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Title: Laboratory diagnosis and control


1
Laboratory diagnosis and control of brucellosis
Dr. Ihsan M. Ahmad
Central Veterinary Diagnostic Laboratory
2
Introduction
  • Brucellosis is considered as the most wide spread
    zoonosis in the world and it is considered as
    True zoonosis ( That mean it is Basically
    transmitted from animal to human).
  • The importance of this contagious disease is the
    economic impact on livestock industry.
  • Causes sever hazard to human health, through
    either direct contact with infected animals or
    the consumption of contaminated milk and dairy
    products.

3
Causative bacteria of the disease
  • Brucellosis is named after Sir David Bruce, who
    is in 1886
  • isolated the causative agent from a soldier in
    Malta.
  • Brucella species are recognized based on
    the natural
  • animal host to the following species as shown
    in table ( 1).

4
Table (1) show Brucella species
5

Morphology and staining
  • The bacteria are strictly parasitic and prefer
    the intracellular habit.
  • The species of the genus Brucella are small non
    motile, non spore forming, Gram negative rods and
    they do not produce true capsules.
  • They are somewhat resistant to decolonization by
    weak acids and thus stain red by the modified
    Ziehl - Neelsen method.

6
Gram stain of Brucella (Gram ve ) Coccobacilli
7
Antigenic Structure
8
The designation of the antigens in cultures
composed of smooth and rough colonies are shown
in the table 2.
  • The Production of monospecific antisera to A and
    M antigen can be used in the identification of
    the Brucella species.
  • Br.canis and Br.ovis grow as rough colonies that
    do not possess either of the surface antigens A
    and M, but instead of that they have R antigen.

9
Cultural characteristics
  • Culture media
  • There are two major types of media for
    cultivation of Brucella.
  • A. Basal Medium
  • Direct isolation and culture of Brucella are
    usually performed on solid media.
  • This enables the developing colonies to be
    isolated and limit the development of
    contaminants.
  • There is many Kinds of commercial media ,e.g.
    Brucella medium base, Trypticase soy agar,
    Columbia agar, Serum- dextrose agar or
    Glycerol-dextrose agar.
  • The addition of 2-5 Bovine or Equine serum is
    necessary for the growth of strains such as B.
    abortus biovar 2.
  • B. Selective Media
  • Appropriate antibiotics are added in order to
    suppress the growth of organisms other than
    Brucella. The most widely used medium is
    Farrells medium, which is prepared by the
    addition of six antibiotics
  • Polymyxin B sulphate,Bacitracin, Cycloheximide,
    Nalidixic acid, Nystatin, Vancomycin

10
  • 2. Colony Morphology
  • Brucella colonies are visible after 3-5 days
    incubation period at 37 ºC on suitable solid
    media, and they are aerobic or microaerophilic.
  • Cultures should not be discarded as negative
    until 8-10 days have elapsed.
  • Brucella colonies are 1-2 mm in diameter, round,
    entire, smooth, glistening, translucent, and a
    pale honey color when plates are viewed in the
    daylight through a transparent medium.

11
Brucella colonies on blood agar
12
Brucella colonies on blood agar
13

Epidemiology of the disease
1.Transmission of the disease
  • Animal to animal Transmission
  • The oral route, Contamination of the
    udder during milking and contact with aborted
    fetuses and infected newborn lambs are considered
    to be common methods of spread, also the venereal
    transmission of the disease is occur due to
    infected male or contaminated semen.
  • Animal to human Transmission
  • Infected tissues, and contaminated
    materials must be handled under (biosafty 3)
    conditions. Transmission could be either by
    contaminated food, invasion by intact skin,
    inhalation of aerosols containing the bacteria
    and aerosol contamination of the conjunctiva.

14
One of the most important route of animal to
human transmission of Brucella
15
  • Brucella melitensis ( biovar 1, 2 or 3) is the
    main causative agent of caprine and ovine
    brucellosis. Sporadic cases caused by
    B.abortus have been observed. The infection is
    widespread world-wide.
  • Brucella abortus is usually causes bovine
    brucellosis, less frequently by brucella
    melitensis.

16
Brucellosis Edema and swelling of scrotum
17
2.The economic losses of brucellosis
  • Losses due to abortion in the affected animal
    population.
  • Diminished milk production, mastitis and
    contamination of milk.
  • Cull and condemnation of infected animals due to
    breeding failure.
  • Human brucellosis causing reduced work capacity
    of the affected people.
  • Government costs on research and eradication
    schemes.
  • Losses of financial investments.

