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Chapter 5:
Special Bacteriology
BEMS
Syeda Ambreena Batool
Gram Negative Bacilli
Brucella
• Brucellosis is primarily a zoonotic infection
caused by Brucella organisms infecting the
domestic animals.
• The condition is known by various names, such
as Malta fever, Mediterranean fever, undulant
fever
Brucella
• Family : Brucellaceae
• Genus : Brucella
• The genus Brucella consists of seven species,
four of which are human pathogens. These are
Brucella abortus(cattle), Brucella
melitensis(goats, sheep), Brucella suis(swine),
and Brucella canis(dogs).
Morphology
• Brucellae are Gram negative, but
counterstain poorly and require
relatively more time for staining.
• They are small coccobacilli,
measuring 0.5–0.7 x 0.6–1.5μm in
size.
• These are arranged singly or in
short chains.
• The genus Brucella is small,
nonmotile, nonsporing,
nontoxigenic, nonfermenting
aerobic, and Gram-negative
coccobacilli .
Pathogenesis and Immunity
Brucella species have predilection for intracellular growth
hence may be demonstrated inside phagocytes.
Virulence Factor: Brucella produces the following
virulence factors:
a)LPS
b)Intracellular location of the bacterium.
• Pathogenesis :Brucella enter through:
• Abrasion or cut in the skin
• Conjunctiva
• Respiratory tract
• Gastrointestinal tract
• Shortly after gaining entry to the body, brucellae are rapidly
ingested by polymorphonuclear leukocytes, which are
attracted to the site of inflammation. But inside the
leukocytes, the brucellae are not killed due to the presence of
enzyme superoxide dismutase, O polysaccharide of LPS, and
nucleotide-like substances produced by the bacteria.
• Brucellae that are not killed by leukocytes spread from the
site of infection to the local lymph nodes which drain the site
of infection. Inside the lymph glands, the bacteria multiply
and are released to the blood stream following rupture of the
cells and are phagocytosed by the macrophages.
• Bacteria present in the macrophages are then carried to the
organs of reticuloendothelial system, such as liver, spleen,
bone marrow, lymph nodes, and kidney. In these organs,
brucellae multiply in phagosomes of macrophages due to
production of adenine and guanine monophosphate, which
inhibits the phagolysosome fusion, oxidative burst activity,
and tumor necrosis factor production.
• Host Immunity : Immunity in brucellosis, like other
obligate intracellular pathogens, is primarily cell-mediated
immunity.
• The humoral immunity is characterized by production of
antibodies against LPS of the bacteria.
• Clinical Syndromes:
• B.melintensis is the most virulent species. It causes the most
severe and acute cases of brucellosis. It is also the most
common cause of brucellosis (survive in phagocytic cells
and multiply in them, it produces a large number of
bacteria.)
• B.suis is associated with a prolonged course of illness often
with suppurative, destructive lesions.
• B.abortus causes mild to moderate sporadic disease that is
rarely associated with complications.
• B.canis infection is associated with an insidious onset, more
relapses.
◗ Brucellosis
• The incubation period may range from 3 days to several
weeks. Human infections may be of three types, as
follows:
• (a) acute brucellosis.
• (b) chronic brucellosis.
• (c) localized infection.
• Acute brucellosis
• Undulant fever.
• Headache.
• Limb and back pains are unusually severe, night sweating
and fatigue are marked.
• Anorexia, weakness, loss of weight, depression.
• Chronic brucellosis This condition develops in incompletely
treated patients.
• It is usually non bacteremic.
• There is lassitude sweating and joint pain.
• Symptoms often last for 3–6 months and occasionally for a year or
more.
• Localized infection
• B. suis is more likely to cause localized and suppurative infection.
Complications include infections of the heart, central nervous
system (CNS), and the skin.
• Brucella endocarditis is the most dangerous complication and is
responsible for 80% of deaths in brucellosis.
• Chronic meningoencephalitis is the usual manifestation of CNS
infection.
Transmission source
• Unpasteurized milk
• The infected animals, such as cattle, goats, sheep,
buffalos, and swine are the important reservoirs of
infections.
• Brucella organism is transmitted to humans by the
following ways:
(b) Ingestion
(a) inhalation.
(c) direct contact.
(d) accidental inoculation.
(a) Ingestion: Brucellosis results primarily by drinking
contaminated unpasteurized milk or milk products. The infection
can also be transmitted by drinking water or eating raw vegetables
contaminated with feces or urine of infected animals and also by
eating meat of infected animals.
(b) Inhalation: The infection is transmitted by inhalation of dust
from wool or other dried material of infected animals. Infection by
inhalation is important among veterinarians and laboratory workers.
(c) Direct contact: The infection is acquired by direct contact
with the infected materials of septic abortion of the animals or at
the time of slaughter of animals. Brucella species present in the
infected materials (placenta, fetuses, vaginal discharge, urine, or
infected carcasses) enter the human host through the mucosa,
conjunctiva, or skin.
(d) Accidental inoculation: Accidental inoculation is a serious
risk in laboratory workers who handle culture of the organism.
