THEME A: GENERAL INTRODUCTION TO MICROBIOLOGY

1. Indicate where microorganisms fit into classification of life on earth

• On phylogenetic tree microorganisms fall under 3 domains 
1. Eukarya
All eukaryotes ▪ Fungi
▪ Protozoa
▪ Algae
All prokaryotes 2. Bacteria
3. Archaea Not a veterinary pathogen
• Viruses are non-living and acellular (not composed of cells)
• Most microorganisms are unicellular, and some are multicellular
• Microorganisms that can cause disease in animals or humans: pathogenic

2. Describe the characteristics that distinguish the prokaryotes and be able to subdivide them
into different domains
• Prokaryotes: Differentiate based on 16S ribosomal sequence

BACTERIA ARCHAEA
• Many vet pathogens • No vet pathogens
• Rigid cell walls containing • Cell walls with no peptidoglycan
peptidoglycan
▪ Both are exclusively microbial and prokaryotic
▪ Do not undergo true reproduction
• TAXONOMY: is the practice and science of orderly classification of organisms into
hierarchical units called taxa
▪ 3 interrelated parts to taxonomy
• Identification
• Nomenclature
• Classification
▪ Why is taxonomy NB in microbiology?
I. Permits accurate identification of organisms
II. Provides precise names that permit efficient communication
III. Groups similar organisms in a way that allows predictions to be
made and hypotheses to be framed with reasonable confidence
regarding members of the same group

3. Tabulate the differences between pro-and eukaryotes

Characteristics PROKARYOTES EUKARYOTES

Size 0.2-2.0m diameter 10-100m diameter
Smaller Much larger
Nucleus No true nucleus True nucleus
nuclear membrane and nucleoli Consisting of nuclear membrane
absent and nucleoli
genetic material not Nuclear membrane
separated from separates genetic
cytoplasm material from cytoplasm
 Membrane enclosed Absent Present
organelles Including:
Lysosomes
Golgi complex
ER
Mitochondria
Chloroplasts
Flagella Simple: Complex:
Consists of 2 protein building Consists of multiple
blocks microtubules
Glycocalyx Present as a slime layer or a Present in some cells that lack a
capsule cell wall
Cell wall Usually present Not present in animal cells
Chemically complex Chemically simple
Contains peptidoglycan Chitin or cellulose
Plasma membrane No carbohydrates and generally Carbohydrates and sterols
lack sterols present
Serve as receptors
Cytoplasm No cytoskeleton Cytoskeleton
No cytoplasmic streaming Cytoplasmic streaming
Ribosomes Smaller size (70S) Larger size (80S)
Smaller size (70S) present in
organelles
Location: Location:
Dispersed throughout Dispersed throughout
cytoplasm cytoplasm
Also attached to ER
Chromosomes Single circular chromosome Multiple linear chromosomes
No histones With histones
Cell division Binary fission Mitosis
Sexual recombination None Meiosis
(transfer of DNA only)

THEME B: GENERAL INTRODUCTION TO BACTERIOLOGY (INCLUDING MYCOPLASMAS)

B1: INTRODUCTION, TERMINOLOGY, STAINING AND VISUALIZATION OF BACTERIA

1. List the different microscopic methods by which bacteria can be visualised and
• LIGHT MICROSCOPES
i. Brightfield
− Standard
− Produces image on bright background
− Requires staining
ii. Darkfield
− Increases contrast without staining
− Produces bright image on dark background
− Useful for viewing live specimens
 iii. Phase contrast
− Uses refraction and interference caused by structures in the
specimen
− To create high-resolution and high-contrast images without staining
− Useful for viewing live specimens, and structures like organelles and
endospores
• ELECTRON MICROSCOPES
i. Transmission electron microscope
− Used for demonstrating viruses in biological material
− And for identifying ultrastructural details of bacterial, fungal and
mammalian cells
ii. Scanning electron microscope
− Used for demonstrating 3D structure of a microorganism

