This document discusses Brucellosis, a zoonotic disease. It was first isolated 120 years ago by Sir David Bruce and Bernhard Bang. Brucellosis is caused by bacteria of the genus Brucella, which includes B. melitensis, B. suis, B. abortus, and others. It is transmitted from animals to humans through consumption of infected animal products. While treatment involves prolonged antibiotic use, control relies on vaccination programs and testing/culling infected herds. The document outlines the history, transmission, diagnosis, and strategies to control Brucellosis in animals and humans.
2. 1st
spp isolated & characterized almost 120 years ago & recently the
complete nucleotide sequences of the genomes of a number of well-
characterized Brucella strains have been determined
Sir David Bruce (1855-1931)
British Army physician & microbiologist
Discovered Micrococcus melitensis
Bernhard Bang (1848-1932)
Danish physician & veterinarian
Discovered Bacterium abortus could infect cattle, horses, sheep, & goats
undulant fever in humans
The zoonotic nature of the brucellosis was demonstrated in 1905 by Zammit
isolating Brucella melitensis from goat’s milk in Malta (Godfroid et al.,
2005)
HISTORYHISTORY
3. Brucellosis is an important re-emerging zoonosis with a worldwide
distribution, in India was recognised first in 1942.
It is still an uncontrolled serious public health problem in many developing
countries including India
Brucellosis in India is yet a very common but often neglected disease
The genus Brucella consist of 8 species according to antigenic variation &
primary host: Brucella melitensis (sheep & goats), B. suis (hogs), B. abortus
(cattle), B. ovis (sheep), B. canis (dogs), B. neotomae (wood rats), & B.
cetacaea and B. pinnipedialis (marine mammals),
Biovar- carbon dioxide, H2S, thionine and digitonin sensitivity
Susceptibility- age, sex , breed, pregnancy status (adult one more susceptible
regardless to sex)
INTRODUCTION
4. The Many Names of BrucellosisThe Many Names of Brucellosis
Human Disease
Malta Fever
Undulant Fever
Mediterranean Fever
Rock Fever of Gibraltar
Gastric Fever
Animal Disease
Bang’s Disease
Enzootic Abortion
Epizootic Abortion
Slinking of Calves
Ram Epididymitis
Contagious Abortion
5. MORPHOLOGYMORPHOLOGY
GENOMEGENOME
They are Gram-negative, partially acid fast, facultative intracellular
coccobacilli or short rods- Proteobacteriacea family, aerobic, Capnophilic,
Catalase positive, Oxidase positive , Urease positive, Not grow on
MacConkey agar (Garrity2001)
The genome contains two circular chromosomes of 2.1 Mb and 1.5
Mb
Eating food & drink with the bacteria
Injured skin coming into contact with the bacteria
TRANSMISSIONTRANSMISSION
6. Species biotypes Species affected
B. abortus 1, 3, 4, 6
1
Cattle
Sheep, goat
B.melitensis 1
1, 2, 3
Cattle
Sheep, goat
B.suis 2 Pig, goat
B.canis - dog
B.ovis Not reported rams
B.neotome - Wood rats
BRUCELLA BIOTYPES ISOLATED
FROM INDIA
7. There are two types of smooth lipopolysaccharide (SLPS) surface
antigens, designated A and M. A antigen predominates in B. abortus and B.
suis, while M is the major antigen in B. melitensis. Numerous outer and
inner membrane, cytoplasmic, and periplasmic proteins have also been
characterized
Other Virulence Factors
Cyclic β-1,2-glucans (CbGs)
Superoxide dismutase
Catalase
Urease
Alkyl hydroperoxide reductase (ahpC&D)
Cytochrome oxidase
Nitric oxide reductase (norD)
Brucella virulence factor A (BvfA)
Brucella virulence factor B (Vir B) encode Type IV secretion system
ANTIGENIC DETERMINANTS
9. Because of the deceptive nature, the disease may be easily
misdiagnosed or diagnosis may be delayed thereby making clinical
diagnosis a challenge
1. Clinical signs
2. Laboratory diagnosis
Diagnostic tools include
Isolation and identification of Brucellae from clinical samples,
Antigen detection
Genome detection
Antibodies detection
Diagnostic Technique For Brucellosis
in Cattle
10. Samples- aborted fetuses (stomach contents, spleen and lung), fetal
membranes, vaginal secretions (swabs), milk, semen and arthritis or
hygroma fluids
a) Staining methods
Stamps’s method- modified zeihl neelson method
Brucella organisms stain red against a blue background
A fluorochrome or peroxidase-labelled antibody conjugate based technique
could also be used
b) Culture
Basal media- tryptose (or tryptone) soy agar (TSA)
B. abortus biovar 2, such as blood agar base or Columbia agar
Serum–dextrose agar (SDA) or glycerol dextrose agar
Biphasic medium, known as Castañeda’s
Selective Medium - Farrell’s medium
IDENTIFICATION OF THE AGENT
11. Serum agglutination test (SAT)
The complement fixation text (CFT)
ELISA (CELISA) , Recombinant OMP28 ELISA
RBPT, MRT , 2-mercaptoethanol test (2ME)
Agar gel immunodiffusion test (AGID)
Fluorescence polarization assay (FPA) etc.
