Brucella

College Project

1. Medical Microbiology Research Made By:

NORSEEN HOSAMELDEEN LOTFY
221101448

Also called Mediterranean fever or Malta fever or
Undulant fever It is called Brucellosis after its
bacterial cause.
It is called Undulant fever because of its rising and
falling like a wave. It’s Zoonotic infection (endemic
in animals). It is infectious disease.

2. Four types are important to human;


MORPHOLOGY
Brucellae are Gram-negative coccobacilli (short rods)
measuring about 0.6 to 1.5 μm by 0.5-0.7 μm. aerobic,
but may need added CO2.
They are non-sporing and lack capsules or flagella and,
therefore, are non-motile. The outer cell membrane
closely resembles that of other Gram-negative bacilli
with a dominant lipopolysaccharide (LPS) component and
three main groups of proteins. The guanine-plus-cytosine
content of the DNA is 55-58 moles/cm.

3. Pic. Brucella abortus SEM

No Brucella species has been found to harbor plasmids
naturally although they readily accept broad-host-range
plasmids.
Brucella colonies become visible on suitable solid media
in 2-3 days. The colonies of smooth strains are small,
round and convex but dissociation, with loss of the O
chains of the LPS, occurs readily to form rough or mucoid
variants. These latter forms are natural in B canis and B
ovis as the LPS of these lack O chains.

4. PREVELANCE
Highest incidences of brucellosis is in the Mediterranean
countries, middle east and tropics
There are about 500.000 new cases diagnosed worldwide
per year.

Brucella suis occurs in most areas in which pigs
are kept. It affects both sexes of swine causing
infertility, abortion, orchitis and lesions of
bones and joints. The prevalence is generally
low except in parts of South America and South
East Asia. In both south eastern United States
and in Australia, particularly Queensland,
populations of feral swine are heavily infected.
Apart from the rare cases in which cows' milk is
infected, infections with B suis in humans occur
in people handling pigs on farms and during
slaughtering and processing - including the
hunting of feral swine. While an eradication
campaign in the United States has made much

5. headway, abattoir outbreaks still have occurred
recently.
Bovine brucellosis, caused by B abortus, has
been eradicated from Canada, Japan, northern
Europe and Australasia. Cases in humans tend
to be sporadic and often stem from
occupational exposure. Infection can be
acquired by drinking unpasteurized milk, but
this a relatively inefficient mode of spread,
while abattoir workers can be significantly
exposed and veterinarians are doubly at risk
from attending parturient cattle and from
accidental inoculation with live vaccine.
Cases of B canis infection in man have tended
to occur only in dog handlers. Close, frequent
contact seems to be necessary for transmission.
Cases of brucellosis have continued to present
in some regions from which brucellosis has
been effectively eradicated. These are caused,

6. usually by B melitensis, in travelers to popular
tourist destinations, like Mexico and the
Mediterranean region, in which this organism is
highly prevalent, and by the importation of
infected dairy products. Although person to
person spread is quite exceptional, the species
of principal importance are all potent causes of
laboratory infections. Hence stringent safety
measures, including adequate containment to
reduce the hazard of aerosol spread, are
essential. The situation has been aggravated by
the failure of certain popular laboratory kits to
identify Brucella spp appropriately.



PREVELANCE IN EGYPT
Serodiagnostic test results for brucellosis in animals, Egypt, 2007*

7. Serum source Location No. tested
Serologic test, no. positive (%)

BAPA RBP SA Rivanol
Cattle

Alexandria 333 17 (5.11) 15 (4.5) 13 (3.9) 13 (3.9)
Behera 374 11 (2.94) 11 (2.94) 10 (2.67) 9 (2.41)
Monofia 280 20 (7.14) 18 (6.43) 17 (6.07) 15 (5.36)
Qalioubia 221 14 (6.33) 12 (5.43) 12 (5.43) 11 (4.98)
Giza 346 15 (4.34) 15 (4.34) 14 (4.05) 14 (4.05)
Benisuef 309 24 (7.77) 22 (7.12) 21 (6.8) 21 (6.8)
Assiut 103 6 (5.83) 5 (4.85) 6 (5.83) 5 (4.85)
Total

