MYCOBACTERIUM
INFECTIONS
• PHYLUM : Actinobacteria
• ORDER : Actinomycetales
• SUBORDER : Corynebacterineae
• FAMİLYA : Mycobacteriaceae
• GENUS : Mycobacterium
More than 100 species
• Aerobic, non-spore, immotile, rod, growing slowly
• Acide-resistance (acid-fast): they are not decolorized with acid-alcohol (1% HCl + methanol) after staining with aryl methane dyes (carbol fucsin)
• Provides resistance to acid: mycolic acid, arabinomannan, arabinogalactan and peptidoglycan acid on the cell wall
• Cord factor: cell wall glycolipid, granuloma formation, intracellular survival
• Nocardia, Rhodococcus and Corynebacterium (coryneform)
• To demonstrate this characteristic, Ziehl-Neelsen staining (under heating - not in Kinyoun) with carbol fucsin (basic fucsin + phenol) are made
• Are considered Grams positive
Classification
• Classical types
M. bovis, M. avium, M. tuberculosis, M. leprae, M. avium subsp. paratuberculosis
• Anonymous, unclassifiable, atypical (Ernest Runyon, 1959)
M. kansaii, M. marinum, M. genaveuse, M.fortuitum, M. ulcerans
According to Runyon classification:
Mycobacteria in the runyon classification are saprophytic and are known as tuberculoids, causing disease in immunosuppressants, not pathogens
Non-tuberculous mycobacteria according to pigmentation (dark and light) and growth rate:
1. Photochromogenes
When contacted with light, yellow colony, M. genaveuse..vs.
2. Skotchromogenes
The yellow colonies without light, M. gordonae..vs.
3. Nonphotochromogenes
Without pigments M. avium-intracellulare complex.
4. Fast growing
Variable pigmentation, fast-growing, M. fortuitum, M. phlei..vs.
Growing slowly
Tuberculosis agents are examined in 3 groups :
1. Mammary Tb agents → M. tuberculosis (human) M. bovis (cattle)
M. microti (field mouse) M. pinnipedii (monk seal)
M. leprae (human-cüzzam-Hansen’s disease) M. lepraemurium (mouse and rat)
2. Poultry Tb agents → M. avium (subsp. avium) (poultry-except psittacine) M. genaveuse (In pet birds)
M. avium subsp. paratuberculosis (bovine aratuberculosis) 3. Coldblooded Tb agents→ M. marinumfish)
M. piscium (frog)
M. chelonae (tortoise)
• Species closely related to each other due to their bacteriological characteristics (micolic acid in cell wall) and DNA similarities are collected under the heading "complex"
• Similar G + C ratio (65%)
• M. tuberculosis complex
– M. tuberculosis, M. bovis, M. microti, M. africanum, M. bovis BCG, M.
caprae, M. pinnipedii
• M. avium complex (non-tuberculosic, saprophytic, immunosuppressive) – M. avium, M. intracellulare, M. chimaera
• Pathogenic species form typical colonies in egg-based media.
• For growing of M. avium subsp. paratuberculosis, the addition of or mycobactin ”(siderophore) is necessary.
• Stays alive at 4+ degree for weeks and at -70 degree for years.
• They die in 20 minutes at 60 and in 15 seconds at 72 (pasteurization temperature).
• Isolation: growth rate, appearence of colony, bacil in Ziehl- Neelsen staining, biochemical profile, molecular methods
• Growth rate is 2 days to 8 weeks (slow-fast species) but usually 3-5 weeks.
• Generating time 2-20 hours.
• Infections caused by MTC (Mycobacterium tuberculosis complex) are called classical
tuberculosis
• M. avium complex is called avian mycobacteriosis
• Technically, other than MTC, M. leprae and M.
leprae murium are mycobacteriosis.