2. The different staining techniques used to demonstrate and identify bacteria and
mycoplasmas
to study bacterial morphology one has to make a bacterial smear on a microscopic
slide, stain the smear an look under the microscope
can obtain a lot of information, inexpensively and quickly

• Wet preparation
o Some microorganisms can be visualised microscopically without the use of
staining techniques
o Can be examined by:
▪ Phase contrast
▪ Dark-field microscopy
▪ High-dry objective of the light microscope with the condenser
slightly lowered
o Fungal material can be viewed in this way

• Smears from tissue lesions

o Hold specimen with clean forceps
o With clean scalpel scrape deep into material
o Small amount of scraping placed on clean microscopic plate
o Another clean slide is used in a scissors action to create thin slide
o Needs to be stained

• Liquid specimens
o Little of a sample placed on slide with sterile swab
o Contents on swab smeared over the surface of the slide with the aim to
have thick and thin areas of the specimen present
o Allow to dry before further processing

• Fixing smears
o Two methods:
Attachment of material to a I. Heat fixation
microscope slide in such a manner
that it is less likely to wash away in
the course of staining and washing.
 • Done by quickly passing the slide smear side up through a
Bunsen flame
• do not overheat
II. Chemical fixation (methanol)
• With absolute ethanol
• Like Giemsa stain
o Reasons for fixing smears:
▪ Killing bacteria (not always endospores) and making them
permeable to the stain
▪ To ensure that the material is firmly fixed to the slide

• Staining smears
o Fixed smears placed on staining rack over a sink
o Staining solutions are flooded over entire solution and left for appropriate
amount of time
o Between each staining reagent the smear is washed under gently running
tap
o Staining methods:
Basic/ simple stains Differential staining
− Contain chromophore − Divides bacteria into
groups (giving the stain its different groups
colour) they can bind to cells

Examples: Examples:
− Methylene blue 1. Gram stain
− Basic fuchsin 2. Ziehl-Neelson
− Crystal violet
− Safranin
− Malachite green
− Eosin

−Stains generally used:

• Simple stain: methylene blue, iodine
• Gram stain:
GRAM NEGATIVE GRAM POSITIVE.
Colony is dried on a slide (heat/dry)

Primary stain: crystal violet

Mordant: iodine fix

Fixation stabilizes crystal violet stain
Complex with primary dye formed
Alcohol dehydrates
peptidoglycan of
Decolourizer: alcohol de-stain gram+
Making them less
permeable to crystal
Counter stain: safranin stain violet than -
Gram positive bacteria are already
Both + and – absorb the counter stain
stained
but only visible in -
 • Ziehl-Neelson or Acid-fast stain

o Stains and staining methods used for smears:

− Staining for morphology and other characteristics:
• GRAM
• Ziehl-Neelson, acid-fast bacteria
− E.g. Mycobacterium
• Stamp/ Giemsa
− Special staining techniques:
• Malachite green for spores
• Indian ink for capsule staining
− Negative stain
• Flagella staining
o Following features can be visualised using different stains:
− CAPSULE:
• Clearly visible under light microscope when negative stains or
special capsule stains are used
• Indian ink: stains organism and surrounding and not the capsule
− ENDOSPORES:
• Does not stain with gram stain
• Malachite green usually used
• Malachite green stain:
− Hot malachite green
Endospores stain green and rest of cell
stain pink
− Water
− Safranin
• Spores are very heat resistant and can survive boiling for hours
• For successful staining of endospores:
− Temperature of stain must be near boiling
− Stain cannot drain out quickly
• Autoclaves must be used for sterilisation
• The position of the spore within the organism is used for
identification purposes
• Under favourable conditions the spore will germinate:
− Gives rise to single vegetative cell
− i.e. sporulation is not a means of replication but rather a
survival mechanism