18

Collection of the samples
  • For serological examinations
  • Serum samples are collected for serological
    diagnosis.
  • Collect 5-10 ml of blood in plain tubes (with out
    EDTA).
  • Avoid shaking of the tubes (which contain blood)
    at transporting to prevent distraction of the
    RBCs and hemolysis.
  • Try to separate the serum from clotted blood as
    possible as you can.
  • The tubes are placed vertically at room
    temperature for 1 hour then refrigerated at
    (4 - 8 C) for 1- 2 hour. Dont refrigerate the
    whole blood immediately after collection.
  • Dont freeze the whole blood.
  • After the serum was separated distribute it
    equally in to two plastic tubes for serum and
    keep it in a refrigerator for short periods or in
    freeze for longer periods.
  • Avoid repeated freezing thawing as this may
    affect the protein structure of the serum.

19
  • The most valuable samples from live animals are
    semen, vaginal swabs, and milk.
  • After necropsy, the preferred organs are
    epididymas, seminal vesicles inguinal lymph
    nodes in rams, and the uterus, iliac and supra
    mammary lymph nodes in ewes.
  • In aborted and stillborn lambs the preferred
    culture sites are abomasal content and lung.
  • Samples for culture should be
    transported to the laboratory on ice as soon as
    possible after collection.

B. For bacterial isolation
20

Classes of Immunoglobulin
  • There are five major classes of antibodies
    which have diverse functions.
  • IgM (Immunoglobulin M)
  • The class of serum antibody first produced
    during an infection. It is a large pentameric
    molecule that is active in agglutination
    pathogens and activating complement.
  • 2. IgG (Immunoglobulin G)
  • The predominant immunoglobulin class in
    serum. Has function such as neutralizing toxins,
    opsonizing bacteria, activating complement and
    crossing the placenta to protect the fetus and
    neonate.

21
  • IgA (Immunoglobulin A)
  • The class of immunoglobulin that is
    present in dimeric form in many body secretions
    (e.g., saliva, tears, bronchial and intestinal
    secretions) and protects body surfaces.
  • 4. IgE (Immunoglobulin E)
  • The class of immunoglobulin that binds
    to mast cell and basophils, and is responsible
    for type I or anaphylactic hypersensitivity. It
    is also involved in resistance to helminth
    parasites.
  • 5. IgD (Immunoglobulin D)
  • The class of immunoglobulin found on
    the surface of many B lymphocytes thought to
    serve as antigen receptor in the stimulation of
    antibodies synthesis.

22
Types of antibody response
  • Primary antibody response.
  • It is stimulated during immunization
    procedures (and also in naturally acquired
    immunity) the type of immunoglobulin is (IgM) and
    it is characterized by low antibodies titer.
  • B. Secondary antibody response.
  • It is characterized by producing the
    (IgG) type of immunoglobulin with high level and
    affinity for the antigen. It depends on the
    formation of the memory B cells.

23
Diagram show the primary and secondary antibody
response and the types of immunoglobulin
24
The most common serological test
  • Rose Bengal Test (RBT).
  • Complement Fixation Test (CFT).
  • Enzyme Linked Immuno Sorbent Assay (ELISA).
  • Milk Ring Test (MRT).
  • Standard Agglutination Test (SAT).

25
Rose Bengal Test (RBT)
  • A. Principle
  • The buffered acid antigen stained with
    Rose Bengal is used for the early detection of
    Brucella specific agglutinins.
  • B. Reagents
  • Rose Bengal Antigen.
  • Positive and Negative controls.
  • Important notes
  • Shake before use.
  • Do not freeze.
  • Store at 4 C in a dark place.

26
  • C. Equipments
  • Precision pipettes calibrated to 30 µl.
  • Enamel plate or disposable agglutination cards.
  • Disposable stirring sticks.

27
  • D. Procedure
  • Allow reagents and serum samples to reach room
    temperature (18-25 C).
  • Gently shake the reagents.
  • Check the reagent against positive and negative
    controls (as follows).
  • Place a drop (30 µl) of undiluted serum onto a
    circle of the slide.
  • Add a drop of the reagent (Rose Bengal Brucella
    antigen) next to the drop of the serum.
  • Mix both drops by the disposable stirring stick,
    spreading them over the full surface of the
    circle.
  • Rotate the plate (card) manually or with
    mechanical rotator at (80- 100) R.P.M (Round per
    minute) for 4 minutes.

28
  • E. Reading
  • The reading must be carried out exactly 4 minutes
    from the beginning of shacking.
  • Beyond this time nonspecific reaction may occurs
    (False Positive).
  • No agglutination (- Ve) Negative result.
  • Any visible agglutination (even slight) (Ve)
    Positive results (Presence of specific
    antibodies).

29
Positive and negative results for RBT (Rose
Bengal test)
Positive
Negative
30
  • Important Notes
  • The test is very sensitive especially in
    vaccinated animals.
  • Positive samples should be retested by
    confirmatory test such as CFT or ELISA.
  • False Negative reaction may occur and can be
    detected by retesting animals at intervals over a
    period of at least 3 months.