SEROLOGYSEROLOGY
MOLECULAR
METHODS
MOLECULAR
METHODS
PCR,
RFLP and Southern blot
Pulse-field gel electrophoresis has been developed that allows the
differentiation of several Brucella species
12. Tube Agglutination Test
Also known as the standard agglutination test or serum
agglutination test (SAT)
Test serum is diluted in a series of tubes (doubling
dilutions)
Constant defined amount of antigen is then added to each
tube and tubes incubated for ~20h @37°C
Particular antigen clumps at the bottom of the test tube
Test is read at 50% agglutination
Quantitative
17. AMOS
• Set of 8 primers used
• For B. abortus, B. melitensis, B. ovis and B. suis
• Could detect
Biovars 1, 2, and 4 of B. abortus
All 3 biovars of B. melitensis
Biovar 1 of B. suis
Biovar 1 of B. ovis
• Showed 100% agreement with conventional biotyping methods
• Able to differentiate vaccine strain S19 and RB51 from field
strain isolates
Multiplex PCRs
(Yu and Nielsen, 2012)17
18. MLVA
Multiplex PCR using 8 multi-locus variable number
tandem repeat analysis
Able to distinguish B. melitensis from other Brucella
species and allowed strain typing
Bruce-ladder
A mPCR to identify 9 Brucella sp. at genus level,
including 6 terrestrial species, the marine species of
Brucella, and the vaccine strains S19, RB51and Rev. 1
(Yu and Nielsen, 2012)18
22. Guinea pig - most susceptible to brucella. (García-Carrilo 1977).
Infection - non progressive and may recover spontaneously
Inoculation - into thigh
Examination - animal is killed after 6-8 weeks.
Lymph node in the region of inoculation may be
enlarged and should be cultured.
Spleen - should be cultured by rubbing the cut surface on the
surface of SD agar containing bacitracin 10 units/ml ,
polymyxin B 4 uints/ml to suppress the contaminants.
Blood from JV - tested for presence of antibodies to brucella
Guinea pig inoculation
23. Contd……
Guinea pigs (Cavia porcellus) are probably the animals that are
most susceptible to Brucella spp infection. As few as 11 cells of
Brucella spp are sufficient to cause infection (García-Carrilo
1977).
They are very sensitive to infection by any route: subcutaneous,
conjunctival, intraperitoneal, intranasal, intravenous, vaginal, oral
or cutaneous scarification (García-Carrillo, 1990).
Female guinea pigs have generally been preferred for Brucella
spp. studies, even though sensitivity to Brucella spp. Infection is
practically the same for both sexes (García-Carrillo, 1978).
24. Mice
Susceptibility to Brucella infection varies according to genetic
constitution of mice (García-Carrillo, 1990).
Bosseray et al. (1984) suggest mice as the animal model for
titration of biological activity and quality control of anti-Brucella
spp. vaccines.
25. There is no practical treatment for infected cattle or pigs, but long-term
antibiotic treatment is sometimes successful in infected dogs.
Prolonged treatment with clinically effective antibiotics are necessary to
penetrate these facultative, intracellular pathogens
Brucellae are inaccessible to antibiotics - The treatment recommended by
the WHO for acute brucellosis in adults is rifampicin and doxycyclin
However, the use of more than one antibiotic is needed for several weeks,
because the bacteria incubate within cells
Since the treatment of animal brucellosis is very expensive, one should
encourage the mass vaccination of livestock
TREATMENT
26. OIE/FAO Reference Laboratory for Brucellosis in France
Building of laboratory technical staff as well as of strengthening of national
laboratories and good epidemiological knowledge
Since the treatment of animal brucellosis is very expensive, one should encourage
the mass vaccination of livestock
Standard operating procedures for diagnostic tests --RBT, CFT and iELISA
Human brucellosis is best prevented by controlling the infection in animals.