3. Staining and visualization of poor gram stainers, Chlamydiales and Rickettsiales

o Poor gram stain bacteria
− Acid-fast bacteria
• Examples:
− Mycobacterium
− Nocardia
− Corynebacteria
 • Difficult to characterize using standard microbiological techniques
(like gram stain)
• Gram positive bacteria
− But in addition to peptidoglycan the outer membrane or
envelope of the acid-fast cell wall contains large amounts of
glycolipids
− Especially: mycolic acid that resists staining unless heat is
applied to allow stain to penetrate
• Most common staining technique used: Ziehl-Neelson stain
− Acid-fast bacilli: bright red
− Background: blue
ACID-FAST NON-ACID-FAST
Primary stain: hot carbol fuchsin

Decolourizer: acid alcohol

Counterstain: methylene blue

− No cell wall (Mycoplasma)

• Examples:
Only 2 genera of pathogenic
significance − Mycoplasma
In class Mollicutes − Ureaplasma

• Mycoplasmas: smallest free-living prokaryotic cells capable of self-

replication
• Pleomorphic:
− From spherical (0.3-0.9m) to filamentous (up to 1m)
• They cannot synthesize peptidoglycan (or its precursors) so they do
not have rigid cell walls
− They have flexible triple layered outer cell membranes
− This flexibility allows them to pass through bacterial
membrane filters of pore sizes from 0.22m- 0.45m
• Susceptible to:
− Desiccation
− Heat
− Detergents
− Disinfectants
• Resistant to:
− Antibiotics such as penicillin which interfere with the
synthesis of bacterial cell wall
• They require enriched medium to grow
• Cholesterol required for growth of spp. of genus Mycoplasma
• They are dependant on host cell for essential nutrients that they
cannot produce
Intracellular bacteria:
Visualised with:
1. Microscopy:
• Spinal fluids
(meningitis)
• Sputum (TB) • Host specific
• Sensitivity • Facultative anaerobes
poor • Do not replicate in environment
2. Serological • Fastidious in growth requirements when cultured in vitro
identification
• Detecting
o Rickettsiales
antibodies
• Poor gram stainers RAPID DIAGNOSIS WITHOUT CULTURE
against
• Some ATP WHEN AND WHY?
antigen in
1. Organism grows poorly
serum o Chlamydiales
2. Organism can’t be cultures
3. PCR • Require ATP
3. Speed is of essence
• Molecular
detection of
DNA regions

B2: MORPHOLOGY OF BACTERIA

1. name and draw the different morphological forms

− microscopically bacteria have different shapes:
Sphere-shaped Cocci
Occurring in chains: streptococci
Grapelike clusters: staphylococci
Rod-shaped Bacilli
Spiral rods Spirilla

Bacteria usually gain part of their name from their shape

2. describe the use of different morphological characteristics in the identification of bacteria

− most organisms are grouped according to their genotypic and phenotypic
(morphology, metabolism, physiology, cell chemistry and motility) characteristics

B3: LABORATOY IDENTIFICATION OF BACTERIA

1. list the different techniques used to identify bacteria and mycoplasmas in the laboratory
• colony morphology
Examination of smears • gram method
• motility (not fixed stain)
cultural • presence or absence of haemolysis on blood agar
▪ ability to grow on selective media
• biochemical characteristics
i. catalase and oxidase test
ii. oxidation-fermentation:
▪ differentiate organisms that use carbohydrates:
− aerobically (oxidation)
− anaerobically (fermentation)
− and those that do not use carbohydrates (non fermenters)
• phage typing
• molecular techniques
• immunological techniques:
i. serotyping
 ii. IFA
iii. Serology

CRITERIA FOR [RESUMPTIVE IDENTIFICATON OF NACTERIAL PATHOGENS:

1) Colonial morphology and colour
2) Presence or absence of haemolysis on blood agar
3) Appearance when stained by gram method
4) Motillity
5) Ability to grow on MacConkey agar
6) Reaction in oxidation-fermentation tsest
7) Reactions in catalase and oxidase tests

