Actinobacillus equuli is a gram-negative, non-motile rod bacteria from the family Pasteurellaceae.
A. equuli is classified as a bio-containment level 2 organism under the U.S. Public Health Service Guidelines. Biochemical differentiation tests with positive results are: catalase, urease and oxidase tests. Actinobacillus equuli subspecies equuli is not hemolytic and is found in equine and porcine hosts. Another subspecies exists and is classified as Actinobacillus equuli subsp. haemolyticus which is positive for hemolysis and is found in horses and very rarely in rabbits. Occasionally, humans can become infected with A. equuli, more commonly as a result from destruction to the skin barrier, such as a horse bite.
A. equuli can commonly be isolated from the upper respiratory tract and oral cavity of adult healthy horses and can be important for diseases of foals, adult horses and pigs. It is a commensal bacteria unless it multiplies to overwhelming numbers, at which point it will cause disease. Specifically, A. equuli is known to cause neonatal sepsis in foals, known as Sleepy Foal Disease. Less commonly, A. equuli will cause disease in adult horses, resulting in respiratory infections. A. equuli also has the capability to cause kidney damage, arthritis and septicemia in affected pigs. The subspecies A. equuli subsp. haemolyticus results in similar pathogenesis as A. equuli subsp. equuli in horses. Due to these diseases and conditions, A. equuli can result in great economic losses especially in the equine industry.
Taxonomy and genome
Taxonomy It was previously thought that this bacteria was limited to the species level, however it is now divided into two subspecies of Actinobacillus equuli which include: A. equuli subsp. equuli, and A. equuli subsp. haemolyticus. Differentiation between these two subspecies can be done via biochemical tests including a CAMP test, and haemolytic properties. A. equuli subsp. equuli is non-haemolytic and CAMP negative, while A. equuli subsp. haemolyticus is haemolytic and CAMP positive. +Actinobacillus equuli Subspecies Differentiation SummarySubspeciesBiochemical testResultequuliCAMP Haemolytic(−) (−)haemolyticus CAMP Haemolytic(+) (+) Isolation of the subspecies equuli have been found in both horses and pigs, but only subspecies haemolyticus has been isolated from horses.
Genome Actinobacillus equuli genome's size is approximately 1.7 GDa and contains 40% guanine and cytosine in its genetic makeup.
A. equuli type strains include: ATCC 19392, CCUG 2041, GIP 103284, DSM 19655, JCM 2432, LMG 3736, and NCTC 8529. The 16S rRNA gene for A. equuli is AF381186, this helps classification of the bacteria to the species level but not to the subspecies level. This information helped determine that this species is closely related to Actinobacillus suis and Actinobacillus hominus. Another 16s rRNA study determined that an A. equuli type strain was 98.3% similar to A. lignieresii but are of different species.
Isolation of the subspecies equuli in the UK and Australia determined that it contained strains NCTC 8529T, and I 5445/4. A. equuli subspecies haemolyticus contain strains: T 355/87, F 92, Ac 7 F 119, F 154T, and F 490. PCR testing that results in the presence of the RTX gene allows differentiation to be made between the two subspecies, as only A. equuli subsp. equuli contains the RTX gene.
To differentiate similar strains of A. equuli from other genera originally thought to be A. equuli, ribotyping can be performed. The characteristic of being L-arabinose positive was thought to be an important taxonomic characteristic to classify A. equuli strains from others. Many different strains of A. equuli were determined through ribotyping, indicating that this species is diverse.
Epidemiology
Horses A. equuli is found on a variety of mucosal surfaces in the horse; it is considered to be part of the normal flora and an opportunistic pathogen. Despite it being a component of the normal flora, a mean isolation frequency as low as 37% (n =174) in adult horses and foals was detected in one study. However, the isolation frequency varied from 12 to 88% between farms. A. equuli does not have a preference for age, breed or gender, suggesting that husbandry practices may play a role in isolation frequency. An important aspect of the isolation frequency is that a horse or foal will not be culture positive every time it is swabbed, making it hard to determine the correct isolation frequency.
A. equuli is the causative agent in sleepy foal syndrome, which has been documented throughout the world. A. equuli does not only cause sleepy foal syndrome in foals, it is also a causative agent of pneumonia, muscular degeneration and jaundice in foals. On necropsy, foals were found to have the highest distribution of the bacteria in their synovial fluid, followed by the thoracic fluid and the liver.Kamada M., et al. “Isolation of Actinobacillus Equuli from Neonatal Foals with Death in Colostrum-Deficiency or Failure of Maternal Immunity Transfer.” Bulletin of Equine Research Institute, 1985, pp. 38–42. Important risk factors for foals acquiring an infection with A. equuli include failure of passive transfer, unsanitary foaling environments, and the presence of immunosuppressive conditions. Foals can become infected in-utero, during parturition or shortly after birth. Adults horses have similar diseases associated with A. equuli infections, including septicemia, respiratory and bone/joint diseases. Less common forms of infection can manifest as cutaneous abscess, pharyngitis, guttural pouch eustachitis, pericarditis, mastitis, urethritis, and lymphadenitis. Risk factors for developing infections in adult horses differs from that of foals, these factors include: stress, concurrent bacterial and/or viral infections, and discontinuities in the mucosa membranes allowing the bacteria entry into the body's internal compartment.