1,966

107 (5.44)

98 (4.98)

93 (4.73)

88 (4.48)

Buffaloes

Alexandria 137 6 (4.38) 6 (4.38) 6 (4.38) 6 (4.38)
Behera 397 7 (1.76) 5 (1.26) 5 (1.26) 5 (1.26)
Monofia 210 10 (4.76) 8 (3.81) 6 (2.86) 7 (3.33)
Qalioubia 131 7 (5.34) 6 (4.58) 7 (5.34) 6 (4.58)
Giza 198 8 (4.04) 8 (4.04) 8 (4.04) 7 (3.54)
Benisuef 231 16 (6.93) 14 (6.06) 14 (6.06) 14 (6.06)
Assiut 33 1 (3.03) 0 0 0
Total

1,337

55 (4.11)

47 (3.52)

46 (3.44)

45 (3.37)

Sheep

Behera 210 11 (5.24) 10 (4.76) 10 (4.76) 10 (4.76)
Monofia 81 2 (2.47) 0 0 0
Qalioubia 133 6 (4.51) 6 (4.51) 6 (4.51) 6 (4.51)

8. Serum source Location No. tested
Serologic test, no. positive (%)

BAPA RBP SA Rivanol
Giza 172 10 (5.81) 9 (5.23) 9 (5.23) 9 (5.23)
Benisuef 217 15 (6.91) 14 (6.45) 14 (6.45) 14 (6.45)
Total

813

44 (5.41)

39 (4.8)

39 (4.8)

39 (4.8)

Goats Behera 55 1 (1.82) 0 0 0
Monofia 63 4 (6.35) 2 (3.17) 2 (3.17) 2 (3.17)
Qalioubia 103 3 (2.91) 2 (1.94) 2 (1.94) 2 (1.94)
Giza 58 0 0 0 0
Benisuef 87 5 (5.75) 4 (4.6) 4 (4.60) 4(4.6)
Total 366 13 (3.55) 8 (2.19) 8 (2.19) 8(2.19)
*BAPA, buffer acidified plate antigen; RBP, Rose Bengal plate; SA, standard tube agglutination.

Results obtained for different animal groups are
shown in Table 1. Prevalence of brucellosis in cattle
was 5.44% by the BAPA test; highest prevalence
was in Benisuef (7.77%) and Monofia (7.14%).
Prevalence of brucellosis in buffaloes was 4.11% by
the BAPA test; hig0hest prevalence was in Benisuef
(6.93%) and Qalioubia (5.34%). Prevalence of
brucellosis in sheep was 5.41% by the BAPA test;

9. highest prevalence was in Benisuef (6.91%) and Giza
(5.81%). Prevalence of brucellosis in goats was
3.55% by the BAPA test; highest prevalence was in
Monofia (6.35%) and Benisuef (5.75%).

Prevalence of Brucella spp. in milk or tissues of animals, Egypt, 2007*

Buffaloes, no. positive/no. tested


Sheep, no.
positive/no.
tested


Goats, no.
positive/no.
tested

Milk


Tissue

Milk


Tissue

Tissue

Tissue

SRA SNRA SRA SNRA SRA SNRA SRA SNRA SRA SNRA SRA SNRA
Alexandria 2/10 0/11

1/5 0/5

1/6 0/19

1/5 0/5

0 0

0 0
Behera 2/9 0/9

1/5 0/5

1/5 0/20

1/5 0/5

1/5 0/5

0 0/5
Monofia 4/20 0/12

1/5 0/5

1/7 0/18

2/5 0/5

0 0/5

0/2 0/5
Qalioubia 2/20 0/10

0/5 0/5

2/6 0/3

1/5 0/5

1/5 0/5

1/2 0/5
Giza 4/20 0/10

0/5 0/5

1/7 0/6

1/5 0/5

1/5 0/5

0 0
Benisuef 6/20 0/21

2/5 0/5

1/10 1/15

0/5 0/5

1/5 0/5

1/4 0/5
Assiut 1/5 0/7

1/5 0/5

0 0/5

0 0

0 0

0 0
Total 21/104 0/80

6/35 0/35

7/41 1/86

6/30 0/30

4/20 0/25
*SRA, samples from serologically reactive animals; SNRA, samples from serologically nonreactive
animals.
Prevalence rates in cattle, buffaloes, sheep, and
goats were generally higher in Benisuef than in

