GENERAL INTRODUCTION: ANAEROBIC GRAM-NEGATIVE, IRREGULAR RODS

ANAEROBIC GRAM-NEGATIVE, IRREGULAR RODS

A General Introduction has been added to each disease chapter in an attempt to give a brief updated overview of the taxonomic, biological and other characteristics of the virus family or group of bacteria /protozoa that cause disease in livestock and, where relevant, involve wildlife. As the text of the three-volume book Infectious Diseases of Livestock is currently under revision the Editors are aware that there are inconsistencies between the updated introductions to chapters and the content of the chapters themselves. Once the chapters have been updated – a process that is currently underway – these inconsistencies will be removed.

Of the many bacteria within this group only the genera Fusobacterium, Bacteroides, Porphyromonas, Prevotella and Dichelobacter are commonly associated with specific (Table 1) or nonspecific (abscesses in a variety of organs and tissues, dental and oral lesions, chronic pleuropneumonia, and chronic sinusitis) diseases in livestock and humans. Fusobacterium necrophorum is the most important pathogenic species in this group and, apart from the diseases with which it is primarily associated, it may secondarily infect and complicate lesions of infectious diseases in which the mucous membranes of the gastrointestinal tract are involved, such as foot-and-mouth disease, rinderpest and orf. Other important species include Dichelobacter (Bacteroides) nodosus, the agent of ovine footrot, and Prevotella melaninogenica (Bacteroides melaninogenicus), a cause of foot lesions in cattle.

These bacteria are obligately anaerobic, Gram-negative, non-sporeforming bacteria that commonly occur on mucous membranes of the mouth, the upper respiratory tract and the gastrointestinal and urogenital systems of healthy animals and humans.

They often occur in lesions in association with aerobic or other facultatively anaerobic bacteria, e.g. Trueperella pyogenes. Fusobacterium necrophorum and Bacteroides spp. appear, in many instances, to depend on these bacteria to reduce the redox potential in the affected tissues to a level that allows their growth and subsequent tissue invasion.8, 26, 27, 31, 38, 39

Before the advent of the newer methods used in molecular genetics, these bacteria were very difficult to study because they are fastidious in their nutritive and atmospheric requirements, and they are usually found in association with other microorganisms such as T. pyogenes, Pasteurella multocida, Escherichia coli, streptococci and others, and are therefore possibly overlooked.7, 51 In these mixed infections it does, however, seem likely that anaerobic species such as Bacteroides act synergistically with the other bacteria in the production of suppurative and necrotizing lesions.7 This emphasizes the importance that bacteriological examinations of exudates should include both aerobic and strict anaerobic culturing techniques.

Table 1 Most common specific diseases in livestock in which Fusobacterium necrophorum, Dichelobacter nodosus or Prevotella melaninogenica are the primary causative agent. Bacteria that may play a secondary role are indicated in brackets

DISEASE ANIMAL SPECIES AFFECTED INFECTIOUS AGENT
Bovine foot rot Cattle F. necrophorum (P. melaninogenica, D. nodosus, T. pyogenes)
Ovine foot rot Sheep and goats D. nodosus (F. necrophorum, T. pyogenes
Interdigital dermatitis Cattle D. nodosus: benign strains
  Sheep and goats F. necrophorum, T. pyogenes
Digital dermatitis Cattle F. necrophorum?
Heel erosion Cattle P. melaninogenica?
Foot abscess Sheep and goats F. necrophorum, T. pyogenes
Toe abscess Sheep F. necrophorum, T. pyogenes
Hepatic necrobacillosis Cattle, sheep and goats F. necrophorum (T. pyogenes and other bacteria)
Rumenitis Cattle F. necrophorum
Necrotic and ulcerative stomatitis and laryngitis Calves, lambs and kids F. necrophorum
Necrotic rhinitis Pigs (bull nose) F. necrophorum (spirochaetes and other bacteria)
Porcine foot rot Pigs F. necrophorum (T. pyogenes, spirochaetes and other bacteria)
Cara inchada Cattle P. melaninogenica? (T. pyogenes, Bacteroides bivius, F. nucleatum, Actinomyces israelii )

One of the hallmarks of infection by anaerobic bacteria, including Bacteroides, is an exudate with a putrid odour, although the absence of such an odour does not preclude an anaerobic infection.7, 50 The odour is primarily the result of metabolic end-products such as volatile amines, shortchain fatty acids and organic acids.50

Fusobacterium necrophorum

Fusobacterium necrophorum is an obligately, anaerobic, Gram-negative, non-spore-forming, non-motile and pleomorphic bacterium which varies in shape from small cocci (0,5 to 1,75 mm in diameter) to filaments. The bacteria have irregular swellings along their length and blunt or tapering ends.33

On blood agar, colonies of F. necrophorum are convex, translucent to opaque, 1 to 2 mm in diameter, and have a circular outline with scalloped to eroded edges. The colonies are often ridged or uneven.

