GENERAL INTRODUCTION: MYCOBACTERIA

MYCOBACTERIA

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.

Introduction

Tuberculosis is a chronic contagious disease caused by infection with certain acid-fast bacterial species of the genus Mycobacterium. It affects many vertebrate animals, domestic and wild (Table 1), and in bovine species it is typically manifested by the formation, in various tissues, but particularly in the lungs and lymph nodes, of granulomas known as tubercles that consist of a core of caseous necrotic tissue surrounded by a zone of granulomatous inflammation.34 The term ‘tubercle bacillus’ was introduced by Robert Koch who first described the causative agent of tuberculosis in humans in 1882. This organism has subsequently been found to be only one of many very different acid-fast bacilli. For a long time the term ‘tubercle bacillus’ has been applied to not only what is today known as the M. tuberculosis complex8, 81 but also to distinct species such as M. avium. It is therefore necessary to define the term ‘tubercle bacillus’ whenever it is used.14, 30 The characteristic appearance of the lesions, however, differs according to animal species and the respective Mycobacterium sp. with which it is infected. Before effective control measures were adopted, it was one of the major diseases of humans and domestic animals. The disease in humans still remains one of the most important notifiable infectious diseases in the developing world, and is the single biggest public health problem in South Africa,4 hugely complicated by an HIV/AIDS epidemic of shattering dimensions.54 The various veterinary control measures applied at present in South Africa are aimed at eradication of the disease in livestock and the containment of M. bovis infection in wildlife within the infected game reserves.

For many years tuberculosis appeared to prevail over the entire globe,26 and annually caused more deaths in humans than all of the so-called scourges and wars put together. The records of the Parsees, the Mosaic Laws, the Talmud and other ancient documents all refer to a malady that can be identified as tuberculosis of humans.34 Much of the present knowledge of tuberculosis is based on the initial work of scientists like Laennec and Villemin. Robert Koch first described the causative agent in 1882, and tuberculin, which proved to be a valuable diagnostic tool for the detection of the disease, was developed by him in 1891 for its supposedly curative value.42, 43

Aetiology

The mycobacteria are aerobic, considered Gram-positive (although not readily stainable by the Gram’s staining method), non-motile, non-spore forming, straight or slightly curved rods, 1,5 to 4,0 μm long and 0,3 to 0,5 μm wide.17, 84 Their cell walls have a high lipid content, which accounts for their resistance to acids, desiccation and most disinfectants, and also for their slow growth and hydrophobic characteristic in fluid media. Once mycobacteria have been stained with carbol fuchsin, they cannot be decolorized by acid-alcohol — thus their name ‘acid-fast bacteria’. When isolation is attempted, the resistance afforded by the high lipid content of the cell wall allows treatment of samples with acid or alkali to kill microbial contaminants without destroying significant numbers of the mycobacteria in the process.

The genus Mycobacterium consists of well over 120 species, most of which are environmental saprophytes that exist and multiply in a wide variety of substrates such as soil, water and plants, domestic and wild mammals, and birds.5, 32, 46, 63, 87 Some of these saprophytic mycobacteria (opportunistic pathogens) may cause ‘opportunistic’ infections in animals and humans, but others never cause disease.31 Certain members of the genus, such as those that make up the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, M. microti and M. canetti), which are also known as ‘tubercle bacilli’ and which cause tuberculosis in humans and animals, and M. leprae which is the cause of leprosy in humans, are obligate parasites and are usually transmitted only by infected mammalian hosts. They are also susceptible to a number of chemotherapeutic agents, while the opportunistic pathogens are often resistant to chemotherapeutic agents and are not contagious.85

The mycobacteria of the M. tuberculosis complex are characterized by a 99,9 per cent similarity at the nucleotide level and identical 16S rRNA. They differ widely with regard to phenotypes and host tropism. Mycobacterium tuberculosis, M. africanum and M. canettii are human pathogens. Mycobacterium microti causes disease in rodents while M. bovis has a very wide host spectrum, and it was therefore thought for many years that the cause of human tuberculosis evolved from M. bovis. However, the results from a recent study indicate that the common ancestor of the tubercle bacilli resembled modern M. tuberculosis or M. canettii and that a separate lineage, represented by M. africanum, M. microti and M. bovis, branched from the progenitor of M. tuberculosis strains. Successive loss of DNA may have contributed to the appearance of more successful pathogens in new host species as observed for M. bovis.8

Table 1 Differentiation between members of the M. tuberculosis complex on the basis of biochemical attributes. (Adapted from Collins et al.14 and Pfyffer et al.68)

MYCOBACTERIUM SPP TCH NITRALASE OXYGEN
PREFERENCE
PYRAZIN-AMIDE CYCLO-SERINE
M. tuberculosis R + A S S
M. africanum S V M S S
M. bovis S M R S
M. microti*          
M. canetti S + A R ?

