Diagnosis of important bacterial diseases
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Transcript of Diagnosis of important bacterial diseases
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.
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 Agent id
Listeria Agent id
Escherichia coli Agent id
Strangles Agent id
Steps in Diagnosis of Bacterial diseases
Clinical Signs Laboratory examination1- 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
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)
. 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 +
Staphylococcus, Streptococcus, Clostridium, Bacillus
Gram - Enteric, respiratory and others
Acid-fast Mycobacterium
Wall-less Mycoplasma
Unusual Obligate intracellular
Rickettsia, Chlamydia
G+ G- AF WL IC
Bacteria
Bacteria
Gram+ Gram-Acid Fast
IntraCellular
WallLess
Cocci Rod CocciRodSpiral
Staph.
Strep.Non-spore Spore
StraightCurve
+O2 -O2+/-O2 Other
S. a.S. e.S. s.
ABPnVir
Fil Rod
A.i. C.d.L. m.
M.t.
+O2 -O2
B.a.B.c.
C.b.C.t.C.p.C.d.
TreponemaBorreliaLeptospira
NeisseriaMoraxella
P.a. Enteric Bact.
Resp.Zoo
GUBordetella.H. influenzaeLegionella
YersiniaPasteurellaBrucellaFrancisellaStreptobacillus
H. ducreyiGardnerellaCalymmatobacterium
RickettsiaCoxiellaErlichiaChlamydia
Mycoplasma
VibrioCampylobacterHelicobacter
Gram negative
Straight rods Curved rods
Lactose+ Lactose-
Citrate+ Citrate- H2S+ H2S-
Klebsiella E. coli Salmonella Shigella
Campy blood agar42oC+ 25oC-
Campylobacter
TCBS agarYellowOxidase+
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
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.
Further Reading
Questions