Fusobacterium necrophorum produces haemolysin and indole but does not reduce nitrate. Most strains cause either alpha or beta haemolysis on rabbit blood agar. Generally, beta-haemolytic strains are lipase-positive, and alphahaemolytic and non-haemolytic strains are lipase-negative. No lecithinase is produced.29

There is considerable doubt about the validity of conclusions concerning the involvement of F. necrophorum in diseases that appear in publications prior to 1970, because it was referred to by a variety of names (e.g. Fusiformis, Bacteroides, Sphaerophorus).29 In 1970 a subcommittee of the International Committee on Nomenclature of Bacteria published a report recommending that members of the genera Sphaerophorus and Fusobacterium be incorporated into a single genus Fusobacterium and that the genus Bacteroides should be retained because of the inability of its species to produce butyric acid — a feature which distinguishes them from the species of Fusobacterium. 1

Based on cell morphology, haemagglutination properties, haemolytic activities and virulence in mice, strains of F. necrophorum are grouped into biovars (phases) A, AB, B and C.3, 13, 21, 40, 43The pathogenic strains of biovars A, B and AB are haemolytic and produce a leukotoxin (leukocidin); strains of biovar B are, however, less pathogenic than those of biovars A and AB. Biovars A and B have been designated as F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme respectively.43 Although biovar AB is closely related to both subspecies, its taxonomic status is unclear.36 The strains of biovar C are neither haemolytic nor pathogenic and are now known as F. pseudonecrophorum. Strains of F. necrophorum subsp. necrophorum are commonly found in cattle, biovar AB in cases of ovine footrot while those of subsp. funduliforme and F. pseudonecrophorum are more often isolated from humans than from animals.34

Although further study is required of the virulence factors of F. necrophorum its cell wall contains a lipopolysaccharide with endotoxic activities similar to those of the Enterobacteriaceae (see Escherichia coli infections).5, 28, 37 An important virulence factor is leukotoxin which is produced in the late-log and early-stationary phase of growth and is toxic for bovine and ovine neutrophils and bovine ruminal cells.51 A haemolysin which is thought to be a phospholipase A and lysophospholipase is thought to aid the bacterium in acquiring iron from the host and in maintaining an anaerobic environment. Haemagglutinins, the capsule and fimbriae are thought to be mediators of attachment to host cells but their exact role still needs to be elucidated.20, 35, 51

Dichelobacter (Bacteroides) nodosus

The designation ‘Dichelobacter’ means ‘rod of the cloven hoof’ and ‘nodosus’ means ‘knobbed’ which refers to the terminal (sometimes central) enlarged areas of the cells of D. nodosus. Terminal enlargements are more pronounced in bacteria that are present in lesions than in those grown in cultures. Dichelobacter nodosus is more closely related to the Enterobacteriaceae and is now a member of the family Cardiobacteraceae.8 These bacteria are fairly large (1–1,7 × 3,0–6,0 mm), non-motile, straight or slightly curved rods. Varying numbers of pili are found on the surface of different strains of D. nodosus. Reports differ as to whether or not D. nodosus possesses a capsule.29

Surface colonies of the type strain of D. nodosus are 0,5 to 2,0 mm in diameter, smooth, convex, and translucent or semi-opaque. Colonies often etch into the surface of the medium immediately beneath them, producing a sunken appearance. Three basic colony types, namely papillate or beaded (B)-type, mucoid (M)-type, and circular (C)-type, have been described.4, 41

Ten major serogroups, designated A to I, and more recently M,54 have been defined according to the antigenicity of their pili which is determined by employing a slide agglutination technique.11-12, 25, 47 Cross-reactions between strains of specific serogroups have been reported.11 At least 19 different serovars of D. nodosus are recognized among the ten serogroups, and their distribution varies around the world. Serovars that belong to serogroups A, B, C and G have been found in South Africa.2

