Introduction

Pathogenic members of Mycobacterium and related genera are known to cause chronic granulomatous lesions in animals and people. They are characterised by having a thick mycolic acid layer covering their peptidoglycan layer in the outer membrane. This makes them resistant to desiccation and improves their survivability within host cells, especially the monocyte-macrophage. The most important pathogens belong to the Mycobacterium tuberculosis group and include Mycobacterium tuberculosis the agent of human tuberculosis and Mycobacterium bovis, the agent of bovine tuberculosis. Both are reportable diseases in Australia and worldwide. Mycobacteria are very slow to replicate, with the slowest grower being the agent of Johne’s disease in livestock, known as Mycobacterium avium ssp. paratuberculosis. It too, is a reportable  disease in animals in Australia.

As shown in the picture below, mycobacteria are related to Corynebacterium, Nocardia and Prescotella. 

Diseases in animals caused by Mycobacterium and related genera

Many of the diseases in animals are not only economically important and hard to treat, but are also of public health significance. A list of the diseases is shown in the table below.

Morphological characteristics of Mycobacterium and related genera

Bacteria of this genus uniquely have thick walls very rich in mycolic acids. This makes them especially resistant to chemical agents and allows pathogenic strains to survive in the environment for a prolonged period. In the body, they are able to resist acid degradation in the phago-lysosome within macrophages.

The picture below provides a diagrammatic representation of the unique characteristics of the Mycobacterial outer membrane.

Although they have a thick peptidoglycan layer like other gram-positive bacteria, they rarely take up the Gram’s or Romanoski type stains. Heated staining is required to render the wall permeable to stains and unlike other bacteria, they resist a strong acid decolourisation (3% HCl in ethanol) of the primary stain. A special acid-fast stain known as the Ziehl-Neelsen stain will stain them a deep red and any other bacteria in the sample either the blue or green of the counterstain. Occasionally they will partially stain with the Gram’s stain giving a beaded appearance. Note that the beaded appearance on the Gram stain can also occur with other members of the Corynebacteriales, like Trueperella pyogenes.

Pathogenic mycobacteria tend to be slow-dividing. The slowest – Mycobacterium avium subsp. paratuberculosis divides every 12 hours (compare with E. coli which divides every 20 minutes). They are also facultative intracellular parasites where they are able to survive in the cytoplasm as well as in the acidic phago-lysosome.

Diseases caused by mycobacteria usually involve the lymphopoietic system, and are chronic and insidious (they only reveal their presence late in the disease process). Due to the slowly-developing lesions, intracellular nature and poor antigenicity of the cell wall, early diagnosis of disease caused by this genus is difficult.

Activated macrophages i.e. those primed by γ-interferon produced by differentiated T_H1 are able to destroy mycobacteria either via the respiratory burst response or by phagosome acidification and phagosome-lysosome fusion. This is augmented by cytotoxic T-cells that lyse infected macrophages releasing mycobacteria and thus making them accessible to the immune system. Effective immunity against this genus is predominantly cell-mediated.

General pathogenesis of Mycobacterium species

On entry into the body, either via inhalation, ingestion of percutaneously, the local phagocytic cells, i.e. alveolar macrophages in the lungs, phagocytose mycobacteria via a receptor-dependent, not antibody-dependent process.  In this way, the antibody-dependent killing by macrophages is not triggered allowing mycobacteria to survive and divide in phagocytic cells. A mycolic acid in the cell wall known as trehalose dimycolate (TDM) or “cord factor” is believed to assist in protecting mycobacteria from phagocytic killing especially the action of gamma interferon. However, infected macrophages recruit other macrophages to the site of infection ensuring a predominantly granulomatous inflammation.

As more cells are recruited they re-organise into discrete granulomas or tubercules. These granulomas typically consist of a central area of necrosis, sometimes with mineralisation surrounded by giant and epitheloid cells, lymphocytes and fibroblasts encased in a collagen layer.

Sometimes a T_H2 rather than a T_H1 is initiated. One of the manifestations of the T_H2 response is the lepromatous response, where the lesions have a low cellularity and are predominantly liquid. The non-protective T_H2 immune response can result in haematogenous spread of the bacteria with the consequence of disseminated tuberculosis. Disseminated tuberculosis is often recognised by the presence of numerous small granulomas (miliary tuberculosis) in a number of internal organs i.e. the lungs, liver, bone and spleen.

