Test 3- Mycobacterium Flashcards
Mycobacterium
• Gram positive,
• Acid Fast positive bacteria
• Important human and animal pathogen
Mycobacterium spp.
Bacteria that contain mycolic acid and have a unique peptidoglycan chemotype
stain lightly gram positive
Acid Fast stain positive
Mycolic Acids
Fatty acids in the cell wall
Carbon chain length varies by Genus
Rapid-growing Mycobacterium spp have shortest chains Slow-growing Mycobacterium spp. have Longest chains Corynebacterium has the shortest
mycobacterium have the longest
Properties Attributed to Mycolic Acid in Mycobacteria
Acid fast staining
Drug, chemical and environmental resistance
Immunomodulating activities
(lipoarabinomannan, lipoarabinogalactan, wax D)
Prevent phagocytic killing- why they can survive in the cell
(trehalose dimycolate - cord factor)
(phenolic glycolipid - scavenger of oxygen radicals) (sulphated glycolipids - prevent lysosome fusion)
Acid Fast Stain
A differential stain that uses a lipid permeablizing first step (with heat or chemical solvent);
- Primary stain
- An acidic alcohol decolorizing step
- A counter stain
(Carbol fuschin>decolorizer>Methylene Blue) Examples
Ziehl-Neelsen Stain
Kinyons stain
Auramine Rhodamine stain(Fluorescent based stain- Very sensitive) for TB
Habitat of Mycobacterium spp.
Some species are obligate pathogens- always ass with disease
(M. tuberculosis complex, M. avium subsp paratuberculosis, M. leprae, M. lepraemurium)
- *Opportunistic Pathogens**- present in the soil and water
- *Many species are soil / water saprophytes**
(rapid-growers, M. avium complex, M. kansasii, M. intracellulare, etc.)
Under optimum conditions obligate pathogens can survive in a contaminated environment for extended periods
Identification of Mycobacterium sp.
Runyongroups:
Growth patterns:
Photochromogens (pigmented in the presence of light)
Scotochromogens (pigmented in the absence of light) Non-Chromogens
Rapid growers (\< 7 days) Slow growers (2-10 weeks) Mycobactin dependent (exogenous siderophore)
Biochemical patterns (limited specialty techniques)
Total cell fatty acid analysis
Nucleic acid detection (DNA probes/PCR)
DON’T HAVE TO REMEMBER THIS SLIDE
Mycobacterium Virulence Factors
1. Mycolic acid containing cell wall lipids
Facilitate Survival in macrophage (Facultative intracellular Pathogens) Stimulate cytokine production
Enhance Adjuvant / immunomodulating effects
2. Cell protein antigens
Tuberculin> purified protein derivative > specific tuberculoproteins
Protein exotoxins and extracellular enzymes generally do not play a prominent role in disease pathogenesis
(except M. ulcerans – mycolactone(-mycins antiboditics)/macrolide toxin)
Diseases caused by Mycobacterium spp.
• Mammalian tuberculosis:
Avian tuberculosis:(M. avium subsp. avium serotypes 1-3)
Leprosy: M. leprae (human), M. lepraemurium (cat)
Johne’s disease: M. avium subsp. paratuberculosis
(M. tuberculosis complex –M. tuberculosis, M. bovis, M. africanum, M.microti)
M. tuberculosis
• Humans are the main reservoirs
• Tuberculosis (TB) is second only to HIV/AIDS as the greatest killer worldwide due to a single infectious agent.
Emerging zoonosis and anthropozoonosis
• Dogs, cats, pigs, nonhuman primates
• Psittacine birds and canaries are susceptible to tuberculosis
Elephant-to-Human Transmission of Tuberculosis Endemic infections in some wild life populations
Eg. Banded Mongoose in Botswana
Suricates in south Africa
Drug resistant
Multidrug resistant TB (MDR-TB)
Resistant to at least isoniazid and rifampin
Extensively Drug resistant TB (XDR)
Resistant to isoniazid and rifampin, plus any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin).
Mycobacterium bovis
Cause zoonotic TB
Ingestion, inhalation and, less frequently, by contact with mucous membranes and broken skin.