• M. lepraemurium is the cause of cat leprosy
Leprosy
(Hansen’s disease)
• M. leprae
• Nerve, skin and eye granulomas occur
• Do not feel pain
• Losses and injuries of body parts
• Blindness and weakness
• Interpersonal transmission
• Used rifampicin for 6 months in treatment
• Can be infected from Armadillo
• Infected with coughing, over the upper respiratory tract granulomas
TUBERCULOSIS
History
• Found in a Bison who lived 17,000 years ago
• In the human bones of 4000 BC
• 1882 Robert Koch (M. tuberculosis)
He received Nobel Prize in Physiology and Medicine in 1905
• 1921 Calmette and Guerin (BCG)
Bovine tuberculosis strains were obtained by passaging 230 times in the bile-glycerin- potato medium for 13 years
BCG
• The most successful method of immunotherapy
• Used in the treatment of bladder cancers
• 60% efficacy in tumors that stimulate local immunoreaction
• Provides protection to Tuberculosis for 10-20 years
• Intracutaneous application
• Depending on the genetic variation in the population, its protection changes
• 60-80% in England, decreases as the equator approaches
• New vaccines:
–MVA85A (modified vaccine developed in Ankara 85A, Oxford) –rBCG30 (recombinant)
–72F (fusion protein vaccine, developed by Statens Serum Institute)
• Tuberculosis is a chronic and contagious zoonotic disease characterized by the formation of caseous tubercles in the lungs, in other organs and tissues,
• The agent of bovine tuberculosis is M. bovis
• Gram positive, rod, non-spore, without capsules, immobilized, acidoresistance, aerobic
• Exo and Endotoxins and no known plasmids
• Pathogenic agents grow very slow
• Intracellular
• There is more lipid (60% of cell wall) in the cell wall of the agent than the other microorganisms
• Lipoarabinomannan is an important virulence factor
• Peptidoglycan (murein) provides the bacteria stability
• Mycolic acid is prolonged from the side chain of arabinogalacton and mycic acid is bound to a sugar such as trehalose, thus forming the cord factor
– This structure provides resistance to acids, reproducibility in macrophages, and imperviousness to normal laboratory dyes
• Tuberculosis agents are best stained with the Ziehl-Neelsen method
• In addition, Konrich and Kinyoun are also used for staining
Structure of Mycobacterium Cell Wall
1-out lipid, 2-mycolic acide, 3-polysaccharide (arabinogalactan), 4-peptidoglycan, 5- plasma membrane, 6-lipoarabinomannan (LAM), 7-fosfatidilinositol mannosid, 8-cell wall
Ziehl- Neelsen Staining
• It's an aerobic microorganism
• M. bovis, does not grow on medium which are used general purposes in laboratories
• Sodium pyruvate is added to media to increase the reproducibility of M. bovis
• However, they can grow on coagule serum, coagule eggs or potatoes at 37 ° C, 4-5 weeks
• As solid medium; Loewenstein-Jensen, Stonebrink, Dorset and Petragnani
• As a liquid medium; Dubos, Proskauer-Beck
• Semi-synthetic medium; Middlebrook-Cohn
• As a synthetic medium; Souton and Lockman can used
• Bovine type TB agents do not grow very well in glycerinized media (disgonik)
• The human and poultry type is more abundant in glycerinized media (eugonic)
• The three of them grow by forming a thick,
wrinkled membrane on top of the liquid and
this membrane collapses over time.
M. tuberculosis M. bovis M. avium Colony
morphology Dry, cauliflower-
like (pink-orange) Small, flat, damp,
bright round Widely moist, whitish, sticky
Growing time 5-6 week 4-5 week 2-6 week
Glycerin need + - +
Growing
temperature 37 ºC 37ºC 42-44 º C
Niasin test + - -
Nitrat redüksion + - -
Ürease + + -
M. tuberculosis
(Bread-crumb)
M. bovis M. avium M. tuberculosis
Epidemiology
Cattle, deer, llama, cat, dog, pig common; very rare in sheep, goat and horse Can survive for 6 months in pasture, soil and fertilizer
It was only one on OIE's B list, nowadays is still on list
Zoonose is widely spread all over the world, congenital, digestive, respiratory, genital and skin-transmitted, mastitis endogenous
In rare cases, M. tuberculosis transmission is possible from human to bovine Transmission is mainly in 5 ways:
1. Kongenital path: Before the offspring are born, they can receive the disease through the mother and umblical vena. In this way the microorganism can pass on their infants and cause infections
The offspring are either killed or born normally. However, the offspring of babies die as a result of generalize tuberculosis
2. Alimenter way: Getting infection from their mom that has breast
tuberculosis or infected by sucking milk
3. By inhalation: Animals with pulmonary tuberculosis are important
in aerogen transmission
In very frequent or crowded barns, infection occurs by inhalation of the microbial droplets that come out as a result of coughing or tearing of animals with tuberculosis
Since some of the droplets that are coming out are big, they fall down and infect the food
4. Genital tract: Animals that has tuberculosis in their bulls can infect healthy females with microbial sperm by natural and artificial insemination.