10. other governorates. Variations in infection in
different governorates may be attributed to
environmental factors and stress, which may
modulate susceptibility to infection.
All Brucella isolates were B. melitensis This finding
is consistent with reports of B. melitensis,
particularly biovar 3, being the main cause of
brucellosis in animals and humans in many
countries
We recently reported prevalence of human
brucellosis in Egypt as high as 8% in high-risk
populations. Our findings emphasize the need for
continuous national surveillance programs for
control and prevention of brucellosis in Egypt and
other affected countries. Measures should be
established to control spread of brucellosis,
especially in mobile flocks.

Who is at risk?

11. Brucellosis is found globally and is a reportable
disease in most countries. It affects people of all
ages and both sexes. In the general population,
most cases are caused by the consumption of raw
milk or its derivatives such as fresh cheese. Most of
these cases are from sheep and goat products.
The disease is also considered an occupational
hazard for people who work in the livestock sector.
People who work with animals and are in contact
with blood, placenta, foetuses and uterine
secretions have an increased risk of contracting the
disease.
This method of transmission primarily affects
farmers, butchers, hunters, veterinarians and
laboratory personnel.
Human-to-human transmission is very rare.


MODE OF INFECTION

12. infection is transmitted to human mainly by
Direct contact with an infected animal
Ingestion of unpasteurized (raw) milk from
infected animals, uncooked meat, less
frequently via respiratory tract, abraded skin,
animal urine, faeces may act as source of
infection
Inhalation of aerosols
Occupational exposure can occur
They travel in the lymphatics and infect lymph node,
this is followed by haematogenous spread with
localization in reticulo – endothelial system
Brucellosis acute infection lasts few days to few
months
After acute Brucellosis, infection, symptoms persist
in some patients for more than one year in patients
with Chronic brucellosis
Very rarely, the bacteria may spread from person to
person. Breastfeeding moms with brucellosis may
pass the bacteria to their baby and
also Brucella may be spread through sexual contact.
The bacteria can enter your body:

13.  Through a cut or scratch in the skin.
 When you are breathing in contaminated
air.
When you eat or drink something contaminated
with the bacteria, such as flavored milk or
undercooked meat. Brucellae are facultative
intracellular parasites, multiplying mainly in
monocyte-macrophage cells. This characteristic
dominates the pathology, clinical manifestations
and therapy of the disease.
Oral entry, by ingestion of contaminated animal
products (often raw milk or its derivatives) or by
contact with contaminated fingers, probably
represents the most common route of infection
even though this portal may not be the most
vulnerable one. Inhalation of aerosols
containing the bacteria, or aerosol
contamination of the conjunctivae, is another
route. Inhalation probably underlies some
industrial outbreaks. Percutaneous infection
through skin abrasions or by accidental
inoculation has frequently been demonstrated.

14. Invasion of body  phagocytized by PMN or
Macrophages  spread to regional L/Ns  Blood
stream  formation of granulomas with
epitheloid cells, giant cells, lymphocytes and
plasma cells in liver and RES


Pathogenesis

15. The Brucella organisms penetrate the skin or
mucous membranes, are engulfed by
polymorphonuclear leukocytes, and then enter the
lymphatics and the bloodstream. The Brucella
organisms penetrate the skin or mucous
membranes, are engulfed by polymorphonuclear
leukocytes, and then enter the lymphatics and the
bloodstream. Brucella multiplies within the
polymorphonuclear leukocytes and lyses them
(facultative intracellular parasite). Brucella
multiplies within the polymorphonuclear leukocytes
and lyses them (facultative intracellular parasite).
Brucella can also multiply within the macrophages
of the reticuloendothelial system inducing small
granulomas and abscesses. Periodic release of
Brucella into the blood induces recurrent chills and
fever. Antigen-specific activated macrophages are
able to kill Brucella with T lymphocytes.

