Dr.

Shehab Ahmed Lafi

PARVOBACTERIA
Parvobacteria

This group includes heterogeneous small Gram

. negative fastidious coccobacilli
They are non motile non spore forming aerobic
.and microaerophilic bacteria
It includes Haemophilus , Bordetella and
.Brucella
Haemophilus

This genus includes small gram negative coccobacilli

that require enriched media usually containing
blood or its derivatives for its isolation . These
. factors are known as X factor which is hemin factor
V factor which is Niacin Adenine Dinucleotide
NAD .
Some of them resembles flora of conjunctiva and
.mucous membranes while others are pathogenic
 Pathogenic species are :

 Haemophilus influenza
 Haemophilus ducreyi
 Haemophilus aegypticus
Haemophilus influenza

Organisms within this genus are Gram v-e

coccobacilli ,it requires both X and V factors
. for their growth
.Their mean size is about 1.5 x 0.3 ᶬ
 Non pathogenic strain of this species is non
capsulated and it is known as Haemophilus
influenza Type A , it is found on the mucous
membrane of the upper respiratory tract in
humans .
 While the pathogenic strain is known as Haemophilus
influenza type B , it is capsulated and it is considered as
an important cause of meningitis in children and
occasionally cause respiratory tract infection in
children and adults . Also it causes conjunctivitis ,
septicemia , subacute endocarditis and septic arthritis .
Growth Characters
 It is fastidious organism and needs specific
requirements for growth like X and V factors.
It grows much larger around Staphylococcus
aureus colonies and shows what is known as
Satellitism phenomenon due to V factor
synthesis by Staphylococcus.
Antigenic Structure

Capsular Antigen -1:

 There are two types of Haemophilus

influenza :
 Type A which is non capsulated

 Type B which is capsulated and the capsular

antigen is Poly saccharide (Polyribose Ribitol

Phosphate PRP) ,Regarding this antigen there
are 6 types known as A, b , c ,d, E& F.
.Somatic Ag-2
 Somatic [Link] protein in nature and the
endotoxin which is lipopolysaccharide in
nature .
 Type B is usually causes 95% of disease while
other types are rarely causing disease.
Pathogenicity and Clinical Findings
 Normally the non capsulated organisms
inhabit human respiratory tract and
nasopharynx . Capsulated type produces
primary infection and may cause sinusitis ,
laryngeo - tracheitis ,epiglottitis ,otitis and in
young children it causes meningitis.
 The organisms reach blood in case of untreated
patients leading to septicemia, the more
complicated case is meningitis and it may
establish joints causing septic arthritis.
 Haemophilus influenza meningitis occurs in
children between 5 months – 5 years .before 5
months baby has maternal immunity and
above 5 years bacterial antibody will develop so
the disease is rare in adults.
 Lab diagnosis
 Clinical specimens depends on the site of
infection and the type of the clinical
symptoms.
 Nasopharyngeal swab , sputum ,blood ,CSF
and pus in case of sinusitis and otitis. Each
specimen undergo investigation by :
Direct Investigation Using:

A- Gram stained smear , positive findings

show gram negative coccobacilli
B-Fluorescent antibody test for smears
C-Capsule swelling test using capsular
antibody in the same way of that of S.
pneumoniae capsular quelling reaction .
 Indirect investigation
 Indirect investigation testing through
cultivation of the specimen on chocolate agar
or other media enriched with X and V factors
for 24 hours at 37c . Colonies are small
glistening like due drops.
 Molecular diagnostic methods can be used like
PCR.
 Haemophilus aegypticus
 It Is formerly called Koch's week bacillus and
it has been associated with highly
communicable form of conjunctivitis (pink eye
) . It can be recover from pneumonic patients .
 
 Haemophilus ducreyi
 It causes soft chancre , a sexually sex
transmitted disease STD .The chancroid lesion
consists of ulcer on the external genitalia with
marked swelling of regional lymph nodes and
 It must be differentiated from syphilis. It
requires only X factor for growth. Treatment
with sulfonamide or oral erythromycin often
results in healing within 2 weeks.
 Haemophilus parainfluenza
 It is found normally in the respiratory tract of
human and it resembles Haemophilus
influenza in characters. It requires V factor
for growth. It has been encountered in disease
rarely and mainly infective endocarditis.
Bordetella
 There are three species of Bordetella :
 
 Bordetella pertusis
 Bordetella parapertusis
 Bordetella bronchoseptica
Bordetella pertusis
 It is gram negative coccobacillus resembling
Haemophilus influenza but do not require X
and V factors for its growth.
 Bipolar staining technique with toluidine blue
dye and metachromatic granules can be
detected. Capsule is present .
Cultural characters

