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
Aminoglycosides
+
+
Beta-lactams
+
Chloramphenicol
+
+
+
+
Lincosamides
+
+
Macrolides
+
+
+
Pleuromutilins
+
+
+
Tetracyclines
+
+
+
+
Quinolones
+
+
+
+
Sulfonamides
+
+
Trimethoprim
+
Protozoa
+
+
+
+
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
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
Effects
 Bacteremia
 Blood
 Septicemia

Harmful waste products in blood
 Toxemia
 Toxins in blood
 Toxico infection
 Intoxications
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.

Diagnostic tests may be performed by a
technician.
 Diagnostic techniques, history, clinical examination,
and other information considered.
 Diagnostic techniques: radiography, anatomical
pathology, necropsy, microscopic examination of
tissue sections, clinical pathology, microbiology,
hematology, blood chemistry, immunoserology,
parasitology and urinalysis
Diagnosis
 Pen-side
 At clinic
 At laboratory
Recommended Diagnostics
Disease
Diagnostic tests
Anthrax
Demonstration of organism, Agent id
Leptospirosis
MAT, Agent Id
Brucellosis
BBAT, CFT, FPA, MRT, STAT, ELISA, Agent
id, FAT, Brucellin
TB
Tuberculin test, ELISA, Agent id
JD
Johnin test, Agent id, ELISA
Q-fever
CFT, Agent id
Tularemia
Agent id
Salmonellosis
STAT, ELISA, Agent id
CBPP
CFT, ELISA, Agent id
CEM
Agent id
Glanders
CFT, Agent id, Mallein
Campylobacteriosis
Listeria
Agent id
Agent id
Escherichia coli
Agent id
Strangles
Agent id
Steps in Diagnosis of Bacterial
diseases
 Clinical Signs
 Laboratory examination
1- Microscopy
2- Culture techniques
3- Biochemical reactions
4- Serological identification:
5- Molecular biology techniques
6- Bacteriophage typing
Pre-requisite for laboratory
Examination
 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
slid
b- 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)
. Mordant (Grams Iodine mixture)
. Decolorization (ethyl alcohol)
. Secondary stain ( Saffranin)
Ziehl-Neelsen stain: staining acid fast bacilli
. Apply strong carbol fuchsin with heat
. Decolorization (H2SO4 20% and ethyl alcohol
. Counter stain (methylene blue)
Sample for Bacterial Isolation
 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
Bacteria are of many types
 With Cell Wall
 Gram +


Gram 

Enteric, respiratory and others
Bacteria
Acid-fast


Staphylococcus, Streptococcus,
Clostridium, Bacillus
Mycobacterium
Wall-less

Mycoplasma
 Unusual
 Obligate intracellular

Rickettsia, Chlamydia
G+
G-
AF
WL
IC
Bacteria
Gram-
Gram+
Cocci
Rod
Strep.
Staph.
Non-spore Spore
Fil
Rod +O2 -O2
Spiral
A
B
Pn
Vir
Cocci
Treponema
Borrelia
Leptospira
A.i. C.d. B.a. C.b.
L. m. B.c. C.t.
C.p.
C.d. +O
2
S. a.
S. e.
S. s.
Rod
P.a.
Enteric
-O2
Intra Wall
CellularLess
Mycoplasma
M.t.
Neisseria
Moraxella
Rickettsia
Coxiella
Erlichia
Chlamydia
Curve
Straight
+/-O2
Acid Fast
Other
Vibrio
Campylobacter
Helicobacter
Bact.
Resp.
Bordetella.
H. influenzae
Legionella
H. ducreyi
Zoo
GU
Gardnerella
Yersinia
Pasteurella Calymmatobacterium
Brucella
Francisella
Gram negative
Curved rods
Straight rods
Lactose+
Citrate+
Klebsiella
Lactose-
Citrate- H2S+
E. coli
Salmonella
Campy blood agar
42oC+ 25oC-
TCBS agar
Yellow
Oxidase+
Campylobacter
Vibrio
H2SShigella
Animal pathogenicity
* Animal pathogenicity test:
Animals commonly used are guinea pigs, rabbits, mice
* Importance of pathogenicity test:
- Differentiate pathogenic and non pathogenic
- Isolation organism in pure form
- To test ability of toxin production
- Evaluation of vaccines and antibiotics
Serological identification
A- Direct serological tests:
- Identification of unknown organism
- Detection of microbial antigens by using specific
known antibodies
- Serogrouping and serotyping of isolated organism
B- Indirect serological tests:
- Detection of specific and non specific antibodies
(IgM & IgG) by using antigens or organisms
SEROLOGICAL DIAGNOSIS OF
INFECTIOUS DISEASES