Spread of Brucella in the body

16. In humans, the tissue lesions produced
by Brucella species consist of minute granulomas that are
composed of epithelioid cells, polymorphonuclear
leukocytes, lymphocytes and some giant cells. In cases of
infection with B melitensis these granulomas are
particularly small although the toxemia associated with
this organism is great. Necrosis is not common, and
abscesses do not form, except in B suis infection. The fact
that humans rapidly develop hypersensitivity to brucellar
antigens suggests that many of the symptoms of human
brucellosis result from the reaction of the host defenses.
Host Defenses
The specific host defenses against brucellae resemble
those against other intracellular bacteria and are both
humoral (antibody-mediated) and cell-mediated.
Passively administered monoclonal antibody directed
against LPS has been shown to reduce the numbers of
brucellae surviving in the spleens and livers of
experimental mice, indicating a role for antibody in
protection. However, the principal component in defense
against brucellae is cell-mediated. Macrophages have
been shown to process brucellar antigen and present this
to T lymphocytes which produce lymphokines. These
agents, of which interferon is the most active in this

17. context, activate the formerly ineffective macrophages
to greater bactericidal potency. Depletion of gamma
interferon makes experimental animals vulnerable to
infection. T cell-derived lymphokines are also involved in
attracting cells to the foci of infection. This leads to
granuloma formation. While this contributes to the
pathology, it also delivers the activated macrophages to
the site where they are needed. This inflammatory
response is enhanced by cytokines, such as the colony-
stimulating factors, tumor necrosis factor and
interleukin-1, produced by a number of cell types.
Mice which have survived brucellosis are protected
against further challenge, and there is clinical evidence
that complete recovery from a natural infection is
associated with at least a degree of residual resistance in
humans.

18. Symptoms in humans include:
 continuous or intermittent fever.
 headache.
 weakness.
 profuse sweats.
 chills.
 joint pains.
 aches.
 weight loss.
 Enlarged lymph nodes

Brucellosis Clinical Features

Incubation period 1-3 weeks ( days )
Acute infection is characterized by undulant
fever (high swinging temperature), rigors,
lethargy, headache, joint and muscle pain

19. Onset may be insidious, with malaise,
headache, weakness, generalized myalgia and
night sweats
Hepatosplenomegaly – is common
Pancytopenia (Leukopenia, Thrombocytopenia,
Decreased RBC ) Arthritis, Sacroilitis,
Osteomyelitis Endocarditis can occur

The presentation of brucellosis is
characteristically variable. The incubation
period is often difficult to determine but is
usually from 2 to 4 weeks. The onset may be
insidious or abrupt. Subclinical infection is
common.
In the simplest case, the onset is influenza like
with fever reaching 38 to 40°C. Limb and back
pains are unusually severe, however, and
sweating and fatigue are marked. The
leukocyte count tends to be normal or reduced,
with a relative lymphocytosis. On physical
examination, splenomegaly may be the only
finding. If the disease is not treated, the

20. symptoms may continue for 2 to 4 weeks. Many
patients will then recover spontaneously but
others may suffer a series of exacerbations.
These may produce an undulant fever in which
the intensity of fever and symptoms recur and
recede at about 10 day intervals. Anemia is
often a feature. True relapses may occur
months after the initial episode, even after
apparently successful treatment.
Most affected persons recover entirely within 3
to 12 months but some will develop
complications marked by involvement of various
organs, and a few may enter an ill-defined
chronic syndrome. Complications include
arthritis, often sacroiliitis, and spondylitis (in
about 10 percent of cases), central nervous
system effects including meningitis (in about
5%), uveitis and, occasionally,
epididymoorchitis. In contrast to animals,
abortion is not a feature of brucellosis in
pregnant women. Hypersensitivity reactions,
which may mimic the symptoms of an infection,
may occur in individuals who are exposed to

21. infective material after previous, even
subclinical, infection.