Primary isolation requires enriched medium

known as Bordet –Gengou Medium , it contains
glycerol, potato extract , glycerol and agar, in
.addition to that 0.5 ᶬg/ml of penicillin
A charcoal containing medium Similar to that
used for the isolation of Legionella
pneumophilia. Suitable incubation period is 3-7
days at 35-37C in moist environment
 Mercury drop colonies are produced on
Bordet – Gengou Medium.
 A narrow zone of beta hemolysis on blood
agar is associated with virulent Bordetella
pertusis.
Antigenic structure:

1-Pertusis toxin : It is the major virulence factor

and elicits immunity.
2-Histamine sensitizing Antigen : It is
responsible for paroxysmal cough .
3-Haemagglutinins : They are fimbrial
Haemagglutinins , leucocytosis promoting
factor promotes lymphocytosis .

Phase-1 usually contains large amount of

protective antigen than other phases.
 
Pathogenesis :
 Bordetella pertusis is an obligate human
pathogen causing whooping cough which is an
acute child respiratory disease.
 The organisms survive for short period outside
the body , transmission is mainly by
respiratory route from early cases and
possibly through carriers .
 The organism adheres to the epithelial cells of
trachea and bronchi and multiply rapidly on the
site of adhesion . It interferes with ciliary action .
Bacteria liberate toxins and substances that
irritate surface cells , later necrosis and
lymphocytosis with peri-bronchial inflammation
.and interstitial pneumonia
 . Secondary invaders like Staphylococcus
aureus and H. influenza may give rise to
bacterial pneumonia . obstruction of smaller
bronchioles by mucous plugs results in
atelectasis and diminished oxygenation of the
blood and this contributes convulsion
frequency.
Clinical findings :

 After an incubation period of about 2 weeks ,

pertusis occurs in three stages :
A- Catarrhal stage :
 It is characterized by mild cough and

sneezing , the patient is highly infectious in this

phase due to high number of bacteria in the
droplets .
 B- Paroxysmal stage :
 Cough increases in severity and show its

explosive characters and whoops in inhalation.

It may be associated with vomiting , cyanosis
and convulsion. Whoop is prominent in infants
while paroxysm dominates in older children
and adults.
 
C- Convalescent stage :

 Frequency and severity of cough starts decrease

gradually . Several types of adenoviruses and
Chlamydia trachomatis can produce clinical picture of
Bordetella pertusis.
: Diagnosis

 Specimens like nasopharyngeal swab or cough

droplets can be cultivated on to Brdet-
Gengou agar or blood- charcoal agar.
 Direct examination of the specimen using
fluorescent antibody technique ( FAT ).
 Serology also like ELISA can be used to
diagnose pertusis.
: Treatment &Prophylaxis
 Erythromycin , Ampicillin and Tetracycline
are effective against this infection .
 Prophylaxis : Killed ( smooth phase-1 )vaccine
is quite protective, every baby should receive
injection of killed phase -1 vaccine combined
with tetanus and diphtheria a triple vaccine .
: Bordetella parapertusis
 It is similar to whooping cough , it is isolated
from patients with acute respiratory
infections. Colonies are larger, it is sharing
somatic antigen with Bordetella pertusis so
that they give cross agglutination reaction with
Bordetella pertusis.
Brucella:
These are obligate pathogens for animals and humans,
they are intracellular pathogens and are causing
. undulant fever or brucellosis

The most important species associated with zoonotic

infections is Brucella melitensis typically infects goats
and first isolated from spleen of patient in Malta who
.was dying from fever
 Brucella abortus , it infects cattles primarily and
causes contagious abortion for them.
 Brucella suis , it infects swine
 Brucella canis ,it infects dogs.
 Brucellosis is primarily an animal disease occasionally
infects humans as zoonotic disease through
consumption of unpasteurized milk products and meat
or contact with infected animals. People at high risk
are farmers , workers in slaughter houses and
veterinarians.
Morphology and cultural characters :
 These organisms are Gram negative
coccobacilli non motile , non spore forming
aerobic except certain species needs CO2 for
there growth like [Link] .
 capsule can be demonstrated in smooth and
mucoid variants. Brucella needs complex
nutritional requirements , they grow on
Trypticase Soy agar , blood agar and
Castaneda medium.
 They are catalase , oxidase positive while H2S
is released by some strains . Some variants
grow in the presence of basic dyes .
 Colonies are small convex appear on enriched
media within 2-5 days. Brucellae are
moderately sensitive to heat and acidity . they
are killed in milk by pasteurization.
: Antigenic structure

 There are two antigens A &M , Brucella

abortus contains antigen A more than antigen
M while Brucella melitensis contains more
antigen M. In addition to these antigens , a
superficial L antigen has been detected
resembles Vi antigen of Salmonella.
 Species differentiation among Brucella is made
by their sensitivity to dyes and H2S production.
: Pathogenesis and pathology

