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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 Aminoglycosides Beta-lactams Chloramphenicol Lincosamides Macrolides Pleuromutilins Tetracyclines Quinolones Sulfonamides Trimethoprim Activity of antimicrobial against Bacteria
+

Mycoplasma
+

Rickettsia

Chlamydia

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 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 animals body May harm the animal by feeding off the body cells or secreting a material known as a toxin

Effects
Bacteremia

Blood
Harmful waste products in blood

Septicemia

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
Anthrax Leptospirosis Brucellosis TB JD Q-fever Tularemia Salmonellosis CBPP CEM

Diagnostic tests
Demonstration of organism, Agent id MAT, Agent Id BBAT, CFT, FPA, MRT, STAT, ELISA, Agent id, FAT, Brucellin Tuberculin test, ELISA, Agent id Johnin test, Agent id, ELISA CFT, Agent id Agent id STAT, ELISA, Agent id CFT, ELISA, Agent id Agent id

Glanders
Campylobacteriosis Listeria Escherichia coli Strangles

CFT, Agent id, Mallein


Agent id Agent id Agent id 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 Gmve 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 . Lofflers serum 3- Selective media: allow needed bacteria to grow . LowensteinJensen medium . MacConkeys 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, Robertsons 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 MacConkeys 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 Kovacs 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 proskaurs 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 +

Staphylococcus, Streptococcus, Clostridium, Bacillus Enteric, respiratory and others


Bacteria

Gram

Acid-fast

Mycobacterium Mycoplasma
G+ GAF WL IC

Wall-less

Unusual Obligate intracellular

Rickettsia, Chlamydia

Bacteria
Gram+ GramAcid Fast Intra Wall CellularLess Mycoplasma Rickettsia Coxiella Erlichia Chlamydia

Cocci Strep. Staph.

Rod

Spiral

Rod

Cocci

M.t.

Non-spore Spore Fil Rod +O2 -O2

Treponema Borrelia Leptospira Straight +/-O2 Enteric -O2 Bact. Other

Neisseria Moraxella Curve

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.

Vibrio Campylobacter Helicobacter

A B Pn Vir

P.a.

Resp. Bordetella. H. influenzae Legionella

Zoo Yersinia Pasteurella Brucella Francisella

H. ducreyi GU Gardnerella Calymmatobacterium

Gram negative
Straight rods
Lactose+ Lactose-

Curved rods

Campy blood agar 42oC+ 25oC-

TCBS agar Yellow Oxidase+

Citrate+
Klebsiella

Citrate- H2S+
E. coli Salmonella

H2SShigella

Campylobacter

Vibrio

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

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