Laboratory Diagnosis
• Specimens Blood,bone marrow, and sometime synovial
fluid and pleural fluid is the specimen of choice and is
collected for culture and for serological test.
• Microscopy Gram staining is not useful for
demonstration of Brucella organisms in clinical
specimens due to their small size and intracellular
location.
• Culture brucella is isolated from a blood culture on
Castaneda medium or from bone marrow. Prolonged
incubation (up to six weeks) may be required, as they are
slow-growing, but on modern automated machines, the
cultures often show positive results within 7 days.
Idenfication of Brucella
• Brucellae are strict aerobes.
• Produce small, moist, translucent, and glistening colonies
after 3 or more days.
• Growth in the presence of aniline dyes, such as basic fuchsin
dyes or thionine.
• Oxidase positive.
• Agglutination with specific antibrucella sera.
• Susceptibility to lysis by bacteriophage.
• They grow at a temperature range of 22–40°C and pH range
of 6.6–7.4 .
• Grow best in medium enriched with glucose and animal
serum.
Serological Tests
• Serological tests are useful for diagnosis of subclinical
brucellosis and for cases of acute and chronic brucellosis by
demonstration of specific antibodies in patient’s serum.
• Specific brucella antibodies, both IgG and IgM antibodies,
appear in the serum 7–10 days after infection. IgM antibodies
persist for up to 3 months after which these antibodies
decline. Then IgG and IgA antibodies appear after 3 weeks of
infection and persist for longer time.
• A number of serological tests have been developed and
tested in serodiagnosis of brucellosis. Common methods are:
• (a) standard tube agglutination tests.
• (b) indirect immunofluorescent tests.
• (c) enzyme-linked immunosorbent assay (ELISA).
Treatment
• Tetracycline
• Streptomycin + tetracycline
• Rifampicin + doxycycline
• The combination of antibiotics is recommended whenever
possible.
Prevention and Control
• Pasteurization or boiling of milk
• Use of protective clothing and gloves by persons
handling or coming into close contact with animals.
• Vaccine: Vaccination of animals is effective to control
brucellosis in animals. Live attenuated B. abortus vaccine
containing B. abortus 1019 strain has been used
successfully to prevent infection in cattle. Vaccines are
not available against either B. suis or B. canis. No vaccine
is available for humans.
THANK YOU

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BRUCELLA (Gram negative bacteria) zoonotic infection

  • 3. Brucella • Brucellosis is primarily a zoonotic infection caused by Brucella organisms infecting the domestic animals. • The condition is known by various names, such as Malta fever, Mediterranean fever, undulant fever
  • 4. Brucella • Family : Brucellaceae • Genus : Brucella • The genus Brucella consists of seven species, four of which are human pathogens. These are Brucella abortus(cattle), Brucella melitensis(goats, sheep), Brucella suis(swine), and Brucella canis(dogs).
  • 5. Morphology • Brucellae are Gram negative, but counterstain poorly and require relatively more time for staining. • They are small coccobacilli, measuring 0.5–0.7 x 0.6–1.5μm in size. • These are arranged singly or in short chains. • The genus Brucella is small, nonmotile, nonsporing, nontoxigenic, nonfermenting aerobic, and Gram-negative coccobacilli .
  • 6. Pathogenesis and Immunity Brucella species have predilection for intracellular growth hence may be demonstrated inside phagocytes. Virulence Factor: Brucella produces the following virulence factors: a)LPS b)Intracellular location of the bacterium.
  • 7.
  • 8. • Pathogenesis :Brucella enter through: • Abrasion or cut in the skin • Conjunctiva • Respiratory tract • Gastrointestinal tract • Shortly after gaining entry to the body, brucellae are rapidly ingested by polymorphonuclear leukocytes, which are attracted to the site of inflammation. But inside the leukocytes, the brucellae are not killed due to the presence of enzyme superoxide dismutase, O polysaccharide of LPS, and nucleotide-like substances produced by the bacteria.
  • 9. • Brucellae that are not killed by leukocytes spread from the site of infection to the local lymph nodes which drain the site of infection. Inside the lymph glands, the bacteria multiply and are released to the blood stream following rupture of the cells and are phagocytosed by the macrophages. • Bacteria present in the macrophages are then carried to the organs of reticuloendothelial system, such as liver, spleen, bone marrow, lymph nodes, and kidney. In these organs, brucellae multiply in phagosomes of macrophages due to production of adenine and guanine monophosphate, which inhibits the phagolysosome fusion, oxidative burst activity, and tumor necrosis factor production.
  • 10.
  • 11. • Host Immunity : Immunity in brucellosis, like other obligate intracellular pathogens, is primarily cell-mediated immunity. • The humoral immunity is characterized by production of antibodies against LPS of the bacteria. • Clinical Syndromes: • B.melintensis is the most virulent species. It causes the most severe and acute cases of brucellosis. It is also the most common cause of brucellosis (survive in phagocytic cells and multiply in them, it produces a large number of bacteria.) • B.suis is associated with a prolonged course of illness often with suppurative, destructive lesions. • B.abortus causes mild to moderate sporadic disease that is rarely associated with complications. • B.canis infection is associated with an insidious onset, more relapses.