Various factors appear to influence the pathogenicity of D. nodosus. Earlier studies suggested that increased virulence is correlated with increased production and activity of protease (of which there are two forms, thermostable and thermolabile), the presence of large numbers of pili, increased degree of motility of the bacteria, and specific types of colony morphology on culture. Variation in colony morphology has also been linked to the degree of piliation of D.nodosus. 14–16, 19, 24, 45, 48, 52

Recent studies, however, indicate that the virulence of D. nodosus strains appears to be coupled with the combined effect of the thermostable protease and the degree of motility of D. nodosus, and that it is not strongly correlated to the total protease activity, colony morphology (other than size) or the degree of piliation.4, 16, 53 Based on these studies it has been suggested that strains of D. nodosus may be grouped into two major categories:

  • benign strains which have thermolabile, extracellular protease and a low degree of motility. These strains cause benign foot rot of sheep and interdigital dermatitis in cattle and goats; and
  • intermediate and virulent strains which produce thermostable protease. These cause intermediate and virulent foot rot in sheep, which varies in severity according to the degree of motility of the strain.

The type IV fimbriae coded for by FimA gene are considered essential in the virulence of F. necrophorum. 32 Agglutinating antibodies against pili of D. nodosus are the most important in protective immunity induced by killed whole-cell vaccines.22, 25, 47, 49, 53

The gene regions associated with virulence have been mapped with FimA coding for the type IV fimbriae and brpV and aprV5 for the thermostable proteases and brpB and bprB5 for the thermolabile protease.8, 30

The isolation of D. nodosus is often unsuccessful notwithstanding the application of correct procedures in the collection and transportation of tissue specimens and the use of recommended culturing media and methods. To ensure optimal results when attempting to isolate D. nodosus, specimens should be collected by detaching pieces of affected skin or hoof from active and untreated lesions. These are placed in a suitable transport medium such as Thorley’s medium. Stringent anaerobic conditions should be maintained during transportation of the specimens to the laboratory. Best results are obtained when hoof agar plates are inoculated in the field and then placed in an anaerobic jar with an attached anaerobic gas-generating kit, in which the agar plates are transported to the laboratory.4 Dichelobacter nodosus has fastidious growth requirements. Variable success has been achieved when media such as Thorley’s, Stuart’s and modified Stuart’s containing L-cystine are used to isolate D. nodosus. 4, 18 It grows well in solidified trypicasearginine-serine (TAS) medium at 37 °C under anaerobic conditions.53

Dichelobacter nodosus is an obligate parasite of the skin of the feet of sheep, goats, cattle and deer and cannot survive in the environment for more than 14 days.46

Other Bacteroides spp. and related genera (Porphyromonas and Prevotella)

Because many previous studies of organisms called Bacteroides melaninogenicus included strains that might have been members of any nine currently recognized species, earlier literature is difficult to correlate with present designations.29

Pigmented species of Bacteroides have been reclassified into the genera Porphyromonas and Prevotella. 41, 42 For example, Bacteroides melaninogenicusis now known as Prevotella melaninogenica. 23, 42

Members of Bacteroides, Porphyromonas and Prevotella are all Gram-negative pleomorphic rods, which produce as fermentation products succinate, acetate, lactate, formate or propionate and only rarely small amounts of butyrate. This distinguishes them from those of Fusobacterium spp. in which butyrate is a major product.29

Historically, the predominant differentiating characteristic of P. melaninogenica (B. melaninogenicus) was its production of darkly pigmented colonies on a blood-containing medium. This pigmentation is the result of the production of a dark brown to black pigment, protohemin, and not of melanin as was originally thought. Surface colonies of P. melaninogenica on blood agar are 0,5 to 2,0 mm in diameter, circular, entire, convex and shiny. They are usually darker in the centre of the colony with the edges being grey to light brown. The colonies become darker as they age and the pigmentation usually develops more rapidly when laked blood, rather than blood containing intact red blood cells, is used. All strains produce pigment when cultured on agar containing rabbit blood, while only certain strains produce pigment when cultured on agar containing horse blood. A few strains are beta-haemolytic on rabbit blood agar.29

Bacteroides, Porphyromonas and Prevotella spp. cause a variety of purulent conditions of soft tissues, e.g. liver abscesses and may be present in infected bite wounds. These infections are generally polymicrobial (i.e. more than one species of bacterium are involved): Trueperella, which requires oxygen for its multiplication, is often present in lesions.

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