Wide host range and geographic distribution
GI tract is the main portal of entry
MI and Texas- DEER
M. bovis: Host range
host range: Ireland & UK(badgers), New Zealand(
Maintained primarily in bovine species but has the broadest host range of all TB organisms and can infect several wildlife species (especially cervids in US)
Usually survives only a few weeks outside of host
Causes disease indistinguishable from that of M. tuberculosis in humans
Transmission of M. bovis
cattle- any organ
cats- get via ingestion
dog- get from humans or ingestion
birds
TB in wild deer
can also be in the lymphnode as shown below
THESE ARE GRANULOMAS
Other reservoirs of Mycobacterium bovis
- Badgers in UK
- Feral Brush tail possums: New Zealand
M. bovis infection (bovine tuberculosis)
Chronic, progressive and latent infections
Disease is seldom apparent until it has reached advanced stages (months)
Some infected livestock appear in prime condition showing no evidence of infection until slaughtered and then may be so severely affected that the carcass is condemned
In some cases the organisms remain dormant in the body for lifetime without causing progressive disease
M. bovis: Transmission
• Aerosol transmission most common among
cattle (greatest risk occurs in enclosed or crowded areas eg. barns,, markets, shared watering/feeding places)
- *• May be shed in milk** (most human infections prior to widespread pasteurization resulted from drinking or handling contaminated milk)
- *• Can enter body at any site**
AEROSOL AND INGESTION- MOST COMMON
M. bovis: Clinical Signs
Signs vary greatly with extent of exposure and site of infection
Enlarged regional lymph nodes and generalized wasting (cachexia) are seen in advanced disease stages. Pulmonary forms may be associated with chronic cough
M. bovis: Pathology
May cause lesions in any organ
In early stages lesions difficult to find at necropsy
Some early lesions may grossly appear abscess-like
In later stages firm, nodular lesions become evident in target organs and associated lymph nodes (lungs, head, gastrointestinal)
MAIN LESION IS GRANULOMA IN MYCOBACTERIA(INCLUDING TB)
Pathogenesis
Bacilli are phagocytosed by macrophages>Infected macrophages secrete TNF-alpha and IL-12>T-helper 1 lymphocyte activity and >secretion of INF-gamma and
IL-2 >Cell mediated immunity and destruction of bacilli
If the bacilli survive, infected macrophages are killed following stimulated release of macrophage-derived cytotoxins and enzymes (type IV hypersensitivity or Delayed type hypersensitivity), which leads to tissue destruction and caseous necrosis WITH MINERALIZATION
Liquefaction and cavity formation result from enzymatic action.
Rupture of these cavities allows dissemination.
Lesions
Tubercles are the classic lesion of tuberculosis.
These are well-organized granulomas of varying size with central areas of either solidly packed epithelioid macrophages (“hard” tubercle) or caseous necrosis (“soft” tubercle) surrounded by epithelioid cells and scattered multinucleated giant cells(Langhan’s cells).
A rim of lymphocytes and fibrous connective tissue surrounds mature lesions. In advanced stages, cavity formation may occur.
Acid fast bacilli are seen within macrophages or extracellularly within a caseous core.
Elements of a Tubercle
Granuloma formation as a result of Cell mediated immune response
Activated macrophage/epithelioid cells
Coagulative /caseous necrosis
Fibrosis (fibroblasts/collagen)
Mineralization (liquefaction may also occur)
Granulomas in the lungs
Langhan’s giant cells- multi-nucleated in the center with macrophages
M. bovis: Diagnostic Tests
Tuberculin hypersensitivity skin tests
(low sensitivity in some species, eg. cervids)
• Read reaction at injection site after 72 hrs - TYPE IV DELAYED HYPOSENSITIVITY(cell-mediated immune response)
- screening test in caudal tail fold with single-strength
dose of M. bovis PPD
- suspect reactors in screening test are subsequently
tested by paired cervical tests with double strength M. bovis PPD and M. avium PPD in separate areas of the neck
Caudal Fold Tuberculin Test (screening test)
• single strength bovine PPD is injected intradermally into the caudal tail fold
SENSITIVE BUT NOT SPECIFIC( lots of Mycobacterium found in the soil)
Comparative Cervical tuberculin skin test
(confirmatory test with double strength bovine + avian PPD injected intradermally into different sites of skin on the neck
skin thickness is measured before and after
MAKE SURE IT”S MYCOBACTERIUM BOVIS
M. bovis: Diagnostic Tests
In vitro
• In vitro whole blood tests for cell-mediated immunity:
(generic test description = interferon gamma release assays) - Bovigam - bovine sp. only, contains PPD
- QuantiFERON-TB Gold – human/primates only,
contains mixtures of synthetic peptides derived from 2
tuberculoproteins, ESAT-6 & CFP-10
- lymphocyte proliferation assays (can be used in zoo & wild sp.)
