Brucellosis

Dr. RAJAT VARSHNEY

P-1772

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

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

The Many Names of Brucellosis

The Many Names of Brucellosis

Human DiseaseMalta FeverUndulant FeverMediterranean FeverRock Fever of

GibraltarGastric Fever

Animal DiseaseBang’s DiseaseEnzootic AbortionEpizootic AbortionSlinking of CalvesRam EpididymitisContagious Abortion

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

Species biotypes Species affected

B. abortus 1, 3, 4, 61

CattleSheep, goat

B.melitensis 11, 2, 3

CattleSheep, goat

B.suis 2 Pig, goat

B.canis - dog

B.ovis Not reported rams

B.neotome - Wood rats

BRUCELLA BIOTYPES ISOLATED FROM INDIA

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

Phagosome maturationPhagosome maturation Oxidase killing Oxidase killing

Preferentially localization of Gravid uterus (unknown factors – Allontoic factors Erythritol)

PATHOGENESIS

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

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

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

Tube Agglutination TestAlso 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

13

Tube Agglutination Test

No agglutinationAgglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 1/40

Tube Agglutination Test

RBPT and MRT

Blood

Serum

BrucellaBrucellaserologyserology

Blood Culture Medium

IsolateEnvironmental

Sample Isolation

Biochemical Tests

Dye Tolerance

Slide Agglutination

Gel Formation

Requirement of CO2

Tb Phage Lysis

H2S test

Molecular TestsReal Time PCRMultiplex PCRSequencing of conserved genes

16S rRNARecARpoB

MLVA: Strain IdMLSA: SubtypingWhole genome sequencing:

Genetic relatedness

Contd..

16

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

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

Loop-mediated isothermal amplification (LAMP) test

Highly specific Rapid (60 min.) Works at constant temp. 61 .C Sensitive 100 fold than PCR

(Karthik et al., 2014)

19(Karthik et al., 2014)

20

(Karthik et al., 2014)

BrucellaAb tests

21(ICAR – NIVEDI, 2014)

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

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).

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.

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

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

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

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

Strategies to Fight Brucella Strategies 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

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

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.