B4: BASIC CONCEPTS OF GROWTH AND GROWTH REQUIREMENTS

1. Briefly describe the fundamentals of the methods to obtain pure cultures of bacteria and
mycoplasmas and the use thereof
− Pure culture: consist of a population of cells all derived from a single parent cell,
therefor a single type of bacteria
• Need a pure culture in order to characterize an individual species
• Not necessarily genetically pure
For obtaining − Samples are streaked on incubation plates that are incubated
isolated colonies • Not uncommon for cultures to contain more than one bacterial species
on agar medium • Mixed cultures are purified by streaking single colonies on different plates
• Plate inoculation technique:
1) With sterile inoculation loop a sample of the specimen (the inoculum)
in spread over a small area at the edge of the plate, the well (1)
2) The inoculum is spread from the well sequentially over 3 contiguous
areas of the plate (2, 3, 4)
3) The loop is sterilized by flaming before inoculation of each area
4) Results in reduction of number of bacterial numbers @ each step
5) In area 4 discrete bacterial colonies can be recognized after
incubation
6) Loop should be sterilized by flaming after inoculation to ensure no
pathogens survive on the loop

− Identification involves observation of these colonies for:

• Size
• Colour hemolysis reaction
• Texture
− Media can be enriched to permit the growth of certain bacteria
− Differential and selective media can be used for this purpose
− Colonies are gram stained
− Individual cells viewed under microscope
− Bacterium is identified (genus and or species level) using biochemistry tests using
the pure culture colonies

2. List the most important types of media used for the growth of bacteria and mycoplasmas
− Blood agar: enrich media
 • Supports growth of most pathogens
• Appropriate for routine primary isolation
• Allows recognition of bacterial haemolysin production

− MacConkey: selective media

• Used for particular organisms
• Selective medium containing bile salts
• Especially useful for isolation of enterobacteria and some other gram-
negative bacteria
• Allows differentiation of lactose fermenters (pink) and non-lactose
fermenters

− Nutrient agar
• Basic medium
• Non-fastidious bacteria can grow
• Suitable for demonstrating colonial morphology and pigment production
• Also used for viable counting method

− Selenity broth, Rappaport-Vassiliadis broth

• Selective enrichment media
• Isolation of salmonellae from samples containing other gram negative
enteric organisms

− Edwards medium
• Blood agar-based selective medium
• Used for isolation and recognition of streptococci

− Chocolate agar
• Heat treated blood agar
• Supplies special growth requirements (X and V factors)
• For isolation of haemophilus spp.
• Culture of Taylorella equigenitalis

− Brilliant green
• Indicator medium
• For presumptive ID of Salmonella spp. (red)
− Etc.

3. Define aerobic, anaerobic, and microaerophilic environment for the growth of bacteria and
mycoplasmas
− Bacteria preference for oxygen divide them into 4 major groups namely:
OBLIGATE AEROBE: I. Aerobes
An organism that can grow • Able to grow in the presence of atmospheric oxygen
ONLY in the presence of II. Anaerobes
oxygen • Able to grow in the absence of free or atmospheric oxygen

OBLIGATE ANAEROBE:
An organism growing only in
an anaerobic environment, not
a microaerophilic environment,
a CO2 incubator or air
 • Facultative aerobe:
▪ Organism normally anaerobic but can also grow in the
presence of oxygen

III. Facultative anaerobes

• Organism that can grow well both in the absence of oxygen and in a
Illustration of growth of bacteria with level of oxygen equivalent to that in an air atmosphere (21%)
varying response to oxygen • Some capable of growing aerobically by respiring oxygen and
Surface directly exposed to environment: anaerobically by fermentation
aerobic • Other have strict fermentative type of metabolisms and do not
respire with oxygen
IV. Microaerophiles
• Requiring oxygen but at a level lower than atmospheric