 Although each species of Brucella has a

preferred host, all can infect a wide range of
animals and humans .
 The main routes of infection are :
 1- Oral route through ingestion of infected
milk and milk products and its most common
in humans. Cheese made from unpasteurized
milk is common source.
 2- Mucous membranes of respiratory tract
through inhalation of contaminated droplets as
in risky group infections.
 3-Skin abrasions through contact with infected
animal tissue as an occupational risk factor .
 4-Man to man contact is very rare .
 The organisms progress from the portal of
entry , via lymphatic channels and regional
lymph nodes to the thoracic duct and the
blood stream ,which distributes them to the
paranchyamtous tissue. Granulomatous
nodules may develop into abscess form in
lymphatic tissue , liver , spleen , bone marrow
and other parts of reticuloendothelial system .
Organisms are seen intracellular. Osteomyelitis
meningitis or cholecystitis also occasionally
occur The main histologic reaction in brucellosis
consists of proliferation of mononuclear cells ,
fibrin exudates , necrosis and fibrosis . the
granulomas consists of epitheoid and giant cells
. with central necrosis and peripheral fibrosis
Pathogenicity of Brucella melitensis is more
severe than other species. B. abortus usually
causes mild disease without suppurative
complications and non caseating granulomas
of reticuloendothelial system . Placenta and
fetal membranes of cattle , swine , sheep and
goats contain erythritol , a growth factor for
.Brucellae
 The proliferation of organisms in pregnant
animals leads to placentitis and abortion in
these species .
 There is no erythritol in human placentas and
abortion is not part of Brucella infections of
humans.
: Clinical findings

. The incubation period is 1-6 weeks

The acute onset is characterized by fever , malaise ,

weakness , aches, and sweats, the fever is undulant .
There may be gastrointestinal and nervous
symptoms. Spleenomegaly , hepatomegaly and
enlarged lymph nodes . Deep pain and disturbance of
.motion , particularly in vertebral bodies
 The chronic stage may develop and
characterized by weakness ,aches and low
grade fever with psychoneurotic symptoms.
Brucellae cannot be isolated from the patient
at this time but aggluntition titers may be high
. 
 Acute brucellosis :
Site of entry → Lymphatic → Blood↓ → Undulant fever
(Incubation period is1-6 weeks ) ↓
Reticuloendothelial system
( Spleen ,Liver , Bone Marrow )
Clinical or Subclinical ↓
 
↓
Chronic : Chronisty
 
Weakness , Fatigue , sweating , joint pain
(Culture is negative but agglutination titers will be high )
 
Diagram showing Brucellosis course of the disease
Lab diagnosis :
 A-Specimens:
 Blood for culture should be taken during
febrile stage of illness .
 Biopsy material for culture could be taken
from bone marrow and lymph nodes .
 Serum is used for serological tests.
B-Culture :
soy medium with or without 5% sheep blood ,brain
heart infusion medium and chocolate agar. blood
culture media. All cultures should be incubated in 5-
10% CO2 at 35-37 C and should be observed for 3
weeks before being discarded as negative , liquid
Brucella agar was designed to culture Brucellae
. species. The medium is highly enriched
 Brucella species grow on commonly used Trypticase
media should be blindly subcultured during this time
on Trypticase Soy agar . Bone marrow and blood are
the specimens from which Brucellae are most often
isolated.
 Media used in semiautomated and automated blood
culture systems readily grow Brucellae. isolated
organisms are typed by H2S , dye inhibition using
( Basic Fuchsin 1/50000).
C-Serology :
 Serum is used for serological tests , IgM
appears early in the disease while IgG appears
later.
 A- Agglutination test :
 Rose- Bengal test : It must be done with
standardized antigen . IgG titers above 1: 80
indicates active infection .
 B- Mercaptoethanol test :
The addition of 2Me destroy IgM and leaves
IgG for agglutination reaction, so it is useful in
detection of chronic infections.
 Blocking Antibodies : These are IgA
molecules that interferes with agglutination by
IgM and IgG and cause negative serologic
reactions in low serum dilutions ( Prozone
phenomenon ) in spite of positive infection .
 ELISA test
 IgG, IgM , IgA antibodies may be detected
using ELISA assay ,which use cytoplasmic
proteins as antigens. This test tends to be more
sensitive and specific than agglutination test.
3-skin test :

Brucellogen is a protein extract When

injected intradermally , edema , erythema and
induration develop within 24 hours in case of
positive Brucella infection .
Treatment :
 There are different lines for Brucella
treatment , different types of antimicrobial
agents are dependent her like :
 Tetracycline, doxycycline , Streptomycin ,
Refadin and Sulfonamide.
Control :

Vaccination of humans and animals in endemic

areas.
 
THE END