Swine Infections in swine are more common in European countries compared to the North American Countries. Infections in the United States are extremely rare with only 2 cases reported prior to 2008 and there was a 30-year period leading up to 2008 in which there were no cases documented. There has only ever been one case reported in Manitoba, Canada in 2010. However, infections may have occurred but were unreported. An infection with A. equuli in swine commonly yields septicemia, metritis, and can result in abortion. It was once thought pigs acquired the infection directly from horses, but there has been reports of swine developing infections on farms that housed horses prior to housing swine, and there has also been one case in which the swine infected had no association with horses. Since the exact source of the infection in swine is not known, the risk factors are also relativity unknown.
Microbiology
Morphology and culture growth Actinobacillus equuli are rod-shaped bacteria that measure an average of 1 μm in length. According to a study of the complete genome of A. equuli, this microbe can be cultured on blood agar at an optimal temperature of 37 °C, but is able to grow in temperatures ranging from 20 °C to 44 °C. Two studies noted that the colonies of A. equuli are circular, smooth in texture and produce a slimy, viscous material that gives the colonies a gelatinous and glossy appearance. In addition to its mesophilic properties, A. equuli can grow at a pH of 6.0 to 8.4 which allows the organism to inhabit the intestine and survive in various structures such as the heart and adrenal gland. A. equuli is a facultative anaerobe and is moderately fastidious, therefore culture growth in the laboratory may be challenging.
Biochemical tests
As part of the genus Actinobacillus, Actinobacillus equuli is gram negative. A. equuli displays negative results after inoculation into motility agar indicating a lack of motility. A. equuli is equipped with catalase and urease enzymes and therefore yield positive results for these tests. Although facultatively anaerobic, A. equuli can survive in environments exposed to oxygen as it contains the enzyme cytochrome oxidase for use in the electron transport chain. Differentiation between A. equuli subsp. equuli and A. equuli subsp. haemolyticus can be made using sheep's blood agar or the CAMP test upon the absence or presence of beta-haemolysis, respectively.
A. equuli has varying results for sugar fermentation. A. equuli subsp. equuli is not capable of fermenting cellobiose, whereas the opposite is true for A. equuli subsp. haemolyticus, indicated by a negative and positive result for the cellobiose fermentation test, respectively. However, both subspecies are positive for lactose fermentation, which is indicated by using phenol red lactose broth. Gram negative lactose fermenters typically display positive results on MacConkey agar, however, a study on equine actinobacilli resulted in the lack of growth on MacConkey agar, which may suggest that A. equuli is too fastidious to grow on this medium. To add, A. equuli ferments D-glucose as indicated by a positive result for the oxidative-fermentation glucose test performed using Hugh-Leifson medium. Both subspecies yield a positive result for both raffinose and D-galactose fermentation and displays mixed results for D-mannitol fermentation, where only some biovars of A. equuli subsp. haemolyticus displayed a negative result while others were positive for D-mannitol fermentation.
A. equuli is negative for the methyl red test, which indicates that this microbe does not use the mixed acid fermentation pathway. A. equuli also displays a negative Voges-Proskauer test because A. equuli does not use the 2,3-butanediol fermentation pathway resulting in the absence of acetoin production.Swedish University of Agricultural Sciences- Faculty of Veterinary Medicine and Agricultural Science. Actinobacillus equuli subsp. equuli. Veterinary bacteriology: information about important bacteria. September 21, 2020. Accessed October 2020. https://www.vetbact.org/?artid=107 As demonstrated by a negative Simmon's citrate test result, A. equuli is not able to use citrate as its sole carbon source for energy. +Biochemical Properties of Actinobacillus equuliBiochemical TestTest ResultGram Stain -Motility Agar -Catalase +Urease +Oxidase +Sheep's Blood AgarA. equuli subsp. equuli: γ-haemolysis A. equuli subsp. haemolyticus: β-haemolysisCAMPA. equuli subsp. equuli: - A. equuli subsp. haemolyticus: +Cellobiose Fermentation A. equuli subsp. equuli: - A. equuli subsp. haemolyticus: +Lactose Fermentation +MacConkey AgarNo growthOxidative/Fermentation Glucose +, absent gas productionRaffinose Fermentation +D-galactose Fermentation +D-mannitol FermentationA. equuli subsp. equuli: + A. equuli subsp. haemolyticus: +/-Methyl Red -Voges-Proskauer -Simmon's Citrate -
Pathogenesis
Horses The major host for A. equuli is horses and under physiological conditions, A. equuli is part of horses' mucosal microbiota and does not normally cause clinical symptoms and/or lesions. However, when mucous membranes become compromised (wound penetration, ulcers/erosions) it allows for resident A. equuli to establish infection in the horses' internal environment. A. equuli's penetration into the body's internal environment has been reported to cause septicemia in newborn foals (also called sleepy foal disease) and localized infections in adult horses or in more severe cases acute or chronic peritonitis. It is unknown at this time if there are certain strains of A. equuli that have a greater virulence for adult horses and/or foals.