Obligate Facultative Aerotolerant Obligate Microaerophile

aerobe anaerobe anaerobe anaerobe

4. Draw the growth curve of bacteria and describe the different stages
− Bacteria replicate by binary fission
− Bacterial requirements for growth:
• Oxygen (or absence)
• Energy
• Nutrients
• Optimal temperature
• Optimal pH
− Growth curve
B5: PHYSIOLOGY OF BACTERIA

1. describe and draw the different structures of the bacterial cell

2. define the functions of the different structures

STRUCTURE CHEMICAL COMPOSITION FUNCTION

Cell wall Gram positive: − protects the cell

− peptidoglycan o from mechanical
− teichoic acid damage
Gram negative: o osmotic lysis
− lipopolysaccharide − gives shape
(LPS) o peptidoglycan gives
− protein rigidity
− phospholipid
Outer membrane − − protects cell against some
antibiotics
o only present in gram
negative cells
Cell membrane − phospholipid − regulates movement of material
bilayer into and out of cell
− contains enzymes important for
cellular respiration
o oxidative phosphorylation

Cytoplasm − − contains DNA, ribosomes, and

organic compounds required to
carry out life processes

Chromosome − DNA − carries genetic material

inherited from past generation

Plasmid − − contains some genes obtained

through genetic recombination

Capsule and slime layer − usually − protects cell (desiccation)

polysaccharide o situated outside cell
− polypeptide in envelope
Bacillus anthracis − assists in attaching cell to other
surfaces
Endospore − − protects cell against harsh
environmental conditions
o heat
o drought
Pilus (pili) − protein called pilin − assist cell in attaching to other
surfaces
− NB in genetic recombination
Flagellum − Protein called − Moves the cell
flagellin

Cell membrane+ cell wall= cell envelope

3. Describe and illustrate the bacterial cell wall and be able to distinguish between the Gram-
positive and Gram-negative bacteria.
• Cell wall= tough, rigid walls of bacteria
• Protect them from mechanical damage and osmotic lysis
• Peptidoglycan
− Polymer unique to prokaryotes
− Imparts rigidity to the cell wall
• Based on colour when stained with gram method we get two major groups
− Get colour reaction due to differences in composition of cell wall

GRAM POSITIVE GRAM NEGATIVE

Relatively thick, uniform cell wall More complex structure
Outer membrane:
Peptidoglycan • Protein-containing, asymmetrical
Teichoic acid lipid bilayer
• Inner surface:
o Resembles cytoplasmic
membrane
• Outer surface:
o Composed of
Lipopolysaccharide molecules
(LPS)
o LPS is responsible for endotoxic
effect
o Only released after cell lysis
o LPS structure:
I. Lipid A
• Where endotoxic
activity resides
II. Core structure
III. O-specific side chain
• Polysaccharides
• Stimulate
antibody
production
Periplasmic space (containing relative small
amount of peptidoglycan)
 • Mycoplasmas: important group of bacteria without cell walls

4. Describe the different bacterial appendages and their functions.

• Flagella
− Bacteria with flagella are motile Treat moving bacteria differently to
− Many gram negatives stagnant bacteria
• Pilli (fimbriae)
− Composed of pilin
− Attached to cell walls
− Most common in gram negatives
− Functions in gram negatives:
1. Adhesion to host tissue (adhesin at tip)
2. Unique type of pilus (F type: sex or conjugation pilus)
o Conjugation: transfer of DNA from donor cells (male) to
female recipient cells
− In gram positives:
1. Also adhesion to host tissue

5. Describe the formation of bacterial endospores and their functions illustrated with
examples.
• Endospores: dormant highly resistant bodies formed by some bacteria to ensure
survival during adverse environmental conditions
• Number of gram- positive bacteria
• Normally produced during unfavourable conditions and protects the organisms
against:
− Lack of food
− Severe temperatures
− UV radiation
− Chemical disinfectants
− Desiccation
• Two genera of pathogenic bacteria that form endospores:
− Bacillus
Does not stain with GRAM stain
− Clostridium
• Because of resistance and impermeability of the spore coat, special staining
procedures which employ heat are required to demonstrate endospores
• Hard shell encloses the spore and it can remain viable for years
• No signs of biochemical activity apparent