BOVINE TUBERCULOSIS (mYCOBACTERIUM TUBERCULOSIS VARIANT BOVIS INFECTIONS)

Mycobacterium tuberculosis variant bovis (M. bovis) is the cause of a chronic debilitating disease of  bovids and other mammals that affects the lymphohaematogenous system, with primary lesions being common in the lower respiratory tract. It belongs in the tuberculosis cluster which includes: M. tuberculosis var. tuberculosis (human adapted), M. bovis, M. africanum, M. tuberculosis var. caprae (goat adapted), M. pinnipedii (seal adapted), M. microti (voles), and M. canetti. Of these, only M. tuberculosis and M. bovis are of huge clinical significance.

In many countries, including Australia, bovine tuberculosis is notifiable disease.  This not only because it is a chronic debilitating disease, but also because the agent can spread to people. Due to a massive eradication campaign in and 1980s and 1990s, last recorded cases was in an Asian buffalo in NT in 2002, when abattoir monitoring of granulomas ceased in 2010. Below is a map of the current global status of bovine tuberculosis.

Host range

Although bovids, such as cattle and buffaloes, are the primary maintenance hosts, the bacterium is able to infect a wide range of mammals including humans, dogs, badgers, possums, lions, antelopes and pigs. When the infection rate is high enough and animals live in large, overcrowded colonies, the infection can become self-sustaining in non-cattle. Non-cattle maintenance hosts include, the badger population in Great Britain, the brush-tailed possum and red (farmed) deer populations in New Zealand, the bison in North America, the African buffalo and lions in South Africa and the wild pigs in Spain.

Dead-end hosts, include people (ZOONOSIS), dogs and horses. Note that outbreaks of M. bovis have been recorded in dogs and cats who have fed on infected offal.

Pathogenesis and Clinical signs

Transmission is by inhalation or by ingestion where the bacteria are ingested by macrophages and transported to the nearest lymph node. Inflammation at the site of entry and the regional lymph node is known as a primary complex. In immune animals, the infection usually remains localised and may resolve over time. In the face of incomplete immunity, the lesion/s will enlarge slowly eventually resulting in sufficient tissue damage and clinical disease. Sometimes bacteria remain viable in these lesions, only to reactivate years later where they can disseminate throughout the body and cause numerous small areas of caseous necrosis. This is known as miliary disease. The route of entry determines the site of lesions and ultimately the clinical presentation of this disease.

Most diseased animals will shows signs of gradual weight loss. Infection of the lungs may result in a persistent cough that is especially noticeable on exercise or when the animal gets up after a period of rest. Lymph nodes will enlarge and the superficial ones such as those on the head and above the mammary gland will become palpable. The pictures below show examples of the lesions caused by M. bovis. Whilst in bovids it is often the lungs and associated lymph nodes that are affected any organ can develop granulomas.

In people (Public Health component)

In countries where there is a high prevalence of M. bovis infections in domestic animals human cases are common. Most infections are transmitted by the ingestion of unpasteurised or inadequately pasteurised infected cow’s milk. However, droplet inhalation is increasingly being reported i.e. dairy workers. Most infections in humans remain localised i.e.  in a lymph node of the head or thorax. However, in immune compromised individuals the infection may spread further and be confused with human tuberculosis. In children granulomas may form in the vertebral canal. Infected humans are usually treated with anti-tuberculous drugs.

Mycobacterium tuberculosis Variant tuberculosis

This bacterium is the cause of human tuberculosis which is pandemic. It is highly related to M. bovis, but has a much more limited host range where it will only spread from humans and non-human primates. In cattle the disease is usually restricted to the regional lymph nodes. However, in some mammals severe disease due to M. tuberculosis has been recorded. Since infection of animals with this bacterium can lead to positive test results and consequently the slaughter of positive animals, it is important to confirm a diagnosis in cattle and other mammals to M. tuberculosis.

The clinical signs, pathogenesis, pathology and diagnosis of this agent in humans is similar to that of M. bovis.

Confirming the diagnosis of bovine tuberculosis

Since the clinical signs of the disease are non-specific, the diagnosis of the disease in a living animal is difficult. It is usually based on the presence of a specific immune response. The most sensitive test is the intradermal tuberculin test, where an infected animal will show a localised area of inflammation 2 to 3 days after a purified protein derivative (PPD) suspension of the bacteria was injected into the skin, usually in the neck or caudal skin fold. This is typical of the type IV hypersensitivity response. Other mycobacteria such as the one that causes human tuberculosis, M. tuberculosis, M. avium subsp. paratuberculosis and environmental mycobacteria may also cause a false positive response in this test.  To rule out non-tuberculous bacteria (MOTT) and those belonging to the M. avium complex a comparative intradermal test may be used using PPD from M. avium. MOTT will cause a larger response to M. avium antigen. A laboratory based test, known as the interferon-gamma test which measures the type IV hypersensitivity response in whole blood can also be used, but tends to be less robust than the intra-dermal test. Animals in the late stages of tuberculosis may become insensitive or anergic to the intradermal test. These animals usually have measurable antibodies in an ELISA or are easier to recognise clinically (thin, enlarged superficial lymph nodes, cough).