M. bovis: Diagnostic Tests
Post mortem examination/Histopathology slaughter checks
Post mortem examination/Histopathology slaughter checks
(most effective in low prevalence areas)
• Granulomatous lesions in lymph nodes and major organs
MOST COMMON
M. bovis Diagnostic Tests- SMALL ANIMAL
• Imaging techniques
• Imaging techniques (radiograph, CT, etc) live animal – rarely used for bovine TB dx
M. bovis: Diagnostic Tests Direct Examination of Tissues (from live or dead animals)
Acid fast stained smear (sensitivity may be low due to low numbers)
Nucleic acid detection
PCR on fresh or fixed lymph node tissues
Fatty acid methyl ester (FAME) detection by HPLC (rarely used for bovine TB dx)
M. bovis: Diagnostic Test
• Culture
• Culture (special procedures /media required) - conventional tube media slants
- liquid media used with automated,
continuously monitored incubators
M. bovis: Treatment
- Treatment of bovine TB is not practical or efficacious
- Optimum human treatment may achieve success rates of >95%
Anti-tubercular Drugs
Anti-tubercular Drugs (always used in combinations to treat active tuberculosis)
Primary Drugs
• Isoniazid
• Rifamycins
(rifampin, rifabutin, rifapentin)
- Ethambutol
- Pyrazinamide
Secondary Drugs- for Multidrug resistant TB
•Aminoglycosides
( streptomycin, kanamycin, amikacin, capreomycin)
•Fluoroquinolones
( levofloxacin, moxifloxacin,
gatifloxacin)
- Ethionamide
- Cycloserine
- 4-Amino Salicylic
acid (PAS)
Problems with TB Treatment
(human)
Single drug activity limited
(bacteria present within a lesion can be extracellular, intracellular, grow in logrithmic phase and exhibit quiescent, stationary phase physiology)
Multiple drug resistance (MDR/ XDR)
Prolonged treatment needed
Lesions walled off, poor drug distribution (reactivation)
Elements of bovine TB eradication programs
- Disease surveillance
- Pre and post movement cattle testing
- Removal of cattle exposed to bTB
- Tracing the potential source of infection
- Wildlife control (including culling/vaccination trials)
USDA-APHIS Accredited-Free Status
Applied to States or herds
No confirmed cases for 5 years
States must have laws/rules in place governing bovine TB
Herds must adhere to a schedule of whole herd tuberculin testing
Maintain records for trace back
M. bovis: Vaccination
There are no vaccines currently available for TB in cattle; experimental vaccines are being investigated for cattle, wildlife and people- DON”T WANT A FALSE POSITIVE WHEN YOU DO INTRADERMAL TEST
Human vaccine (BCG(used to treat bladder cancer) – originally of M. bovis phenotype) is used in high prevalence areas:
Vaccine is considered suboptimum and efficacy is questioned. Also interferes with diagnostic tests. Not used in the U.S.
