1. 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 , India

2. Activity of antimicrobial against
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

3. 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

4. Bacteria Definition Invade tissues May produce
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

5. 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

6. Effects Bacteremia Septicemia Toxemia Toxico infection Intoxications
Blood
Septicemia
Harmful waste products in blood
Toxemia
Toxins in blood
Toxico infection
Intoxications

7. 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

8. 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

9. Antibiotics Principles of Antibiotic use
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.

10. 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

11. 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

12. Diagnosis
Pen-side
At clinic
At laboratory

13. 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
Glanders
CFT, Agent id, Mallein
Campylobacteriosis
Listeria
Agent id
Escherichia coli
Strangles

14. 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

15. 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

16. Site of sampling Sterile sites Non-sterile (normal flora) 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

17. 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)

18. 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

19. 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.

20. 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

21. Types of solid media Nutrient agar
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

22. 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)

23. 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

24. 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

25. 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

26. Bacteria are of many types
With Cell Wall
Gram +
Staphylococcus, Streptococcus, Clostridium, Bacillus
Gram -
Enteric, respiratory and others
Acid-fast
Mycobacterium
Wall-less
Mycoplasma
Unusual
Obligate intracellular
Rickettsia, Chlamydia
Bacteria
G G- AF WL IC

27. Bacteria Gram- Intra Cellular Wall Less Gram+ Acid Fast Mycoplasma
Cocci
Rod
Spiral
Rod
Cocci
M.t.
Rickettsia
Coxiella
Erlichia
Chlamydia
Strep.
Non-spore
Spore
Treponema
Borrelia
Leptospira
Neisseria
Moraxella
Staph.
Fil
Rod
+O2
-O2
Curve
A.i.
C.d.
L. m.
B.a.
B.c.
C.b.
C.t.
C.p.
C.d.
Straight
Vibrio
Campylobacter
Helicobacter
+O2
+/-O2
-O2
Other A B Pn
Vir
S. a.
S. e.
S. s.
P.a.
Enteric
Bact.
H. ducreyi
Gardnerella
Calymmatobacterium
Zoo
Resp. GU
Bordetella.
H. influenzae
Legionella
Yersinia
Pasteurella
Brucella
Francisella
Streptobacillus

28. Gram negative Curved rods Straight rods Lactose+ Lactose-
Campy blood agar
42oC oC-
Campylobacter
TCBS agar
Yellow
Oxidase+
Vibrio
Citrate Citrate-
H2S H2S-
Klebsiella E. coli Salmonella Shigella

29. 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

30. 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

31. 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
which will then be

32. 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:

33. Advantages Reduce reliance on culture Faster More sensitive
More definitive
More discriminating
Techniques adaptable to all pathogens

34. 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

35. 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

36. 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?

37. 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:

38. 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:

39. 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:

40. 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.

41. Questions