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