GENERAL | RESISTANCE | SURVIVAL Relationship to other mycobacteria, taxonomy and nomenclature Environmental distribution Host range Virulence factors Colonial morphology Cellular morphology Cell wall chemistry Biochemical characteristics Molecular genetics		M. paratuberculosis, although similar to other mycobacteria in many ways, is also quite unique. The challenge of working with this extremely slow growing organism has limited research and thus the amount known about the organism.

Relationship to other mycobacteria, taxonomy and nomenclature

On a genetic basis, M. paratuberculosis is virtually identical to Mycobacterium avium. Phenotypic characteristics of M. paratuberculosis are, however, different from those of M. avium: M. paratuberculosis grows much more slowly, requires an iron-transport chemical known as mycobactin for in vitro growth, forms rough colonies on solid agar media, and infects mammals instead of birds. Consequently, the most appropriate taxonomic classification and proper name for M. paratuberculosis has been under debate. An opinion supported by the International Association for Paratuberculosis is that M. paratuberculosis should be reclassified as a subspecies of M. avium and thus renamed M. avium subspecies paratuberculosis (abbreviated M. avium subsp. paratuberculosis). This subspecies designation appears in many recent publications concerning the organism. For simplicity, the name M. paratuberculosis is used throughout this web site. Similarities and differences between M. paratuberculosis and M. avium will be discussed throughout this section on the biology of M. paratuberculosis.

While sharing many genetic similarities with M. avium, M. paratuberculosis is less closely linked genetically to pathogenic mycobacteria in the TB complex: Mycobacterium tuberculosis, the cause of tuberculosis in humans, and Mycobacterium bovis, the cause of tuberculosis in cattle and other animals. M. paratuberculosis also is not closely related to the cause of leprosy in humans, Mycobacterium leprae. M. paratuberculosis, and the disease it causes, Johne's disease, does however share certain biological characteristics in common with these mycobacterial pathogens. Scientists often draw parallels among these mycobacterial organisms to try and understand their basic mechanisms for causing disease.

A partial phylogenetic tree showing the relationship of pathogenic mycobacteria based on 16S rDNA sequencing is shown on the adjacent figure (adapted from Rastogi et al. Rev. sci. tech. Off. int. Epiz., 2001, 20:21-54).

Environmental distribution

M. paratuberculosis bacteria are not thought to be free-living (able to grow and multiply) in the environment. Because of its inability to produce mycobactin (a chemical needed to transport iron), unique among members of the mycobacterial family, M. paratuberculosis can grow only inside animal cells where it "steals" iron from its host's cells, most often immune cells called macrophages. Thus, it is an obligate parasitic pathogen of mammals. This means infected animals are the only place in nature where growth and multiplication of M. paratuberculosis can occur. If found in soil or water samples, it can be assumed that M. paratuberculosis is simply persisting in those places (not multiplying) after being deposited there through fecal contamination from an infected animal.

The environmental distribution of M. paratuberculosis is markedly different from that of M. avium. M. avium can produce mycobactin and thereby acquire iron essential for growth and survival from the environment. Mycobactin production allows M. avium to grow and multiply outside a host animal. M. avium is commonly found in lakes, streams and domestic water supplies. Certain acidic soil types, notably peat bogs, contain higher than average numbers of M. avium. A tenuous association between the occurrence of Johne's disease and geographical regions with acidic soils has been reported (see discussion below under "survival in soil"). The strength of this association and the biological basis of this association remain to be determined.

Host range

M. paratuberculosis has a broad host range. Ruminants (animals with a four-chambered stomach that chew their cud) are the type of animal most commonly infected. These include: cattle, sheep, goats, deer, elk, antelope, bison, camels, llamas, and alpacas (these latter three species are technically called pseudo-ruminants as they have a three-chambered stomach). There are also infrequent reports of M. paratuberculosis infections in non-ruminant species such as horses, pigs, chickens, rabbits, fox, non-human primates and people with Crohn's disease.

If you wish to focus on particular species, select it at the left of your screen. You will then be able to choose the particular Johne's disease topic in which you are interested.