* Information on M. microti is not given as it has only been reported in the vole (Microtus agrotis) 

S = sensitive 

R = resistant 

A = aerobic

 M = microaerophilic

 V = variable 

TCH = Thiophen-2-carboxylic acid hydrazide 

+ = positive

 − = negative 

? = not documented

The two most important members of the M. tuberculosis complex are M. bovis and, M. tuberculosis which are distinguished from one another either on the basis of biochemical attributes (Table 1) or by advanced molecular tools involving the polymerase chain reaction (PCR),16, 22, 64 restriction fragment length polymorphism (RFLP), spoligotyping or variable numbers of tandem repeats (VNTR).45 Mycobacterium tuberculosis was one of the first mycobacteria to be described. 90

Mycobacterium bovis, which is closely related to M. tuberculosis, causes tuberculosis mainly in cattle but may also affect other species, including humans, if there is contact with tuberculous cattle or their products. The most important mycobacterial species causing disease in livestock are M. bovis, serovars of the M. avium complex, M. avium subsp. paratuberculosis (see Paratuberculosis), and M. farcinogenes, which causes bovine farcy, a purulent inflammation of superficial lymph vessels and nodes in tropical countries.11, 12, 32 In terms of the variety of animal species that it may infect, M. bovis is the most universal pathogen among the mycobacteria and produces progressive disease in many domestic and wild animals as well as in humans.39

The susceptibility of different animal species to infection with M. bovis, M. tuberculosis and serovars of M. avium complex varies (Table 2).27 Of the livestock species, cattle, goats, sheep, horses and pigs are susceptible in varying degree to M. bovis infection (Groups II and IV in (Table 2). Cattle can, in particular, serve as maintenance hosts for M. bovis from which spillover infections to less susceptible species can occur. In wildlife the maintenance host status for M. bovis has been shown for African buffalo (Syncerus caffer).21 As far as M. tuberculosis is concerned, the statement by Griffith in 1928 that animals suffering from tuberculosis are most often observed to live in close contact with humans or domestic animals and that the prevalence is highest among animals living in captivity, such as in zoological collections, and lowest in those with rare contact with civilization, still holds truth.33

Lesions contain moderate numbers of bacilli. Horses, donkeys and mules are quite resistant to tuberculosis caused by all Mycobacterium spp.19, 27 The response to infection by the mycobacteria varies widely among different animal species, and to a lesser degree among individuals of the same species.19

Mycobacterium africanum was initially isolated from sputum of tuberculous humans in equatorial Africa.9 It is phenotypically intermediate between M. tuberculosis and M. bovis. Reports of infection with M. africanum in animals are rare, but it has been isolated from the tissues of a monkey, 77 and an outbreak of M. africanum infection in cattle and pigs in Norway was described in 1992.1

Mycobacterium microti, the vole bacillus, is mainly infectious for rodents and is not considered an important pathogen in either domestic or wild animals.85 Cases have mostly been reported in voles, wood mice and shrews10, 36 but infections have also been described in a cat, pigs, llama, a rock hyrax and a ferret.36, 67, 82 The recent use of spoligotyping to characterize this microorganism, however, suggests that M. microti might be more widespread among different hosts than previously assumed.44 Recently, studies have been conducted to investigate the potential use of M. microti as a live tuberculosis vaccine.20, 51

A novel pathogenic taxon of the M. tuberculosis complex was described in 1997.81 Canetti had first isolated the variant of M. tuberculosis in 1969 followed by two other case reports in 1993 and 1998.68 Mycobacterium canettii has been classified, but not validated, as a member of the M. tuberculosis complex on the basis of the 16S rRNA gene sequence but phenotypically and genetically it differs from the other members of this complex. All three isolates were obtained from patients living at least temporarily in Africa which raises the question of the geographic distribution of this organism. Mycobacterium canettii can be distinguished from the other M. tuberculosis complex organisms by PCR restriction analysis.29

