Diagnosis of Important Bacterial Diseases Effective treatment can be initiated sooner if diagnostic results can be made quickly available to the clinician treating a disease outbreak. Bhoj R Singh Section of Epidemiology, CADRAD, IVRI, Izatnagar- 243122, India
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Diagnosis of Important Bacterial Diseases
Effective treatment can be initiated sooner if diagnostic results can be made quickly available to the clinician treating a disease outbreak.
Bhoj R Singh
Section of Epidemiology, CADRAD, IVRI, Izatnagar-243122, India
Activity (range) of various antimicrobial classes (Prescott and Baggot)
Group of Antibiotics
Activity of antimicrobial against
Bacteria Mycoplasma Rickettsia Chlamydia Protozoa
Aminoglycosides + +
Beta-lactams +
Chloramphenicol + + + +
Lincosamides + + +
Macrolides + + +
Pleuromutilins + + +
Tetracyclines + + + +
Quinolones + + + +
Sulfonamides + + + +
Trimethoprim + +
Scope
Bacterial infections affect the skin; the eye; the ear; the mouth; the nose
the reproductive system the digestive system
the respiratory system the urinary system the nervous system
the circulatory system the locomotion organs
the appendages
Bacteria
Definition Single-celled microorganisms which can exist
either as independent (free-living) organisms or as parasites (dependent upon another organism for life)
Invade tissues May produce
Pus Harmful or poisonous waste
Live in a wide range of conditions Live on and in the bodies of all animals More numerous than the cells of the body Useful in production of foods such as cheese
and sauerkraut Many can be harmful Invade the cells of an animal’s body May harm the animal by feeding off the body
cells or secreting a material known as a toxin
Bacteremia Blood
Septicemia Harmful waste products in blood
Toxemia Toxins in blood
Toxico infection Intoxications
Effects
Types of Bacteria
Cocci: Round spherical shaped bacteria Some forms of pneumonia and sepsis are caused by this bacteria
Bacilli: Rod shaped Single, pairs, or arranged in chains Cause many serious diseases in animals
Spirila Shaped like spirals or corkscrews Very motile Require moist atmosphere to live Live very well in the reproductive tracts of animals Leptospirosis Vibrosis and spirochetosis
Why diagnosis is needed?
To administer the treatment For prognosis To initiate appropriate control measures To take suitable preventive steps To understand epidemiology To know the disease history For certification in International trade
To export For import
To know who is at risk
Antibiotics
Once thought to be able to eliminate/ cure all pathogenic Bacterial infections.
MDR in pathogens lead to failure. Antibacterial drug resistance is more natural than
induced.
Principles of Antibiotic use 1. Either not use or try to avoid unless very much essential. 2. Not use many at a time. 3. Use specific antibiotics rather than broad-spectrum. 4. Complete the course. 5. Never use antibiotics reserved for human use.
What is needed for diagnosis
Sound knowledge about the diseases Knowledge about the host animal Knowledge about the environment Sound clinical experience Right material (Sample) Diagnostic facilities Laboratory expert
Diagnosis responsibility and need of a veterinarian.
Suitable sample Proper dispatch of sample to reach the
laboratory along with all the relevant history of disease (morbidity, mortality, contagiousness etc.), signs and treatment.
In-time arrival at Laboratory Proper laboratory facility In-time processing at the Laboratory by the
trained personnel
Site of sampling
Sterile sites Blood Cerebrospinal fluid (CSF) Body fluids (Peritoneal and pleural)
Non-sterile (normal flora) Respiratory tract Ear, eye and mouth Skin (wound and abscess) Urine (mid-stream) Feces
Microscopy
Microorganisms can be examined microscopically for:a- Bacterial motility: Hanging drop method: A drop of bacterial suspension is placed between a cover slip and glass
slidb- Morphology and staining reactions of bacteria: Simple stain: methylene blue stain Gram stain: differentiation between Gm+ve and Gm–ve bacteria . Primary stain (Crystal violet)
Prevent drying of the sample or swab. Culture container must contain fluid/ semisolid
transport medium to keep bacteria alive for 24 hrs. Some media for swab transportation:
Liquid Liquid transport medium
Campylobacter transport medium
Brucella transport medium
Semisolid Stuart transport medium
Carry and Blair transport Medium with and without charcoal
Amies transport medium
Culture for bacteria
Sample is inoculated for culture and identification either in pre-enrichment or selective enrichment for broth culture. Incubated at suitable temperature for suitable time in proper environment
Streaked on either selective, differential or both type of agar media for suitable time in proper environment
Individual colonies are picked and grown as a pure culture.