Positive cases on intradermal tests

Positive animals are slaughtered and examined for any granulomatous lesions. Particular attention is paid to the lymph nodes of the head, thorax and udder. The lungs are also examined visually and by palpation. Lesions are collected and submitted for organism detection by culture and qPCR. Since the bacterium grows slowly in culture (up to 2 months), radioisotope broths that specifically detect mycobacteria have been used to speed up the process as well as qPCR. Histology will reveal a typical granulomatous response with the presence of numerous giant and epitheloid cells and a central area of caseous necrosis that may contain areas of mineralisation. Acid-fast bacteria may be present in the lesion in varying numbers and should be carefully sought.

Control in countries free from Mycobacterium bovis infections

In countries, such as Australia, that are declared by the WOAH to be free of the disease, routine surveillance is done at the abattoir by meat inspectors, where any suspect lesions are submitted for laboratory confirmation. Should a carcass test positive, trace-backs and trace-forwards will be done – where intensive testing will be carried out, positive farms placed in quarantine and positive animals slaughtered. However, this is a slow process necessitating long quarantine periods. Therefore, the relevant veterinary authority may opt to eradicate the disease by slaughtering all cattle and other susceptible animals on a property; so-called “stamping-out”. These countries also enforce strict importation regulations which will include permits declaring that imported animals or their semen and embryos originate from regions or farms free from tuberculosis and that the imported animals have been tested free from the disease. Note that according to the WOAH 99.8% of herds have to be free of the disease to ensure disease-free status, so sporadic cases may be detected from time to time without the disease-free status being withdrawn.

An AUSVETPLAN published in 2009 is available online on how to manage incidences of bovine tuberculosis.

johne’s disease in ruminants and alpacas

Mycobacterium avium ssp. paratuberculosis (Mptb) is the agent of paratuberculosis or Johne’s disease,  a slowly progressive granulomatous disease of the intestine that affects predominantly ruminants. Other animal species including rabbits, deer, horses and humans are also susceptible. It is present in some sheep flocks, alpacas, goats herds and cattle herds in Australia. The disease caused by sheep strains of Mptb are endemic in many States and Territories in Australia.  Less common are the cattle strains of Mptb. The most recent reported cases in cattle were  in 2021 in Western Australia. The most recently reported cases in Queensland were initially at a Red Brahman stud near Rockhampton with trace forwards identifying a further 97 potentially infected farms. This outbreak has been eradicated. It is a NOTIFIABLE DISEASE in Australia.

Mycobacterium avium subsp. paratuberculosis is usually transmitted to calves either in utero (26%) or to calves and other young animals within the first few weeks of life, mainly via the faecal-oral route, but occasionally via maternal milk. Repeated exposure to the agent is required for infection. The organism can under ideal  (high moisture, low pH) conditions survive for more than one year in soil and up to 2 two years in water. It is resistant to acids and alkalis and will survive in faeces for up to one year. It is destroyed by moderate heat and 5% formalin. Since it is a very slow developing disease it will go unnoticed in a herd for many years and only be recognised once the disease reaches a high prevalence rate.

Oral infection with this slow-growing mycobacteria results phagocytosis by macrophages in the lymphoid tissue of the small intestine. The bacteria will then spread haematogenously. However, a diffuse granulomatous inflammatory response occurs only in the distal intestine, especially the ileum, intestinal lymphatics and associated lymph nodes. The incubation period is incredibly long, usually between 15 to 18 months.  The progressive granulation of the intestinal mucosa gradually leads to malabsorption, diarrhoea, hypoproteinaemia, emaciation and death.

Only one in 20 infected animals will show clinical evidence of disease. Affected animals suffer from emaciation, bottle jaw and will be extremely hungry. Cattle may show profuse diarrhoea. In sheep, the stool is only slightly pasty.