Pasteurisation of milk from infected animals
Also, the Government of India has made it mandatory to regularly screen all the
breeding bulls from artificial insemination centres for brucellosis and to use
brucellosis free bulls for semen production ( Renukaradhya et al 2002)
Major OIE Recommendations
27. Education about risk of transmission
Farmer, abattoir worker, butcher, consumer, hunter, public
Wear proper attire if dealing with infected animals/ tissues
Veterinarian- Gloves, masks, goggles
Avoid consumption of raw dairy products
Immunize in areas of high prevalence
Young goats and sheep with Rev-1
No human vaccine
Eradicate reservoir
Identify, segregate, and/or cull infected animals
contd
28. Japan, Canada, some European countries, Australia, New Zealand, and
Israel, has been eradicated
B. abortus persists in wildlife hosts in some regions, including the Greater
Yellowstone Area of North America
It is a notifiable disease
Australia declare itself free of bovine brucellosis in 1992 (Bunn, 2002)
Canada declared their cattle herd brucellosis-free on September 19, 1985
and Ireland was declared free of brucellosis on 1 July 2009
The nation of Trinidad and Tobago was classified “free of bovine
brucellosis” by the (OIE) in 1984 (Blajan and Melendez, 1984)
Eradication of Bovine
Brucellosis
Eradication of Bovine
Brucellosis
29. Strategies to Fight BrucellaStrategies to Fight Brucella
Control the infection :
Source of Infection or Transmission of infection, restricting Movement of
animals, Natural service by bulls ,Transmission by carnivores animals and
through milk
Test and slaughter method :
No effective treatment, so diagnose, if +ve kill the animals until no reactor
animal for three consecutive tests, carried out at three-month interval is found
(Mathur et. al., 1974) Financial compensation to farmers
Vaccination Programme :
Vaccination Programme Increases resistance and decreases the source of
infection. Different vaccine against the B. abortus are Live B. abortus Strain-
19 vaccine, Killed adjuvant B. abortus 45/20 vaccine, B. abortus vaccine
RB51.
Make calfhood vaccination compulsory and avoid vaccination of adult
animals
30. contdcontd
Live B. abortus Strain-19 vaccine :
Calves are vaccinated once at the age of 4-8 month
Disadvantages: Induces abortion in pregnant animals Excreted in milk
Killed adjuvant B. abortus 45/20 vaccine :
Not giving lasting immunity
Two initial vaccinations 1st at 3-4 month of age and then repeat after 6 month
and an annual booster (Plommet, 1991)
B. abortus strain RB51 vaccine :
Compared with S-19 vaccine, RB51 vaccine causes less abortion (Cheville et
al., 1996) Protective effect is similar to that of S-19
B. melitensis strain Rev 1 vaccine :
Partially attenuated vaccine for sheep and goat
B. melitensis H38 vaccine :
Killed vaccine for sheep and goat
31. CONTROLCONTROL
Control of brucellosis is difficult
due to absence of defined control
strategies, long life span of
infected animals, uncertainty of
epidemological reservoir and
bacteria species involved.
Editor's Notes
Culture methods - ‘gold standard’ test
(Nielson et al., 2010)
Presumptive evidence of Brucella - by modified Ziehl-Neelsen & modified Koster staining of organisms in abortion material or vaginal discharge, especially supported by serological tests
(OIE, 2009)
Immunohistochemical technique - complementary to bacteriology and serology for the diagnosis of Brucella infection
Multiple-locus Variable Number ... - MLVA
Analytical sensitivity of LAMP and PCR. (A) Resolution of PCR amplicons in agarose gel. Lanes 1 7: 700 ng/ m l DNA,
70 ng, 7 ng, 700 pg, 70 pg, 7 pg and 700 fg. (B) Resolution of LAMP products. Lanes 1 7: 700 ng/m l DNA, 70 ng, 7 ng, 700 pg, 70 pg,
7 pg and 700 fg. M-100 bp ladder. (C) and (D) SYBR green-based detection under day light and UV light, respectively. Tubes 1 7:
700 ng/ m l DNA, 70 ng, 7 ng, 700 pg, 70 pg, 7 pg and 700 fg.