Professional Documents
Culture Documents
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
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)
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
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
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.
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
Laboratory examination 1- Microscopy 2- Culture techniques 3- Biochemical reactions 4- Serological identification: 5- Molecular biology techniques 6- Bacteriophage typing
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)
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
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
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
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
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
Gram
Acid-fast
Mycobacterium Mycoplasma
G+ GAF WL IC
Wall-less
Rickettsia, Chlamydia
Bacteria
Gram+ GramAcid Fast Intra Wall CellularLess Mycoplasma Rickettsia Coxiella Erlichia Chlamydia
Rod
Spiral
Rod
Cocci
M.t.
A B Pn Vir
P.a.
Gram negative
Straight rods
Lactose+ Lactose-
Curved rods
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
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
Molecular Diagnosis
Ribotyping
(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
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
To identify point sources for hospital and community-based outbreaks To predict virulence
Culture confirmation
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
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