31
Complement Fixation Test (CFT)
  • The complement fixation test (CFT) is widely used
    for the diagnosis of brucellosis in cattle, sheep
    and goat.
  • This test is relatively insensitive to antibody
    produced in response to vaccination with the
    living attenuated vaccine (for Br. abortus, Br.
    melitensis).
  • whilst being highly sensitive and specific in
    animals naturally infected with brucellosis.
  • The test is some what complex and in mass testing
    a screen test such as Rose Bengal test, is often
    used to reduce the number of samples that need to
    be tested by (CFT).

32
  • Principle
  • In the complement fixation reactin an
    antigen-antibody reaction is demonstrated by
    binding of complement.
  • The reaction can be visualized with the aid of an
    indicator reactin (hemolytic system).
  • The hemolytic system consist of sheep
    erythrocytes and antiserum to sheep erythrocytes
    (amboceptor).
  • If no antigen-antibody reaction develops with
    the consumption of complement, the erythrocyte
    will be lysis completely and the hemoglobin will
    be released.

33
Complement fixation test
34
  • B. Reagents

C. Results The test is read by the eye
Sedimentation of the sensitized RBC Positive
result. Complete lysis of the sensitized RBC
Negative result.
35
Milk Ring Test (MRT)
  • The Ring test method is used for the detection
    of brucella antibody in milk samples. This
    technique is very easy to perform it especially
    in dairy herds.
  • A. Principle
  • It is uses the principle of agglutination
    between antibodies contained in milk and dyed
    colored bacterial antigen of brucella to form
    antigen-antibody complexes that are progressively
    carried by the fat towards the surface of the
    milk and formed a blue violet ring
  • B. Reagents
  • Inactivated bacterial culture of Brucella abortus
    S 99 inactivated by heat and phenol and stained
    with hematoxyline.
  • Positive control for milk ring test.

36
  • C. Procedure
  • Keep the antigen for 1 hour at room temperature,
    and shake it gently before the beginning of the
    test.
  • Carefully mix the tested milk, and put 1 ml in a
    test tube.
  • Add 50 µl of the antigen and mix carefully.
  • Place in incubator for 1 hour at 37 OC, after
    that for (18-20 hours) at 4 OC, and then read the
    result.
  • D. Results
  • Ring of cream equal or more colored than the
    underlying milk
  • Positive result.
  • Ring of cream less colored than the underlying
    milk
  • Negative result.

37
Types of Brucella vaccines and vaccination
  • Vaccination increases animal resistance to
    systemic infection and in infected animals
    decreases the probability of placental infection,
    abortion and massive shedding of infections
    organisms.
  • Live vaccines induce a long lasting immunity and,
    are normally administered to the young animals.
    Adults should also be vaccinated with reduced
    doses or by the conjunctival route in order to
    restrict the serological response.
  • The following are the recommended vaccination
    procedures of the 3 live vaccines for B.
    melitensis and B. abortus.

38
  • 1. Brucella vaccines
  • A. Brucella melitensis (Rev-1 Vaccine)
  • Freeze-dried of live B. melitensis Rev-1 strain.
  • For sheep and goats.
  • At 3-6 months as a single S/C dose.
  • Conjunctival route (reduced dose)
  • 5. Disadvantages
  • Persistent serological response.
  • Abortion if given to pregnant animals.
  • Human risk.

39
  • B. Brucella abortus (S19)
  • Freeze-dried of live B. abortus strain 19.
  • For cattle.
  • At 3-6 months as a single S/C dose.
  • Conjunctival route (reduced dose)
  • Disadvantages
  • Persistent serological response.
  • Abortion if given to pregnant animals.
  • Human risk.
  • C. Brucella abortus (RB51)
  • Freeze-dried of live B. abortus strain RB51.
  • Lesser abortafecient effect than S19.
  • At 4-12 months as a single S/C dose.
  • Does not produce persistent Ab titers.

40
  • 2. Specifications of the Ideal Brucella Vaccine
  • Effective (solid and durable immunity) but
    without inducing a long lasting vaccine
    infection.
  • Not interfering with diagnostic tests (Compatible
    with eradication plans).
  • With no limitation of its use, e.g. in pregnant
    animals.
  • Safe for man when performing the vaccination.
  • Not expensive and readily available world wide.

41
  • 3. Brucella vaccine quality control testing
  • Identity test for the bacterial and colony
    morphology.
  • Absence of contaminating organisms.
  • Number of viable organisms.
  • Dissociation test.
  • Stability test.
  • Virulence in guinea pigs and/ or mice.
  • Safety.

42
Diseases to be considered in the differential
diagnosis
  • Vibriosis.
  • Salmonellosis.
  • Toxoplasmosis.
  • Leptospirosis.
  • Enzootic abortion of ewes.
  • Rift Valley fever.
  • Laboratory identifications of the
    causative organisms a requirement for the
    diagnosis.

43
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