Mycobacterium leprae
Leprosy or Hansen’s disease
Chronic granulomatous debilitating disease
Anaesthetic skin lesions(lost of sensation), peripheral neuropathy, and nerve thickening
Transmission from shedding through nose not from skin
Nine banded Armadillo in southern united states is the only known animal reservoir
Can not be cultured in vitro
Properties of mycobacteria include all except;
a. presence of mycolic acid
b. positive acid fast staining
c. presence of lipid compounds in the cell wall
d. may survive in the macrophages
e. all members are obligate pathogens
E
some are opportunitistic pathogens
some live in the soil
Properties of mycobacteria inlcude all except:
A. variable mycolic acid length depending on species
B. complete of partial acid fast staining
C. Presence of lipid compounds in the cell membrane
D. May survive at lower pH
E. some members are saprophtic
C
Because they don’t have a cell membrane!
Which of the following staining method is not an acid fast stain?
A. Kinyoun’s
B. Ziel-Neelsen
C. Auramine rhodamine
D. Gemisa
D.
Floursent microscope- auramine
Mycolic acid present in the cell wall of Mycobacteria facilitate Phagocytosis and removal of mycobacteria
T/F
False-
mycolic acids helps the bacteria to survive inside the phagocytic cell
Which of the following mycobacterium is a common cause of zoonotic Tuberculosis?
M. Bovis
All of the following animals are reservior of M. Bovis in certain geolocations except
A. Elephants
B. Cattle
C. Badgers
D. Brush tail possums
Elephants- reserviors of M. Tuberculosis which they can transfer to humans
Which of the following is incorrect about mycobacterium infections?
A. induce granulomatous inflammation
B.Multinulceated macrophages are common in lesions
C. Caseous necrosis and mineralization may be present in the lesion
D. Humoral immunity helps in protecting from infections
D. It’s Cell mediated (T cells)
Combination antimircobial thearpy is essential in the treatment TB to kill multiple species mycobacterium present in a lesion.
FALSE-
Combination therapy is used because of resistance and the organism can be intercelluar or extracellular.
Most of the time, these are single TB infections
Tuberculoid leprosy
Tuberculoid leprosy (paucibacillary)- few AFB- few bacteria
good cell mediated response
• Lepromatous leprosy (multibacillary)-
Lepromatous leprosy (multibacillary)- No cell mediated response, severe disease with numerous bacteria
MORE INFECTIOUS
Mycobacterium lepraemurium
- Feline and Murine Leprosy
- Solitary or multiple cutaneous nodules or ulcerated lesions
- Very fastidious organism(hard to culture)
• Granulomatous Dermatitis Panniculitis(inflammation of the Sq tissue)
Canine Leproid granulomas
Diagnosis
Direct staining (Geimsa or Acid Fast staining)
In Geimsa or Gram stain-Negative stained bacilli are generally observed
Culture
Biopsy and Histopathology
PCR (Genus specific PCRs)
Treatment
- Surgical excision
- Rifampin, Clarithromycin, Clofazimine
• Combination therapy ?- always USE! otherwise you get resisitance
• Long term therapy
• In dogs, Doxycycline can be effective.
Saprophytic Mycobacterial Species
Rapid-growing species: eg. M. fortuitum, M. abscessus, M. chelonae, M. smegmatis
Slow-growing species: eg. M. avium, M.kansasii, M. marinum
Granulomatous to pyogranulomatous host response- ALL CAN CAUSE
Chronic, non-healing cutaneous lesions (panniculitis, cutaneous ulcers) and Lack of response to common antibacterial treatments
Internal organs(lung, Liver, spleen, intestines)
Solitary masses, diffuse lesions
Lesions can be “multibacillary” or “paucibacillary”- depends on the immune response
Mycobacterium avium complex (MAC group)
Mycobacterium intracellulare
M. avium subsp. avium.- Avian tuberculosis
M. avium subsp. Sylvaticum- Non-tuberculous (atypical) mycobacteriosis
M. avium subsp.‘hominissuis’ (pigs and immunocompromised humans)
M. avium subsp. paratuberculosis -Johne’s disease in ruminants
Generals about MAC
• Wide spread in soil and water, including treated municipal tap water
(low pH and high temperature tolerance i.e. hot tubs)
• Phenotypically indistinguishable members
VERY RESISTANCE!
