Title: Laboratory diagnosis and control
1Laboratory diagnosis and control of brucellosis
Dr. Ihsan M. Ahmad
Central Veterinary Diagnostic Laboratory
2Introduction
- 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.
3Causative 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).
4Table (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.
6Gram stain of Brucella (Gram ve ) Coccobacilli
7 Antigenic Structure
8The 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.
9Cultural 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.
11Brucella colonies on blood agar
12Brucella 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.
14One 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.
16Brucellosis Edema and swelling of scrotum
172.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.
18Collection 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
20Classes 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.
22Types 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.
23Diagram show the primary and secondary antibody
response and the types of immunoglobulin
24The 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).
25Rose 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).
29Positive 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.
31Complement 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.
33Complement fixation test
34C. Results The test is read by the eye
Sedimentation of the sensitized RBC Positive
result. Complete lysis of the sensitized RBC
Negative result.
35Milk 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.
37Types 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.
42Diseases 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.
43Thank you