Animals with tuberculosis lesions in the uterus and vagina infect the environment with streams coming from them 5. Through the skin: It is common in humans. The lesions are
more commonly found in the lower parts of the legs and subcutaneous tissues
The resulting nodules become ulcerated over time
Pathogenesis
• Life and proliferation in macrophages (Cell Wall structure)
• The macrophage accumulates in the first region where the agent enters
• Live phagocytes spread the agent
• Macrophages, T-cells, Neutrophils, B-cells and fibroblasts accumulate around necrotic (caseinous necrosis) tissue to form granulomatous (tuberculous) tissue
• Cytokines (IFN gamma, TNF alpha) secreted by immune system cells provides this accumulation
Pathogenesis
• PRIMARY EFFECT: Starting point
• PRIMARY COMPLEX: It is caused by spreading of the agents from the initial focus to the lymph
nodes with free or macrophages.
• INCOMPLETE PRIMARY COMPLEX: No initial lesion
in the mucosa and lesions in the lymph nodes
• MILIER TUBERCULOSIS: The appearance of small and diffuse lesions (especially in KC, spleen and kidney, which is the indication of the agent spreading through the blood)
• PLEURAL TUBERCULOSIS: In lymph nodes of
the costal pleura
SYMPTOMS
• Clinical signs may not be seen
• Chronic attenuation, loss of condition and cough are the most basic findings
• In pulmonary tuberculosis; coughing and prescapular with fever, swelling of the neck and mandibular lymph nodes
• Breast blindness, swelling in supramammary lymph nodule
• Epididymitis in bulls
• Hemoptysis (blood spitting)
• The disease is understood in the slaughterhouse
DIAGNOSIS
A) CLINICAL DIAGNOSIS Wheezy breathing
Dry and painful cough
Swelling of lymph nodes and fever
DIAGNOSIS
B) Necropsy findings
• The tubercles are seen in the organs where the infection is located
• Mesenteric lymph glands are swollen and become caseous
• In the center there is caseinous necrosis and a cheese-like structure
Lesions that become calcified become encapsulated
Microscope; epithelioid and langhans type giant cells are seen
LANGHANS TYPE GIANT CELL
LABORATORY DIAGNOSIS
1-Bacterioscopy
• After Ziehl-Neelsen stainimng red elements are seen on the blue background 2-Culture:
• Urine, crayon, uterine flow, semen, lymph, milk, lesion tissue
• 2 parts of 4% NaOH is added to some of the morbid substances from the lesioned area. It is homogenized in a sterile mortar. 30 min at 37 ° C. Expected. The
suspension was taken in a sterile centrifuge tube and centrifuged at 3500 rpm for 15-20 min. centrifuged. After transferring the upper liquid into a disinfectant
container, add 1-1.5 ml of distilled water onto the sediment. The pH is adjusted to 7,2. 0,05-0,1 ml is added to these prepared inoculum media. For this purpose, the Löwenstein-Jensen nutrient is used. The seed is planted in both glycerin and
glycerin-free media, incubated at 37-40 ° C for 8-12 weeks
• Bovine type Tb agents are not well grown in glycerin medium they culture on glycerin-free medium
• Atypicals grow in 2 weeks
• M. tuberculosis nitrate and niacin positive
3-Animal experiment
• From the prepared inocula, 0.21-0.3 cc are
injected subcutaneously near the inguinal lymph nodes of two guinea pigs and the animals are
observed for 6-8 weeks
• Grades, attenuation, loss of appetite, are checked daily
• If there is a swelling on injected location and in
lymph nodels, should be checked
4-Allergic tests (PPD test, Montoux test, Tuberculin test)
• The reaction described in 1890 by Koch, Felix Mendel found test in 1908 and Charles Mantoux gave the name
• PPD (purified protein derivative) tuberculin is used
• The only antemortem allergic test based on baseline delayed type hypersensitivity to mycobacterial proteins
• It can be detected 30-50 days after infection in cattle
Avian PPD tuberculine → poultry type and paratuberculosis type Mammalian PPD tuberculine → human and mammalian type
Purified Protein Derivative (PPD)
• Both PPD are prepared in free-flowing vapour by heating and filtering the M. bovis or M. avium
cultures
• The active filtrate is obtained by precipitation with trichloroacetic acid (40%), washed and dissolved in the liquid
• It is stored in sterile glass bottles
Single Intradermal Application
The best application site in cattle is neck (medium 1/3). After the neck is shaved, the pliant is measured and disinfected.