22. (complicated brucellosis case)
Rashes, skin biopsy and X-ray films of the patient’s joints. a-d Rashes before
treatment. There were intensive brown-red papules and a partial scar on the
face, trunk and limbs. Some scabs and scars, pustules at the tip of the
finger. e X-ray of the right hand. The bone density of all bones was reduced
unevenly, the interphalangeal space became narrow, and the soft tissue was
slightly swollen. f X-ray of the bilateral hip joint. The cortex of the left upper
femur was slightly thickened, and the density of the medullary cavity was not
uniform. g X-ray of the bilateral knee joint. The joint space was narrowed. h-
i Skin biopsy of the right upper limb. H&E staining (h: 40× magnification, i:
200× magnification): diffuse and dense infiltration in the dermis, which was
mainly neutrophil infiltration and nuclear fragmentation, irregular
proliferation of the upper epidermis, and a superficial scab. j-m Rashes after
glucocorticoid treatment for 10 months. Acne occurred on the face and back.
Rashes on the extremities were resolved and pigmentation subsided

23. Most common complications
of brucellosis
Endocaditis
Arthritis
Hepatosplenomegaly (most common)
CNS infection neurobrucellosis

24. Brucellosis Diagnostic tests
Definitive diagnosis depend on isolation of organism Blood or
bone marrow cultures are positive during acute phase of illness
in 80% of patients caused by B. melitensis

 Complete blood count
Leukocytosis is rare in brucellosis, Anemia is reported in 75% of
patients.

 Liver enzymes
A slight elevation in liver enzyme levels is a very common
finding. These elevated levels may reflect the severity of
hepatic involvement and correlate clinically with hepatomegaly.

 Arthrocentesis
Although significant joint effusion is uncommon, arthrocentesis
may occasionally be needed toexclude septic arthritis.

 Chest radiograph
Should be obtained if respiratory symptoms are present or if a
source of infection is not apparent

 Serologic tests

25. Increased serum IgG signifies recent infection Neurobrucellosis-
CSF culture is Positive in 30%
Include tube agglutination and enzyme-linked
immunosorbent assay (ELISA), agglutination test 4 fold or more
rise in titre over a 4 week period
EIA (enzyme immunoassay) tests, designed to differentiate
between specific IgM and IgG antibodies
The standard serum agglutination test (SAT) has been
augmented by the modified Coombs' (antiglobulin) technique
and the use of 2-mercaptoethanol to separate the actions of
specific IgG and IgM.

Blood test and blood/tissue cultures
Blood Culture Specimen Blood (10 ml volumes) Inoculate blood
culture tubes or bottles (glucose-serum broth) Incubate in 10%
CO 2 Cultures should be retained for at least 6-8 weeks before
being discarded as negative

CT scan or MRI
Ultrasound, Electrocardiogram
PCR
Lumbar puncture, Joint aspiration

26. Colonial morphology of Brucella on blood agar, showing round,
translucent, with pearly appearance colonies.


Gram stain of a positive aerobic BACTEC blood culture vial
showing Brucella melitensis microcolonies

27. TREATMENT
Doxycycline 100 mg orally twice daily for six
weeks
Rifampin 600 to 900 mg (15 mg/kg) orally once
daily
Streptomycin 1 g IM once daily for the first 14
to 21 days
Gentamicin (5 mg/kg) Both drugs are
administered for six weeks
Alternative agents:
Fluoroquinolones (Ciprofloxacin 500 mg twice
daily or Ofloxacin 200 mg twice daily) in
combination with doxycycline or rifampin, but
are not appropriate first line agents they may
be useful in the setting of drug resistance with
cotrimoxazole replacing doxycycline in
children. Azithromycin has shown promising
results in experimental models.

28. Prevention of brucellosis
 Pasteurization of milk is important for the
prevention of transmission to humans to reduce
your chances of achieving it, you are encouraged
 Avoid consuming raw meat or unpasteurized milk,
cheese, and ice cream.
 Cover any open wounds on your skin when coming
in contact with animal blood
 Wear protective clothing and gloves when handling
animals or animal tissues occupationally exposed
can be protected to some extent by wearing
impermeable clothing, rubber boots, gloves and
face masks and by practicing good personal hygiene
or when helping animals give birth
 Brucellosis may be prevented via vaccination, which
is effective for cattle, sheep, and goats
 Unfortunately, there is no vaccine for brucellosis in
humans. That’s why it’s important to take other
steps to protect yourself from the bacteria