Infectious Disease Indicators, Non-specific


Acute phase reactants
Limulus lysate assay



Detects trace amounts of endotoxin from all gram (-)
bacteria
Presence in CSF = gram (-) bacterial meningitis
Rapid clearance from blood makes serum test
unreliable
Molecular Diagnosis
 Ribotyping
 Restriction fragment length polymorphism
(RFLP)
 DNA hybridization
 PCR, RT-PCR and RAPD
 Nucleic acid sequence analysis
 PFGE
 Phage-GFP (TB)
 Plasmid profile analysis:
Advantages
 Reduce reliance on culture
 Faster
 More sensitive
 More definitive
 More discriminating
 Techniques adaptable to all pathogens
Leading uses for nucleic acid
based tests
 Nonculturable agents
 Fastidious, slow-growing agents
Mycobacterium tuberculosis
 Legionella pneumophilia
 Highly infectious agents that are dangerous to culture
 Francisella tularensis
 Brucella species
 In situ detection of infectious agents
 Helicobacter pylori
 Toxoplasma gondii
 Organisms present in small volume specimens
 Intra-ocular fluid
 Forensic samples

Leading uses for nucleic acid
based tests
 Differentiation of antigenically similar agents
 May be important for detecting specific serovars of
bacteria associated with infection
 Non-viable organisms

Organisms tied up in immune complexes
 Molecular epidemiology
To identify point sources for hospital and
community-based outbreaks
 To predict virulence

 Culture confirmation
Disadvantages of a molecular test?
 Technically demanding
 Relatively expensive
 Provides no information if results are negative
 So specific that must have good clinical data to support
infection by that organism before testing is initiated.
 Will miss new organisms unless sequencing is done as we
will be doing in the lab for our molecular unknowns (not
practical in a clinical setting).
 May be a problem with mixed cultures – would have to
assay for all organisms causing the infection.
 Too sensitive? Are the results clinically relevant?
OIE ad hoc Group on Diagnostic Tests in
Relation to New and Emerging
Technologies
The following new molecular diagnostic methodologies have been
identified:
 Direct diagnostic assays












• PCR-based assays
 o Real time;
 o Rapid detection in a disease outbreak;
 o Multiplex;
 o PCR robotics.
• Isothermal amplification assays;
• Microarray technologies;
• Rapid sequencing technologies, phylogenic analysis/bioinformatics;
• Genomic technologies to determine virulence;
• Complete full length genome sequencing technologies;
• Pen-side test technologies (lateral flow devices);
• Portable PCR technologies for field use;
• Nanotechnology;
• Proximity ligation technologies;
• In-situ hybridisation;
• Proteomics (detection of proteins).
Source: http://www.oie.int/downld/SC/2008/A_BSC_sept2008.pdf
OIE ad hoc Group on Diagnostic Tests in
Relation to New and Emerging
Technologies
The following new molecular diagnostic methodologies have
been identified:
 Indirect diagnostic test (antibody-based assays)








• Bioluminometry;
• Fluorescence polarisation;
• Chemoluminescence technologies;
• Biosensors;
• Biomarkers;
• Recombinant proteins;
• Synthetic proteins;
• Improved monoclonals for enzyme-linked immunosorbent assays
(ELISA).
Source: http://www.oie.int/downld/SC/2008/A_BSC_sept2008.pdf
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.
Source: http://www.wavld.org/Home/tabid/207/Default.aspx
Further Reading
1. McCurnin, D.M. Clinical Textbook for Veterinary
Technicians. W.B. Sanders, Philadelphia, PA, 1994.
2. Pratt, P.W. Laboratory Procedures for Veterinary
Technicians. Mosby, St. Louis, MO, 1996.
3. Singh, B.R. Labtop for Microbiology Laboratory.
Lambert Academic Press, 2009.
Questions