HAEMOPHILLUS

Haemophilus Influenzae: Clinical Infections

Infections caused by typable (encapsulated) strains
Acute epiglottis or laryngotracheal infection in small children Cellulitis/arthritis Meningitis Pneumonia/septicemia (in children) Conjunctivitis

Haemophilus Influenzae: Clinical Infections

Infections caused by Nontypable strains
Otitis media Sinusitis Pneumonia, bronchitis (in adults)

Haemophilus Species
H. influenzae satellitism around and between the large, white, hemolytic staphylococci
Haemophilus species require hemoglobin for growth: X-factor ( hemin): Heat-stable substance V-factor (NAD): Heat- labile, coenzyme I, nicotinamide adenine dinucleotide, found in blood or secreted by certain organisms
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Haemophilus Species

Direct smear of H. influenzae in CSF in a case of meningitis. Note the intracellular and extracellular pleomorphic gramnegative bacilli.
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Haemophilus species
H.influenzae growing on chocolate agar. Notice the semi-opaque, gray-white, mucoid colonies characteristic of encapsulated strains.

Gram stain of H. influenzae from colony

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Haemophilus Species: Identification

This organism would be identified as H. influenzae because it is using both X and V factors.

Haemophilus Species: Identification

This organism would be identified as H. parainfluenzae because it is using V factor only.

Haemophilus Species: Identification

This organism would be identified as H. aphrophilus because it is using X factor only.

Haemophilus Species: Identification

Under ultraviolet light, the organism on the bottom is showing a positive porphyrin reaction, whereas the organism on the top is demonstrating a negative porphyrin reaction.
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BORDETELLA

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Bordetella
Classification the genus contains three medially important species
B. pertussis B. parapertussis B. bronchoseptica

Morphology and cultural characteristics

Small g-cb B. parapertussis and B. bronchoseptica both grow on sheep BA (SBA) in 1-2 days
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Bordetella
B. pertussis for initial isolation (The best clinical specimen is a nasopharyngeal swab.) the organism requires special media with additional nutrients for growth and absorbents to remove toxic substances found in complex media such as fatty acids and sulfides.
Borget-Gengou media contains glycerol, potato infusion, albumin (binds fatty acids), and up to 50% defibrinated SRBCs Charcoal agar supplemented with 10% horse blood with or without cephalexin. May take 3-7 days for growth and colonies are smooth, raised, and glistening (phase 1 colonies).
They are also hemolytic and produce toxin.
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Charcoal-horse blood agar

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Bordetella
Virulence factors (B. pertussis)
Pili for attachment Pertactin, an outer membrane protein also acts as an adhesion Filamentous hemagglutinin is found on the cell surface of and is also secreted.
It attaches to cilia by binding to exposed lactose receptors.
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Bordetella

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Bordetella
Pertussis toxin
Secreted by type IV secretion system Has one A subunit (toxic part), plus four different kinds of B subunits (involved in binding).

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Bordetella
Clinical significance
B. pertussis causes whooping cough
Acquired by inhalation of droplets containing the organism The organism attaches to the ciliated cells of the respiratory tract.
During an incubation period of 1-2 weeks, the organism multiplies and starts to liberate its toxins.

Next the catarrhal stage occurs - the patient has a mild cough and sneezing whereby large numbers of organisms are spread through the respiratory secretions.
This last ~ 2 weeks.
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Bordetella
Next is the paroxysmal stage that lasts 4-6 weeks. The patient has rapid, consecutive coughs with a rapid intake of air between the coughs (has a whooping sound). The ciliary action of the respiratory tract has been compromised, mucous has accumulated, and the patient is trying to cough up the mucous accumulations. The coughs are strong enough to break ribs! Other symptoms due to the activity of the released toxins include: Increased peripheral lymphocytes due to a blocking of homing of lymphocytes to the spleen and lymph nodes. Metabolic alteration such as increased insulin release and the resulting hypoglycemia Increased capillary permeability and increased susceptibility to histamine, serotonin, and endotoxin shock

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Bordetella
Finally there is a convalescent stage during which symptoms gradually subside.
This can last for months.

B. pertussis rarely spreads to other sites, but a lot of damage may occur, such as CNS dysfunction which occurs in ~10 % of the cases and is due to an unknown cause. Secondary infections such as pneumonia and otitis media are common.

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Bordetella
B. parapertussis causes a mild form of whooping cough B. bronchoseptica
Widespread in animals where it causes kennel cough. Occasionally causes respiratory or wound infections in humans.

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Bordetella
Treatment
Erythromyin only effective in early stages of the disease before the toxin(s) have been released Vaccination P part of DPT (killed, encapsulated organism); a subunit vaccine has also been developed (purified pertussis toxin).

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BRUCELLA

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1. 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).
2. The importance of this contagious disease is the economic impact on livestock industry. 3. Causes sever hazard to human health, through either direct contact with infected animals or the consumption of contaminated milk and dairy products.
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Causative 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).

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Table (1) show Brucella species :

NO

Species

Biovar

Natural host

Other animal species affected

Human disease

1. Brucella abortus 2. Brucella melitensis

1-9 1-3

Cattle

Wild animals, water buffalo, camels

Less sever Sever

Sheep Wild ruminant and Goat cattle, camels Sheep (Ram) Swine Dog

3. Brucella ovis
4. Brucella suis 5. Brucella canis 6. Brucella neotomae

1
1-5 1 1

None
Various wild species None

None
Sever ( except biovar 2) Benign None
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Wood rat None

Morphology and staining

1. The bacteria are strictly parasitic and prefer the intracellular habit. 2. The species of the genus Brucella are small non motile, non spore forming, Gram negative rods and they do not produce true capsules. 3. They are somewhat resistant to decolonization by weak acids and thus stain red by the modified Ziehl - Neelsen method.
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Gram stain of Brucella (Gram ve ) Coccobacilli

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Antigenic Structure

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The designation of the antigens in cultures composed of smooth and rough colonies are shown in the table 2. NO. 1. 2. 3. 4. Species Brucella abortus Brucella melitensis Brucella suis Brucella ovis Type of colony Smooth Smooth Smooth Rough Rough
Type of surface antigen

A a A -----

m M m -----

------R R

5.

Brucella canis

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. 32

Cultural characteristics
1. 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 Glyceroldextrose 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, 33 Vancomycin

2. Colony Morphology
Brucella colonies are visible after 3-5 days incubation period at 37 C 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.
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Brucella colonies on blood agar

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Brucella colonies on blood agar

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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. 37

One of the most important route of animal to human transmission of Brucella

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 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.

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Brucellosis: Edema and swelling of scrotum

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