Mycobacterium avium subsp. paratuberculosis (Johne’s Disease)
• A chronic, progressive granulomatous enteritis
• Mycobacterium avium subsp paratuberculosis
• Primary Hosts-Ruminants (Cattle- mainly)
• Infects sheep, goat, llamas, alpacas, deer
• Annual economic loss of 220 million
• Excretion of organism through milk?
• Resistance to pasteurization temperature?
• Possible etiology in Crohn’s Disease
Johne’s problems
Where there is a clinically affected, then there are many more in different stages of infection.
“Iceburg effect!
Symptoms:
Diarrhea and weight loss,
can have bottle jaw due to loss of protein
Pathogenesis of Johne’s Disease ?
Why weight loss and diarrhea?
ultimate effect- wt loss and diarrhea because they can’t absorb the nutrients
Diagnosis of Johne’s disease
Direct fecal culture on Herrold’s egg yolk medium (Gold standard test)
Multistep decontamination process is needed- Lots of contamination
Mycobactin dependency is used for differentiation from other species- differentiation for subspecies paratuberculosis
Time consuming and labor intensive
16 weeks to complete the testing.
Automated Broth Based culture system combined with confirmation by PCR
Host response to infection (ELISA)
Diagnosis of Johne’s
Individual surgical biopsy or necropsy of tissue from ileal-cecal region plus culture/ PCR
(most sensitive test to confirm clinical diagnosis in a herd with no prior confirmed cases)
PCR target-Insertion sequence (IS900)
Culture is the gold standard for dx Johne’s
Control
Control and eradication programs strictly depend on:
- Diagnosis and removal of infected animal
- Management programs to reduce contamination and infection
Who is the most susceptible to Johne’s disease?
Johne’s disease – young animals most susceptible to infection (incubation period is up to two years before symptoms appear)
contaminated maternity pen
BIGGER PROBLEM FOR DAIRY INDUSTRY- because they live longer
Diagnosis other test
• Johne hypersensitivity skin test
• Interferon Gamma Release Assays (IGRA) or Lymphoblast Stimulation Assays
• Serology
ELISA-used for herd testing when there have been prior
confirmed cases or high infection prevalence in herd AGID, CF-used to confirm clinical disease diagnosis
• Nucleic acid detection: (IS 900) PCR on fixed tissues
DNA probes on liquid cultures/isolates
(not widely used, poor sensitivity in some species, eg. cervids)
Johne’s ELISA
Widely used in cattle herds to detect infected animals
Detect humoral immune response to MAP
Low sensitivity in the early stage of diseases
False positives can occur (Specificity issues)
FALSE NEGATIVE in the advanced stages of disease due to a mechanism called anergy( no response)
Treatment/Control of Johne’s disease
we don’t really treat for Johne’s disease
Antimicrobial treatment not recommended
Isolate/slaughter infected animals
(voluntary programs)
Test adult animals annually
Remove animals with clinical signs and high shedders
Cull low to moderate shedders as soon as possible (eg. end of lactation)
Cull calves from clinical cases as soon as possible
Vaccines used rarely in infected herds
Control Management of Johne’s Disease
Avoid introduction through purchased animals (from test negative herds only)
Remove calves from dams immediately
Separate calf-rearingarea
Feed colostrum from test negative cows
Prevent contamination of calf feed, water, bedding by effluent from adult herd
Apply lime to contaminated pastures (more common in areas of low pH & low Ca++)
Reduce total farm exposure to organism (unlike TB, emphasis is on reduction not eradication)
Why it is not remmended to vaccine for Johne’s disease?
because it interferes with M. bovis testing( you will get false positives)
granulomas on vet’s hands
All gram postive bacteria have
thick peptioglycan
Lipolochonric acid
Tuberculosis: Cavitary lesions
associated with lesions toward the end of the infection
M. bovis
use Lowensetin Jensen medium (enriched, egg-based medium)
fatty acid enriched media required for growth of pathogenic species
M. avium subspecies avium
or
Johne’s disease
in a cow
Johne’s disease (thickened, ileal mucosa)
Chronic Granulomatous enterisitis
In a pig
Lawsonia
