation exceeding the body's ability to excrete it as urea (BUN), ammonia will accumulate in toxic levels. Ammonia toxicity can cause all the symptoms that were observed in the horses exhibiting MRLS.     

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.