Table 2 Tuberculosis in a variety of species. (Adapted and reproduced with permission from Francis27)

  SUSCEPTIBILITY TO INFECTION WITH THE THREE TYPES OF TUBERCLE BACILLI
GROUP SPECIES ALLERGY NO.
BACILLI
CASEA
TION
CALCIFI
CATION
FIBROSIS GIANT
CELLS
BOVINE HUMAN AVIAN SPREAD ROUTE
1 Naive human
Monkeys
Guinea pigs
Rabbits
5
2
3
2
1
2
1
2
5
5
5
5
3
3
2
2
2
1
1
1
3
5
4
4
5
5
5
5
5
5
5
5
1
1
1
2
4
4
5
1
1
R 90
R 90
Expr.
Expr.
2 Urban human
Elephants
Cattle
Buffalo
Goats
Sheep
Sheep
Pigs
5
1
2
2
2
2
3
1
3
1
1
1
1
1
5
4
4
4
4
4
4
5
1
5
4
4
4
4
5
5
4
4
3
4
4
5
1
5
4
4
2
2
2
3
4
4
4
3
4
2
3
1
1
1
1
2
1
1
1
1
2
2
2
5
?
5
4
1
1
1
R 90
R 100*
R 90
R 90
R 90
R 80
R 90
3 Fowls 2 4 4 1 3 2 1 5 3 4 A 100
4 Horses
Asses and mules
1
1
3
3
3
3
1
1
2
2
4
4
2
2
11 1
1
1
1
A 90
A 90
5A Dogs 2 2 3 1 2 1 2 2 0 0 R 60
5B Cats 1 3 3 1 2 1 4 1 2 1 A 70
5C Hamsters 0 4 0,5 0 0 0 5 5 1 0 Expr.
5D Mice
Rats
0
0
5
4
0,5
0,5
0
0
0
0
0
0
2
1
2
1
1
0,5
0
0
Expr.

* = little information available

The maximum values for each feature in this table is 5. The figures represent a somewhat arbitrary approximation. A ‘0’ in the columns for allergy or calcification should not be taken as completely precluding the existence of these features in any given species. No species is completely resistant to tuberculosis, and consequently, in the columns on susceptibility to the various types of tubercle bacilli, a ‘1’ has been used for those species that are virtually resistant to infection with a given type. The figures for susceptibility are roughly comparable vertically as well as horizontally: thus those for rabbits are 5, 1, 4, and those for mice, 2, 2, 1. The values for ‘Spread’ represent the ease with which tuberculosis spreads naturally between members of any one species.

The figures under ‘Route’ show the percentage of all infections acquired by the most important route. ‘Expr.’ has been inserted when nearly all information on the tuberculosis in a species is based on experimental work. R = respiratory; A = alimentary; Expr. = experimental. The animal species are grouped according to their reaction to infection with tubercle bacilli. The classification was made primarily on the general characters of the pathological finding after infection with mycobacteria. In the first two groups are such animals in which typical tubercles are formed. The first group consists of highly susceptible species. Naive humans and monkeys in captivity are more susceptible to fatal spontaneous infection than any other species. Spontaneous infection in guinea pigs and rabbits is not known.

The early classification of the ‘opportunistic’ mycobacteria was based on growth rate, pigmentation, and clinical significance.70, 84 Currently the bacteria are classified according to criteria outlined in Table 3. The ‘opportunist’ or nontuberculous group of mycobacteria are also referred to as atypical mycobacteria, ‘mycobacteria other than tubercle bacilli’ (MOTT) or ‘potentially pathogenic environmental mycobacteria’ (PPEM).85 Diseases caused by these mycobacteria are termed mycobacterioses. They are considered to be significant role players in post-surgical infections as well as in opportunistic infections in both immunocompetent and immunosuppressed patients.18, 28, 76 Of the rapid growers, M. chelonae, M. fortuitum and to a lesser extent, M. flavescens, are of clinical importance mostly in non-healing skin lesions in cats and dogs.37 Mycobacterium fortuitum has been isolated from three species of fish7 and a school of sea horses.58 Mycobacterium chelonae has been isolated from nodules in the mesentery and lungs of a snake.35 Mycobacterium smegmatis has been reported as a cause of mastitis in cattle.23, 69