DIAGNOSIS OF JOHNE’S DISEASE

Due to its long incubation period and non-specific clinical signs, the disease in live animals can be difficult to diagnose. Since animals shed bacteria prior to the development of clinical signs and the fact that their appetite is generally unaffected, it is important that infections with this bacterium be identified early in the disease course so that faecal shedding is reduced.

Herd Diagnosis is based on:

1. Farm history
2. Presence of emaciation and diarrhoea in older animals (>2 tooth). Check for the animals that lag in a flock/herd
3. Abattoir surveillance – examine for typical gross pathology within the ileum (see below)
4. Acid-fast staining of rectal swabs and serology to screen animals
5. Pooled culture and qPCR of faeces from older sheep

The advantages and disadvantages of each test used is tabulated below and was obtained from the website of Animal Health Australia.

A review of the diagnostic tests for Johne’s disease is also provided by the OIE in the Terresterial Manual (2014).

Pathology

Although difficult to diagnose in the living animal, the pathology is typical.  The intestinal wall becomes thickened due to the proliferation of large numbers of epitheloid cells and will resemble the gyri of the brain and epitheloid cells plugging the lymphatics result in them becoming twisting and having a glassy appearance = lymphangiectasia. The mesenteric lymph nodes are enlarged. Histology of affected tissue usually reveals a granulomatous response rich in epitheloid cells with the presence of numerous acid-fast cocco-bacilli found in clumps.

Direct acid-fast (Ziehl-Neelsen) stain of faeces, intestine and associated lymph nodes)

Acid-fast staining of faeces or rectal mucosa for the presence of acid-fast cocci usually present in clumps or nests. This result is presumptive that must be followed by culture.

Serological tests

Usually ELISA for cattle & sheep; AGID (agar gel immunodiffusion test) for sheep and goats. There is no certified serological test for alpacas and deer. These tests tend to be positive just prior to clinical signs and are better as herd tests rather than to identify individual animals. The ELISA is used as a screening test on animals over 2 years of age for export. All positive ELISA should be followed up. The AIGD test in sheep yields one false positive every 1000 tests, and the ELISA 2/1000 tests. This is more important in negative herds.

Confirmation tests: Pooled culture and qPCR

• Culture and then identification of the culture by qPCR on faecal samples either individually or a pooled faecal sample. Once a pooled faecal sample tests positive, individual samples are tested. It is important that a laboratory certified to work on Mycobacterium a. paratuberculosis is used as its cultivation and identification require specialised methods and media. A medium rich in fatty acids and one contains an iron-chelating agent known as mycobactin J is used. Due to the slow growth of the bacteria the result can take between 2 to 3 months. Environmental samples containing faeces can be used for continuous monitoring in dairy herds that are known to be negative.
• A high-throughput qPCR can be done directly on faeces, but it can lack sensitivity due to the presence of DNA inhibitors in the faeces. A positive result in a negative or unknown status herd should be followed-up by culture or by post-mortem examination.

The Table below compares the different tests for Johne’s disease in Australia.

 control of johne’s disease

This is a notifiable disease in Australian States and Territories and control is based upon the disease status of the State and whether the individual farm has joined an accreditation scheme.  Johne’s disease is a category 1 restricted matter and under the Biosecurity Act 2014 and suspect cases should be reported to Biosecurity Queensland on 13 25 23 or the Emergency Disease Watch Hotline 1800 675 888.

Most States and Territories no longer place affected properties under movement restrictions. Producers with an infected or suspect herd must manage the disease under their general biosecurity obligation.  Animal Health Australia coordinates and manages control of Johne’s disease.

It is based on the following:

1. Farm and Regional Biosecurity plans. A risk profiling tool known as the Johne’s beef assurance score (J-BAS) and a revised dairy beef score are available to assist beef and dairy producers respectively in determining the risks of Johne’s disease.
2. Producers can decrease their risk rating by joining a quality assurance programme, namely SheepMAP; GoatMAP; AlpacaMAP
3. Vaccination of sheep on endemic farms with the Gudiar’s bacterin vaccine for sheep. Vaccination of cattle on endemic farms more than 3 weeks of age with Zoetis Silirum vaccine for cattle (not registered for use in Western Australia). As this is a notifiable disease, Chief State Veterinarian permission should be sought before using the vaccine. Animals for export should not be vaccinated as vaccinated animals may test false positive on the single intradermal test for M. bovis.
4. Completion of an appropriate Animal health declaration form when selling animals. The forms are available at this website.

Note that all the plans that are in place to control Johne’s disease are determined by the use of approved diagnostic methods. It is in the producers’ best interests to send positive animals to the meatworks. The producer should also be aware that soil can remain contamined with Mptb for at least a year.