Then 0.2 cc from the mammalian PPD tubercle injects
intradermal. The test result is assessed after 72 hours. At the end of this training, the local reactions on skin are carefully examined and the thickness of the skin is calibrated.
According to the Bovine Bovine Tuberculosis Regulation (2009), it is done in 2 ways:
Positive reaction: If there is diffuse or diffuse edema, exudation, necrosis, pain or
inflammation of lymphatic or lymphatic nodules (± 4 hours) and 4 mm or more thickening of skin fold thickness reaction is accepted positive
Suspicious reaction: If there is no clinical evidence on the positive reaction at the site of application, 72 hours (± 4 hours) after injection, the suspicious reaction is if the thickening of the skin fold is more than 2 mm but less than 4 mm, reaction is accepted suspicious
Negative reaction: If there is a very limited swelling at the site of application, 72 hours (± 4 hours) after injection, if the skin fold thickening is not more than 2 mm and if there is no diffuse or diffuse edema, exudation, necrosis, pain or lymphatic channels or lymph, the reaction is accepted negative
Animals with suspicious reactions in a single intradermal test are retested at least 42 days later. Animals that do not produce negative results in the second test are considered to have a positive reaction to the test.
Animals that are positive in the single intradermal test if they are suspected of a misleading positive reaction or interaction reaction may be subjected to a comparative intradermal test.
Intradermal Comparative Application
• The best application site in cattle is neck (medium 1/3). After the neck is shaved, the pliant is measured and disinfected. Chicken (avian) PPD is injected into the upper region of the shaved area, and bovine
(mammalian) PPD into the sub-regional area, with a gap of 12.5 cm between them ,up to 0.2 cc is injected.
The test result is assessed after 72 hours. At the end of this training, the local reactions in the skin are carefully examined and the thickness of the skin is calibrated.
Positive reaction: Clinical manifestations such as diffuse or diffuse edema, exudation, necrosis, pain or
inflammation of lymphatic or lymphatic nodules at the site of application, 72 hours (± 4 hours) after injection, or the skin fold thickness at the site of application of the bovine tubercle is 4 mm and thickness of the skin fold at the site of application of chicken tubercle more than 4 mm is considered as a positive reaction.
Suspicious reaction: If there are no clinically indicated symptoms in the positive reaction at the site of application, 72 hours (± 4 hours) after injection, or if the bovine tubercle is more than 2 mm thick at the
application site and 1-4 mm thicker than the thickness of the skin fold at the application site of the avian tubercle it is considered a suspicious reaction.
Negative reaction: Positive reaction does not reveal the clinical symptoms at the site of application, 72 hours (± 4 hours) after injection, the bovine tubercle is not more than 2 mm or positive if the skin fold thickness at the site of administration of avian tubercle is less then bovine tubercle tikcness the reaction is accepted negative.
In the intradermal comparative test, animals with suspicious reactions are retested after at least 42 days. Animals that do not react negatively in the second test are considered positive for the test.
Tuberculosis Prospect is mandatory !!