Tentative ID made based on colony shape and staining. Definitive ID requires biochemical, serological, and various
tests.
Culture Techniques
* Culture media are used for:
- Isolation and identification of pathogenic organisms
- Antimicrobial sensitivity tests
* Types of culture media:
a- Liquid media:
- Nutrient broth: meat extract and peptone
- Peptone water for preparation sugar media
- Growth of bacteria detected by turbidity
b- Solid media:
- Colonial appearance
- Hemolytic activity
- Pigment production
Types of solid media
1- Simple media: Nutrient agar 2- Enriched media: media of high nutritive value . Blood agar . Chocolate agar . Loffler’s serum 3- Selective media: allow needed bacteria to grow . Lowenstein–Jensen medium . MacConkey’s agar . Mannitol Salt Agar 4- Indicator media: to different. between lact. and non lact. ferment . MacConkey's medium . Eosine Methylene blue Agar 5- Anaerobic media: for anaerobic cultivation . Deep agar, Robertson’s Cooked Meat Medium
Colonial appearance on culture media
* Colony morphology: . Shape . Size . Edge of colony . Color* Growth pattern in broth: . Uniform turbidity . Sediment or surface pellicle* Pigment production: . Endopigment production (Staph. aureus) . Exopigment production (Ps. aeruginosa)* Haemolysis on blood agar: . Complete haemolysis (Strept. pyogenes) . Partial haemolysis (Strept. viridans)* Growth on MacConkey’s medium: . Rose pink colonies (Lactose fermenters) . Pale yellow colonies (Non lactose fermenters)
Biochemical Reaction
Use of substrates and sugars to identify pathogens: a- Sugar fermentation: Organisms ferment sugar with production of acid only Organisms ferment sugar with production of acid and gas Organisms do not ferment sugar b- Production of indole: Depends on production of indole from amino acid tryptophan Indole is detected by addition of Kovac’s reagent Appearance of red ring on the surface e- H2S production: Depends on production H2S from protein or polypeptides Detection by using a strip of filter paper containing lead acetate
Biochemical Reaction (cont.)
c- Methyl red reaction (MR): Fermentation of glucose with production of huge amount of acid Lowering pH is detected by methyl red indicator
d- Voges proskaur’s reaction (VP): Production of acetyl methyl carbinol from glucose fermentation Acetyl methyl carbinol is detected by addition KOH Color of medium turns pink (positive) e- Action on milk: Fermentation of lactose with acid production Red color if litmus indicator is added
Biochemical Reaction (cont.)
f- Oxidase test: Some bacteria produce Oxidase enzyme Detection by adding few drops of colorless Oxidase reagent Colonies turn deep purple in color (positive) g- Catalase test: Some bacteria produce catalase enzyme Addition of H2O2 lead to production of gas bubbles (O2 production) h- Coagulase test: Some bacteria produce coagulase enzyme Coagulase enzyme converts fibrinogen to fibrin (plasma clot) Detected by slide or test tube method i- Urease test: Some bacteria produce urease enzyme Urease enzyme hydrolyze urea with production of NH3
Alkalinity of media and change color of indicator from yellow to pink
World Association of Veterinary Laboratory Diagnosticians
Mission Statement
The mission of the WAVLD is to improve animal and human health by facilitating the availability of quality laboratory testing provided through veterinary diagnostic laboratories around the world. This mission is accomplished by:
Disseminating the latest information relating to the diagnosis of animal diseases through outstanding educational symposia.
Facilitating the organization of associations of veterinary laboratory diagnosticians in all countries of the world.
Providing consulting assistance to countries wishing to build and operate state-of-the-art veterinary diagnostic laboratories.
Supporting other activities to improve the health and welfare of man and animals throughout the world.