Consider the following control measures in a Biosecurity plan

• Estimate the infection rate of a herd. A rough estimate is to find out how many clinical cases there are per year and to assume that for every clinical case there are 5 – 10 sub-clinical cases. Or, more scientifically, determine the percentage of animals with antibodies to M. a. paratuberculosis and knowing that the sensitivity of these tests are about 50% in detecting sub-clinical cases calculate the herd prevalence. This gives an idea as to how many animals are acting as sources of infection.
• Test and cull policy. All positive animals should be culled. To make the most rapid progress but not necessarily is the most cost-effective; the animals should be tested serologically and by faecal cultures every six months with every positive reactor being culled. If funds are not available, annual testing is sufficient.
• Survey management practices e.g. Waste management and period of contact with adult cattle. Ideally calves should be reared in an area free of contamination by faeces and free from adult animals for at least the first 6 months of life.
• Avoid the feeding of waste or mastitic milk. Milk replacers are a better alternative.

• Ensure that the water supply to the calves/lambs/kids is not contaminated.
• Herds/Flocks free of Johne’s disease should ensure that they purchase animals from paratuberculosis-free herds/flocks or test the animals before purchase.
• Vaccination of the Gudair bacterin vaccine to sheep in management zones. Note that JD vaccines can cause severe local reactions including sloughing of tissues, chronic synovitis and tendonitis, if they are accidentally injected into humans. Some cases may require surgery.” The Silirum bacterin-Zoetis vaccine is now available for cattle. It should only be used on confirmed positive farms and NOT on cattle intended for export as these cattle will test positive on serological and intradermal tests. It can only be supplied on approval by the State Chief Veterinary Officer in all States with the exception of Western Australia where it is not registered.

Avian tuberculosis

Avian tuberculosis is a chronic wasting disease of poultry and other birds caused by mainly M. avium subsp. avium, Mycobacterium genavense or occasionally other Mycobacterium species. It has a global distribution, including Australia.

Transmission is usually by the faecal-oral route. Clinical manifestations in birds include emaciation (as noted by atrophy of the breast muscles), depression and diarrhoea. Granulomatous lesions can be seen in the liver, spleen, intestine and bone marrow. Unlike tuberculosis in mammals and man, lesions in the lungs are rare. The disease is a rarity in the commercial poultry sector due to improved farm practices, but occurs in bird collections and backyard avian rearing operations. The bacterium in cattle causes sensitivity to the bovine tuberculin test – thus the comparative intradermal test is preferred in avian tuberculosis endemic areas .

Treatment for this disease is usually unsuccessful and birds either die naturally or are euthanased.

For more information consult:

OIE Terresterial Manual (2014) Chapter 2 Avian tuberculosis.

Wildlife Australia Fact Sheet (2013) Mycobacteriosis in Australian Birds.

Leprosy (Hansen’s disease)

M. leprae and M. lepraemurium are non-cultureable mycobacteria (obligate intracellular pathogens) that cause leprosy in humans and cats respectively. These bacteria infect the skin via wounds causing granulomas and ulcers. They then spread to the peripheral nervous system. Since the advent of antibiotic therapy, leprosy has become a rare disease in humans with only 17 countries (including Indonesia, India and Bangladesh) diagnosing more than 1000 cases per year. Although, not usually considered a zoonosis, it has recently been reported that armadillos present in the southern States of USA can harbour this bacterium and can be a source of infection for humans (Same genotype as a geographic cluster of infections in people. USA has about 10 human cases per year).

The disease is rare in cats.

Rapidly growing (also called saprophytic or atypical) mycobacteria causing infections

Mycobacteria in this group tend to be fast-growing and are common in the soil and water. Occasionally they will infect animals, usually via injuries to the skin, and cause localised granulomas. In animals the most common species identified is Mycolicobacterium fortuitum. This a common granulomatous lesion in cats and dogs in Tropical regions. More recently the more rapidly growing and pathogenic Mycobacteriodes abscessus has been identified in Infections in dogs in Australia, including Townsville. Localised infections with these bacteria can result in false positive intradermal tests in cattle.

Treatment in animals is by the administration of anti-mycobacterial antibiotics and surgical removal of the affected tissue.

Humans can also be infected with the most commonly reported cases due to contaminated substances containing e.g. M. chelonae used in tattooing or skin surgery. In 2018 there were a high number of cases of Mycobacterium infections in people that have undergone heart surgery. This was from a particular brand of a blood warming device.

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