• The herd of tuberculosis and the barn are taken to the quarantine. Animal Health Memorandum is collected, disease outcome is decided and declared
• In the last two tests, all cattle larger than 6 weeks continue quarantine until they are negative
• The first test is 60 days after the positivity is determined and the second test is done at the earliest 4 months and at the latest after 12 months.
• Tuberculous cattle are shipped
• Milk can be used in feeding of animals after heat treatment
• The treatment and inoculation of tuberculosis is prohibited by law !!!
• Tuberculosis compensated disease (local appraisal commission)
– Regulation on Compensation in Animal Diseases-2012; Brucellosis, Bird Flu, Ruam, Rabies, Screed, Newcastle ... etc.
• Those who are positive for the tuberculin test are paid 9/10 of the assets, and those who are found positive in slaughter house are paid ¾ of the value
• Meat, if the lesions are local, it can be consumed as a whole and the food can be consumed from the low meat group and the generalize is destroyed
PARATUBERCULOSIS
(JOHNE DISEASE,
ENTERITIS PARATUBERCULOSA BOVIS)
• M. avium subs. paratuberculosis
• Is seen in domestic and wild ruminants
• Causes Crohn's disease in humans
• Characterized by chronic, infectious, lethal, enteritis
• Short, thick, Gram + rod, Ziehl-Neelsen Staining
• Optimum temperature at 37 ° C, 2-6 weeks
• It requires '' Mycobactin '' to produce or dead M.phlei is added to medium
EPIDEMIOLOGY
• Infection is usually caused by the spreading of the
infected animals through the feces, this effect in the first years of the calf
• Smaller than 1 month calf is very sensitive, usually subclinical
• There is spread by milk
• Venereal infection is unimportant
PATHOGENESIS
• M. avium subs. paratuberculosis; an intracellular pathogen and a cellular immune response for intestinal lesions
• They remain alive in the macrophages (M-cells) and transported to the payer plaque
• As the disease progresses, a granulomatous lesion forms and
lymphocytes and macrophages accumulate in the lamina propria and submucosa
• Macrophage and lymphocyte accumulation cause thickens of the intestinal wall, leading to the inhibition of food absorption, diarrhea
PATHOGENESIS
• As a result of Enteropathy protein, water, food absorption are disrupted
• Numerous mycobacteria are observed in
macrophages in the intestinal wall and
regional lymph nodes
DIAGNOSIS
A) CLINICAL SYMPTOMS:
• The first indications in affected cattle are seen when they are over 2 years old
• DIARRHEA
• The backs of the animals are contaminated with feces. Diarrhea is increasingly exacerbated
• Weakness is seen despite normal appetite
• Animals with prolonged weight loss and loss of
appetite live rarely more than 1 year
DIAGNOSIS
• B) NECROPSY FINDINGS
• The affected parts of the intestines (small intestine end intestinal mucosa) are 4-5 times thicker and have a
brain view
• Mesenteric and iliosecal lymph nodes are enlarged and eudema
• Caseification and calcification are not seen !!
DIAGNOSIS
C) LABORATORY DIAGNOSIS
• Rectum mucosa scrape, gaita, blood for serology
• Ziehl- Neelsen Staining
• Culture on Herrold Egg Yolk medium
• Mycobactin supports growing
• Incubate for 16 weeks at 37 ° C and check every week
• Serology; CFT, ELISA, Gamma Interferon test
• Tuberculin test is performed by applying John's PPD
Colony on Herrold Egg Yolk
Medium
John's Tuberculin Test
• 1978-Regulation on Cattle Tuberculosis
• It is administered similarly to the tuberculin test
• 0.1 ml into the neck
• After 72 hours the thickness is measured
• Blood is taken 2 months after administration of tuberculin
• John is positive if PPD or serology is positive
• Johnin suspicious if PPD or serology is suspicious
• In case of susupicion, test are repeated after 2 months, if they have suspicious results, considered positive
• Positive animals are shipped to the cut
• John's PPD evaluation;
– Thickening up to 3 mm negative – 3-4 mm suspicious
– Positive if it is 4 mm or more