  • 12. ◗ Brucellosis • The incubation period may range from 3 days to several weeks. Human infections may be of three types, as follows: • (a) acute brucellosis. • (b) chronic brucellosis. • (c) localized infection. • Acute brucellosis • Undulant fever. • Headache. • Limb and back pains are unusually severe, night sweating and fatigue are marked. • Anorexia, weakness, loss of weight, depression.
  • 13. • Chronic brucellosis This condition develops in incompletely treated patients. • It is usually non bacteremic. • There is lassitude sweating and joint pain. • Symptoms often last for 3–6 months and occasionally for a year or more. • Localized infection • B. suis is more likely to cause localized and suppurative infection. Complications include infections of the heart, central nervous system (CNS), and the skin. • Brucella endocarditis is the most dangerous complication and is responsible for 80% of deaths in brucellosis. • Chronic meningoencephalitis is the usual manifestation of CNS infection.
  • 14. Transmission source • Unpasteurized milk • The infected animals, such as cattle, goats, sheep, buffalos, and swine are the important reservoirs of infections. • Brucella organism is transmitted to humans by the following ways: (b) Ingestion (a) inhalation. (c) direct contact. (d) accidental inoculation.
  • 15. (a) Ingestion: Brucellosis results primarily by drinking contaminated unpasteurized milk or milk products. The infection can also be transmitted by drinking water or eating raw vegetables contaminated with feces or urine of infected animals and also by eating meat of infected animals. (b) Inhalation: The infection is transmitted by inhalation of dust from wool or other dried material of infected animals. Infection by inhalation is important among veterinarians and laboratory workers. (c) Direct contact: The infection is acquired by direct contact with the infected materials of septic abortion of the animals or at the time of slaughter of animals. Brucella species present in the infected materials (placenta, fetuses, vaginal discharge, urine, or infected carcasses) enter the human host through the mucosa, conjunctiva, or skin. (d) Accidental inoculation: Accidental inoculation is a serious risk in laboratory workers who handle culture of the organism.
  • 16. Laboratory Diagnosis • Specimens Blood,bone marrow, and sometime synovial fluid and pleural fluid is the specimen of choice and is collected for culture and for serological test. • Microscopy Gram staining is not useful for demonstration of Brucella organisms in clinical specimens due to their small size and intracellular location. • Culture brucella is isolated from a blood culture on Castaneda medium or from bone marrow. Prolonged incubation (up to six weeks) may be required, as they are slow-growing, but on modern automated machines, the cultures often show positive results within 7 days.
  • 17. Idenfication of Brucella • Brucellae are strict aerobes. • Produce small, moist, translucent, and glistening colonies after 3 or more days. • Growth in the presence of aniline dyes, such as basic fuchsin dyes or thionine. • Oxidase positive. • Agglutination with specific antibrucella sera. • Susceptibility to lysis by bacteriophage. • They grow at a temperature range of 22–40°C and pH range of 6.6–7.4 . • Grow best in medium enriched with glucose and animal serum.
  • 18. Serological Tests • Serological tests are useful for diagnosis of subclinical brucellosis and for cases of acute and chronic brucellosis by demonstration of specific antibodies in patient’s serum. • Specific brucella antibodies, both IgG and IgM antibodies, appear in the serum 7–10 days after infection. IgM antibodies persist for up to 3 months after which these antibodies decline. Then IgG and IgA antibodies appear after 3 weeks of infection and persist for longer time. • A number of serological tests have been developed and tested in serodiagnosis of brucellosis. Common methods are: • (a) standard tube agglutination tests. • (b) indirect immunofluorescent tests. • (c) enzyme-linked immunosorbent assay (ELISA).
  • 19. Treatment • Tetracycline • Streptomycin + tetracycline • Rifampicin + doxycycline • The combination of antibiotics is recommended whenever possible.
  • 20. Prevention and Control • Pasteurization or boiling of milk • Use of protective clothing and gloves by persons handling or coming into close contact with animals. • Vaccine: Vaccination of animals is effective to control brucellosis in animals. Live attenuated B. abortus vaccine containing B. abortus 1019 strain has been used successfully to prevent infection in cattle. Vaccines are not available against either B. suis or B. canis. No vaccine is available for humans.

Editor's Notes

  1. Castaneda’s method: Castaneda’s media provides both liquid (liver infusion broth) and solid media (3% nutrient agar slope) in one bottle. Since the bottle contains both liquid and solid media, the broth flows over the surface of agar slant when bottle is tilted resulting in automatic subculture
  2. (a)Most common test for diagnosis of brucella.Detects the presence of antibodies against LPS component of Brucella. (b)It is a sensitive method for detection of brucella antibodies in the serum, which may be positive even in agglutination-test-negative cases. (c) ELISA is the most sensitive test for detection of IgM, IgA, and IgG brucella antibodies during acute and chronic brucellosis.