GENERAL INTRODUCTION: ANAEROBIC GRAM-NEGATIVE, IRREGULAR RODS

ANAEROBIC GRAM-NEGATIVE, IRREGULAR RODS

Of the many bacteria within this group only the genera Fusobacterium, Bacteroides, 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 and orf. Other important species include Dichelobacter (Bacteroides) nodosus, the agent of ovine foot rot, and Prevotella melaninogenica (Bacteroides melaninogenicus), a contributing pathogen of foot lesions in cattle, primarily interdigital necrobacillosis.

These bacteria are obligate anaerobic, Gram-negative, non-sporeforming bacteria that commonly occur on mucous membranes of the mouth, the upper respiratory tract, and the gastrointestinal and urogenital tracts of healthy animals and humans. They often occur in lesions in association with aerobic or other facultative anaerobic bacteria, e.g. Trueperella (formerly Arcanobacter) 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.7, 23, 24, 29, 36, 37

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 therefore may be overlooked.6, 49 In these mixed infections it does, however, seem likely that anaerobic species such as Bacteroides act synergistically with the other bacteria in the formation of suppurative and necrotizing lesions.6 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 interdigital
necrobacillosis (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 Treponema spp.?
Heel erosion Cattle (Treponema spp.?, D. nodosus?)
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 rather characteristic putrid odour, although the absence of such an odour does not preclude an anaerobic infection.6, 48 The odour is primarily the result of metabolic end-products, such as volatile amines, short-chain fatty acids and organic acids.48

Fusobacterium necrophorum

Fusobacterium necrophorum is an obligate anaerobic, Gram-negative, non-sporeforming, non-motile and pleomorphic bacterium that varies in shape, ranging from small cocci (0.5–1.75 µm in diameter) to filaments greater than 100 µm long. The bacteria have irregular swellings along their length, and blunt or tapering ends.31

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, whereas alpha-haemolytic and non-haemolytic strains are lipase-negative. No lecithinase is produced.26

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 the organism was referred to by a variety of names (e.g. Fusiformis, Bacteroides, Sphaerophorus).26 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 as a separate entity 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, 10, 18, 38, 41 The pathogenic strains of biovars A, B and AB are haemolytic and produce a leukotoxin (leukocidin); strains of biovar B produce relatively little leukotoxin and, therefore, are 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.41 Although biovar AB is closely related to both subspecies, its taxonomic status is unclear.34 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 foot rot, while those of subsp. funduliforme and F. pseudonecrophorum are more frequently isolated from humans than from animals.32

Although further study is required of the virulence factors of F. necrophorum, it has been established that its cell wall contains a lipopolysaccharide with endotoxic activities similar to those of the Enterobacteriaceae (see Escherichia coli infections).5, 25, 35 A further important virulence factor is the soluble exotoxin, leukotoxin that is produced in the late-log and early-stationary phase of growth and which is toxic for bovine and ovine neutrophils and bovine ruminal cells.49 A haemolysin, which is believed to be a phospholipase A, and lysophospholipase are thought to aid the bacterium in acquiring iron from the host and in maintaining an anaerobic environment. Haemagglutinins, the capsule and fimbriae are assumed to be mediators of attachment to host cells, although their exact role still needs to be elucidated.17, 33, 49

Dichelobacter (formerly Bacteroides) nodosus

The designation ‘Dichelobacter’ means ‘rod of the cloven hoof’ and ‘nodosus’ means ‘knobbed’ which refers to the enlarged, terminal (sometimes central) areas of the D. nodosus organisms. 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.7 The obligate anaerobe bacteria are fairly large (1–1.7 × 3.0–6.0 µm), non-motile, straight or slightly curved rods with rounded and enlarged ends. Varying numbers of fimbriae (pili) are found on the surface of different strains of D. nodosus. Reports differ as to whether or not D. nodosus possesses a capsule.26

Surface colonies of the type strain of D. nodosus measure 0.5–3.0 mm in diameter, and they are smooth, convex, and translucent or semi-opaque in appearance. Colonies often etch into the surface of the culture 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, 39

Ten major serogroups, designated A to I, and more recently M,52 have been defined according to the antigenicity of their fimbriae, which is established by utilizing a slide agglutination technique.8, 9, 22, 45 Additional heterogeneity has been observed in the form of serogroups.9, 14, 27 Cross-reactions between strains of specific serogroups have been reported.8 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 fimbriation of D.nodosus.11, 12, 13, 16, 21, 42, 46, 50

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 fimbriation.4, 13, 51 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 give the D. nodosus bacteria motility in damaged tissue. They are coded for by the FimA gene and are considered essential in the virulence of D. nodosus.30 An additional virulence factor of D. nodosus is extracellular serine protease, which makes it possible for the bacterium to digest the tissue of the host animal in order to get access to free amino acids. Agglutinating antibodies against fimbriae of D. nodosus are the most important in protective immunity induced by killed whole-cell vaccines.19, 22, 27, 45, 47, 51

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.7, 28

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 culture media and methods. The bacteria are slow growing and colonies are difficult to see. Dichelobacter nodosus produces no haemolysis on fastidious anaerobic agar (FAA) culture plates. 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, 15 It grows well in solidified trypicasearginine-serine (TAS) medium at 37 °C under strict anaerobic conditions.51

Dichelobacter nodosus is an obligate parasite of the skin of the feet of sheep, goats, cattle and deer. It cannot survive in the environment for more than 14 days.43

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.26

Pigmented species of Bacteroides have been reclassified into the genera Porphyromonas and Prevotella.39, 40, 44 To illustrate this, Bacteroides melaninogenicusis is now known as Prevotella melaninogenica.20, 40, 44

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.26

Historically, the predominant differentiating characteristic of P. melaninogenica (B. melaninogenicus) was its production of darkly pigmented colonies on a blood-containing culture medium. This pigmentation results from the production of a dark brown to black pigment, protoheme (an heme derivative), and not of melanin as was originally thought. Surface colonies of P. melaninogenica on blood agar are 0.5–2.0 mm in diameter. They are circular, entire, convex and shiny in appearance, and are usually darker in the centre of the colony with the edges being grey to light brown. These black centres fluoresce under UV light. The colonies become darker as they age (one to two weeks) and the pigmentation usually develops more rapidly when laked blood (defibrinated blood that has been processed with freeze/thaw cycles in order to haemolyze the red blood cells), 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.26

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 (formerly Arcanobacter) pyogenes, which requires oxygen for its multiplication, is often present in such lesions.

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