Opportunistic mycobacteria can survive in water and have been isolated from ponds in Europe and New Zealand. They can be responsible for non-specific sensitization of cattle to the purified protein derivative (PPD) of bovine and avian tuberculin and may therefore complicate the interpretation of the tuberculin skin test and whole blood assays for tuberculosis such as the gamma interferon test.13, 38–40, 59, 60

The most important group of opportunistic mycobacteria that cause clinical disease in various animal species besides the obligate pathogens (M. tuberculosis complex), is the M. avium complex, also referred to as the M. aviumintracellulare complex, and is listed as M. avium and M. intracellulare in Runyon’s group III.77, 88 Recently, based on molecular evidence, it was suggested that the designation Mycobacterium avium subsp. avium should be reserved for the bird-type isolates while M. avium subsp. hominissuis should be the name for the human/porcine type of M. avium.61

To date the M. avium complex includes M. intracellulare, M. avium, M. paratuberculosis (see Paratuberculosis) M. lepraemurium (pathogenic for rodents but not for humans),66 and the ‘wood pigeon’ bacillus (M. avium subsp. silvaticum subsp. nov).80, 85 The M. avium-intracellulare complex, as it was known until recently, consisted of 25 serovars, including M. avium (serovars 1, 2 and 3) and M. intracellulare (serovars 4 to 25). Recent developments in the taxonomy based on the structural relatedness of DNA, RNA and protein between M. avium and M. intracellulare have confirmed that they are distinct species. According to the variation that exists, the serovars allocated to the two species are serovars 1 to 6 and 8 to 11 which are assigned to M. avium, and serovars 7, 12 to 17, 19, 20 and 25 to M. intracellulare, while some isolates of serovar 18 are grouped with M. intracellulare and others with M. simiae.85 The division into 25 serovars is based on differences in seroagglutination,72, 89 immunodiffusion53 sensitin typing50 and DNA/DNA hybridization.3, 71, 83

Members of the M. avium complex are the most widespread of the mycobacteria in the environment. Some are saprophytes and others are pathogens of birds and mammals including humans.32, 79 They are a major source of opportunist infection in humans suffering from acquired immune deficiency syndrome (AIDS).32 Serovars 1, 2 and 3 produce progessive disease in poultry and birds under natural conditions. The other serovars, when inoculated into chickens, only produce minimal disease.77 In Europe where ‘fowl tuberculosis’ was of importance, and contact between cattle, pigs and fowls occurred, serovars 2 and 3 were the most frequent ones isolated from porcine lymphadenitis.73 In the USA, serovars 2 and 3 are also found in pigs. Studies in South Africa showed that serovars 1, 4 and 8 are mainly responsible for porcine lymphadenitis41 and that serovars 4 and 8 occur most frequently in the environment, animal feed and bedding, and on plants.41, 63

Table 3 Classification of nontuberculous or opportunist mycobacteria. (Adapted from Runyon70)

GROUP RATE OF GROWTH AND PIGMENT
PRODUCTION
MYCOBACTERIUM SPP. CLINICAL SIGNIFICANCE
I Slow
Photochromogens
M. kansasii
M. marinum
Pulmonary disease in humans
Pathogen of fish and humans
II Slow
Scotochromogens
M. kansasii
M. xenopi
M. flavescens
M. gordonae
M. scrofulaceum
No clinical significance
No clinical significance
No clinical significance
Tuberculous adenitis in children
III Slow
Non-chromogens
M. avium
M. intracellulare
M. gastri
M. nonchromogenicum
M. terrae
M. triviale
M. ulcerans
‘Tuberculosis’ in poultry and birds
Opportunist pathogen in humans,
No clinical significancepigs, cattle, horses
No clinical significance
No clinical significance
No clinical significance
No clinical significance
Ulcers in humans (Buruli in
East Africa)
IV Rapid M. chelonae
M. fortuitum
M. phlei
M. vaccae
M. smegmatis
Opportunist pathogen
Mycobacteriosis in fish
No clinical significance
No clinical significance
See text

Cultural methods and other characteristics of Mycobacterium spp.