Virulence factors

Like other mycobacteria, M. paratuberculosis has the capacity to thrive inside white blood cells known as macrophages. As part of the immune system, macrophages are capable of destroying a wide variety of bacterial pathogens. Mycobacteria, however, are one of the few types of bacteria that not only can survive the antibacterial effects of macrophages, but actually grow and multiply inside them. Bacteria able to avoid being killed and instead replicate inside macrophages and cause disease are referred to as facultative intracellular bacterial pathogens. This picture shows M. paratuberculosis (red) inside macrophages (blue).

Considerable research has been done to try and understand how mycobacteria flourish in what is thought to be the hostile intracellular environment of macrophages. No specific mechanisms have yet been found to adequately explain this effective mycobacterial strategy. In general terms, two properties of mycobacteria explain their resistance to being killed by macrophages: 1) the chemically unique mycobacterial cell wall that is resistant to destruction or penetration, and 2) factors produced by mycobacteria that can neutralize the antibacterial chemicals produced inside macrophages. For detailed information the reader should obtain the references listed at the end of this section on the biology of M. paratuberculosis.

Survival and multiplication in the host animal is a prerequisite to causing disease. As with mechanisms of intracellular survival, mechanisms by which mycobacteria cause disease are also not well understood. Pathology due to mycobacterial infections results in part from the direct action of toxic chemical components of the mycobacterial cell wall. The host animal's immune response to the presence of M. paratuberculosis also contributes to the pathology and dysfunction resulting from the infection.

Colonial morphology

The size, color, and texture of a colony of M. paratuberculosis is dependent in part on the type of bacteriologic medium on which it is cultivated. On Herrold's egg yolk agar medium, one of the most commonly used culture mediums in veterinary diagnostic laboratories, the colonies appear small, somewhat rough and off-white to yellow in color. Pigmented (yellow) strains have been reported in sheep. On Middlebrook agar medium without Tween 80 (a detergent that improves the growth rate) the colonies are very rough in appearance and resemble those of M. tuberculosis. With addition of Tween 80 the growth rate of M. paratuberculosis increases and it's colonial morphology becomes smooth and domed resembling that of M. avium.

Cellular morphology

M. paratuberculosis is a small (0.5 x 1.5 micron) rod-shaped bacterium, roughly the size of the common intestinal bacterium called E. coli, that grows in clumps. It can be seen using a light microscope with 40x or greater power objectives. When stained by the Gram stain, it is blue and so called Gram-positive. When stained by acid-fast stains like the Ziel-Neelsen or Kinyoun's stain, M. paratuberculosis stains red and so is called acid-fast positive. Transmission electron microscopy reveals the waxy rough cell wall of M. paratuberculosis with its trilaminar structure. The intracellular vacuoles or inclusions common to mycobacteria can also be seen.

Cell wall chemistry

The cell wall of mycobacteria is composed of a thick waxy mixture of lipids and polysaccharides. The cell wall of M. paratuberculosis, although not well studied, seems similar in most respects to that of other mycobacteria. One feature is notable, however. While most strains of M. avium produce a surface glycolipid that allows strains to be serotyped (i.e., distinguished using antibodies specific for each glycolipid subtype), M. paratuberculosis strains lack such glycolipid antigens on their surface.

Biochemical characteristics

Biochemical tests used to distinguish among other species of mycobacteria are not used to identify M. paratuberculosis. The tests are taxing to perform due to the extremely slow growth rate of the organism, and test results vary among strains of M. paratuberculosis. Thorel, however, successfully used biochemical tests and numerical taxonomy methods to differentiate among subtypes of M. paratuberculosis-like bacteria.

Molecular genetics

The DNA of M. paratuberculosis is >99% identical with that of M. avium. This is the reason that many characteristics of the two bacteria are similar. The genetic feature of M. paratuberculosis that distinguishes it genetically from M. avium is the presence of multiple copies of a short DNA element called an insertion sequence (IS). Insertion sequences of various types have been reported in mycobacteria. The first to be discovered was the one unique to M. paratuberculosis and named IS900. Genetic probes used for detection of M. paratuberculosis in clinical specimens or identification of M. paratuberculosis in cultures are based on detection of IS900. A second insertion sequence, named IS901, that is approximately 60% similar in DNA sequence to IS900, was recently found in some strains of M. avium. How these insertion elements affect the biology and pathogenic capacity of M. paratuberculosis or M. avium is not understood. Evidence suggests, however, that they play a major role.