29. Case report
A 21-year-old Egyptian male was in his usual good health
until approximately 2 weeks before admission when he
began to complain of anorexia back pain abdominal pain,
weight loss, documented high fever on daily basis 2-3
times associated chills and rigors, vomiting, nausea and
headache. Three days prior to admission he developed
neck stiffness and diplopia and when the symptoms
persisted, he presented to the Abbassia Fever Hospital in
Cairo for evaluation.
On examination, the patient was an alert, thin male with
a moderately ill appearance. His pulse was 75 beats/min,
oral temperature 38 °C, blood pressure 80/120 mmHg
and weight 58 kg. Other significant findings were left
esotropia, bilateral papilloedema and nuchal rigidity. The
remainder of the physical examination, including the
neurological examination, was normal.
Peripheral white blood cell (WBC) count was 8000
cells/mL with a differential count of 80% polymorphs.
Haemoglobin level was 12.2 mg/dL. After admission,
computerized tomography scan of the brain (with and

30. without contrast) was normal, and a lumbar puncture
was performed.

Papilledema
The CSF sample showed 1650 WBC/mL, 95%
lymphocytes, protein 373 mg/dL and glucose 9 mg/dL).
Samples of blood and CSF were sent to the laboratory for
culture. Chest radiograph showed nonspecific
exaggerated bronchovascular markings. The patient was
not tested for human immunodeficiency virus.

Therapy included parenteral chloramphenicol and
penicillin, the Abbassia Fever Hospital standard initial
antibiotic regimen for presumed bacterial meningitis [2].

31. When after 2 days the patient showed no response to
therapy, isoniazid, rifampicin, streptomycin and
pyrazinamide were added to the regimen to empirically
treat tuberculous meningitis. On hospital day 11, Brucella
spp. was isolated from culture of the CSF sample from
admission. Brucella tube agglutination tests performed
on both serum and CSF samples collected at the time of
hospital admission were negative even after high dilution
to avoid the prozone phenomenon. Subsequent
evaluation using an inhouse enzyme-linked
immunosorbent assay (ELISA) test was positive for
immunoglobulin (Ig)G and IgM antibodies in the serum,
along with IgG antibodies in the CSF. Polymerase chain
reaction testing of CSF was positive for B.melitensis. The
patient had some improvement in his clinical condition
after anti-tuberculosis treatment, but once the diagnosis
of Brucella meningitis was made, antibiotic therapy was
changed to anti-brucellosis medication as following:
Medication
Strength and
frequency
Ciprofloxacin 500 mg Q12hr.
Doxycycline 100 mg Q12hr
Rifampicin 600 mg daily

32. trimethoprim/sulf
methoxazole
(TMX/SMP)
2 double-strength
tablets every 12
hours

The patient was given anti-brucellosis therapy
(ceftriaxone + levofloxacin + rifampicin + doxycycline) for
6 months and glucocorticoid therapy
Within a week of initiating anti-Brucella therapy, the
patient became afebrile, had no further vomiting and his
esotropia improved. Two weeks after start of therapy for
brucellosis, his CSF WBC count decreased to 320
cells/mL, protein level dropped to 197 mg/dL and glucose
level rose to 37 mg/dL.

33. REFERENCES:
https://www.emro.who.int/emhj-volume-15-2009/volume-15-issue-
4/case-report-brucella-meningitis-first-reported-case-in-egypt.html
https://www.ncbi.nlm.nih.gov/books/NBK8572/#:~:text=Brucellae%20a
re%20Gram%2Dnegative%20coccobacilli%20(short%20rods)%20measu
ring%20about,therefore%2C%20are%20non%2Dmotile.
https://www.intechopen.com/chapters/49044
https://www.ncbi.nlm.nih.gov/books/NBK8572/
https://www.researchgate.net/publication/23559469_Multicenter_Stu
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https://www.familyhealthcare.co.in/brucellosis-causes-symptoms-
prevention/
https://www.who.int/news-room/fact-sheets/detail/brucellosis
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032053/
https://www.karger.com/Article/Fulltext/503454
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634613/
https://www.researchgate.net/publication/23559469_Multicenter_Stu
dy_of_Brucellosis_in_Egypt
https://www.researchgate.net/figure/Fig-4-Colonial-morphology-of-
Brucella-on-blood-agar-showing-round-translucent-
with_fig2_308029170