Cultural methods are as reliable as animal inoculation methods for the isolation of tubercle bacilli from specimens from infected tissues or material.52 All members of the M. tuberculosis are slow growers and non-pigmented, and fail to grow at room temperature (25 °C). They are catalase- negative after being heated to a temperature of 68 °C. They are sensitive to antituberculous drugs, with the exception of those strains which have developed drug resistance. Members of the M. tuberculosis complex require enriched media for successful culturing, the most frequently used solid media being Löwenstein-Jensen (L–J) medium and Ogawa medium, both containing eggs, phosphate buffer and magnesium salts (L–J medium also contains asparagine), 12 and Middlebrook 7H10 agar, a semi-synthetic medium. The liquid media most often used are Dubos medium, Middlebrook 7H9 and Middlebrook 7H10 liquid medium.64 Most of the other species of mycobacteria grow on ordinary nutrient culture media.

To facilitate the isolation of a member of the M. tuberculosis complex from infected tissues, a specimen that consists of solid tissue should be finely minced and then placed in a small quantity of normal saline solution. This material, as well as fluid specimens such as sputum or exudate, should be decontaminated by treatment with an alkali (e.g. 4 per cent NaOH) or an acid (e.g. 2 per cent HCl); the exposure time must not exceed 30 minutes. The alkali or acid is then neutralized and the specimen is centrifuged in order to concentrate the organisms in the sediment, part of which is then inoculated onto suitable media. Cultures should be incubated at 37 °C. Visible growth of M. bovis may occur on solid media after three to four weeks or may take a longer time to appear. Colonies of M. bovis are white, flat, smooth and moist and are easily emulsified.17, 64 Mycobacterium bovis is microaerophilic, sensitive to thiophene-2-carboxylic acid hydrazide (TCH), niacin production- and nitrate reduction- negative, and resistant to pyrazinamide. Glycerol, which enhances growth of M. tuberculosis but might inhibit that of M. bovis, should be replaced by sodium pyruvate in the medium if the isolation of M. bovis is attempted. These properties distinguish it from M. tuberculosis, which grows more luxuriantly with colonies becoming visible and showing dry, wrinkled surfaces after 10 to 14 days.12

Members of M. avium complex produce visible growth on culture after two to three weeks. They grow at temperatures ranging from 20 to 45 °C and form colonies that are pearly-grey, lemon-yellow or sometimes, bright yellow,32 which emulsify easily. The mycobacteria of this complex are nitrate reduction-negative, tellurite-positive and resistant to most antituberculous drugs.

The development of molecular diagnostic tools such as DNA probes and amplification methods, of which the PCR technique is the most important, have greatly facilitated the diagnosis of mycobacterial diseases. They provide sensitive, rapid and specific tools to replace traditional tests for identifying different species and subspecies of mycobacteria, either from culture isolates or even directly from the clinical specimens.25, 48, 65

More recent methods to determine the natural relationship among the mycobacteria include immunological techniques, comparison of cell wall components, comparison of homologous enzyme sequences, DNA/DNA homology, plasmid profiles, restriction endonuclease analyses, direct 16S rRNA analysis, spoligotyping, insertion sequence analyses, VNTR analysis and whole genome DNA microarrays.2, 8, 6, 15, 74

Mycobacteria are susceptible to sunlight and ultraviolet radiation.17 Protected from daylight, M. tuberculosis can survive in moist or dried, pulverized sputum for weeks or months. Mycobacterium bovis was found to survive for six months in soil, in soil and dung mixtures, and in dung, and for 49 days on grass under moderate climatic conditions.49, 62 In the northern Australian environment, however, the bacillus may only survive in the soil for up to four weeks and exposure to sunlight causes rapid death. In South Africa’s Kruger National Park M. bovis was found to survive for a maximum of six weeks in winter under moist and shady conditions while no viable M. bovis organisms were recovered from naturally infected tissue exposed for longer than three weeks in summer.24, 75 This probably also applies to many parts of southern Africa. Mycobacteria remain alive for over two years in frozen carcasses, and at 9 °C can survive for considerable periods in cheese and butter. With heat treatment, M. bovis is destroyed in meat products at temperatures 6 to 7 °C lower than those necessary for the destruction of the members of the M. avium complex.55–57 Temperatures above 60 °C for 10 minutes destroy M. bovis in Vienna sausages. Disinfection of equipment contaminated by meat emulsions containing M. bovis is achieved by treatment with 2 per cent phenolic disinfectant followed by formaldehyde vapour.57 Disinfectants such as 3 to 8 per cent formalin, 5 per cent phenol or 3 per cent iodophor kill mycobacteria, as does exposure to a temperature of 60 °C for 15 to 20 minutes.17, 64

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