6. Describe the occurrence and function of bacterial capsules.

• External layer: capsule or slime layer
− Capsule: well defined
− Slime layer: not defined
• Outside cell envelope
• Found in bacteria that can synthesise extracellular polymeric material (GLYCOCALYX)
• Usually consists of polysaccharides
 • In vitro: often lost
• In vivo: protective
• Prevents phagocytosis
• Often associated with virulence

7. Describe the occurrence and formation of abnormal forms of bacteria.

??????

8. Describe the origin and characteristics of bacterial exo- and endotoxins.

• Bacterial toxins traditionally seen as one of two types
• Types differ in structure and mode of action

PROPERTY EXOTOXINS ENDOTOXINS

Organisms producing Almost all gram positive Almost all gram negative
Some gram negative
Location in cell Extracellular: excreted into Bound within cell wall:
medium released on death of
bacterium
Chemical nature Polypeptides Lipopolysaccharide complex
Stability Unstable Relatively stable
Denature above 60 Withstand several
And with UV light hours above 60
Toxicity Among most powerful Weak
toxins known Can be fatal in large
doses
Effect on tissue Highly specific Nonspecific
Some act as Systemic effects
neurotoxins Local site reactions
Cardiac muscle
toxins
Fever production Little or no fever Rapid rise in temperature to
high fever
Antigenicity Strong: Weak:
Stimulates antibody Recovery from
production and disease often does
immunity not result in
immunity
Toxoid conversion and By treatment with heat or Cannot be converted to
use chemicals toxoid
Vaccine production Used to immunise Cannot be used to
against toxin immunise
Examples Botulism Salmonellosis
Gas gangrene Tularemia
Tetanus Endotoxic shock
Diphtheria [Link]
Staphylococcus food
poisoning
Cholera
Enterotoxins
 Plague

• Endotoxins
o Lipopolysaccharide (LPS) of the outer leaflet of the outer membrane of gram
negative bacteria
o LPS composed of 3 parts:
1. Lipid A: hydrophobic glycolipid
• Where endotoxicity resides
2. Core oligosaccharide
Hydrophilic polysaccharide
3. O-polysaccharide (O-antigen)
o LPS released when bacterial cell walls are damaged by:
− The complement system
− Phagocytes
− Antimicrobial drugs
o So endotoxins are released during lysis or growth
o Biological and immunological properties of cell wall are due to LPS
o Causes:
− Shock
− Fever
− Intra-vascular coagulation
− Death

• Exotoxins
o Produced by both gram positive and gram-negative bacteria
o Toxins are highly antigenic and can induce the production of protective
antitoxins
o Can be ingested (causing food poisoning: with systemic effects)
o More commonly produced within the host: exert local or systemic effect
o Pharmacology/ pathological physiology unique for each exotoxin
o Can be neutralized by antibody
o Can form toxoid: weakened toxin
o Associated with effect on central nervous system

B6: BACTERIAL GENETICS

1. Explain the central dogma of molecular biology and define basic terms such as mutation,
plasmid, episome and transposons.
o Central dogma:
▪ Shows the flow of genetic information

transcription translation
DNA RNA Protein

o Mutation:
▪ An inheritable change in the base sequence of the DNA of an organism
▪ A stable inheritable alteration of the genome
o Plasmids: (episomes)
 ▪ Extrachromosomal genetic elements of bacteria that are capable of
autonomous replication
▪ carrying a variety of determinants that may permit survival in an adverse
environment or successful competition with other microorganisms of the
same or different species
o Bacteriophage:
▪ Virus that infects a bacterial cell, sometimes ringing about its lysis
o Integration:
▪ The process by which a DNA molecule becomes incorporated into another
genome
o Genetic recombination:
▪ Recombination occurs when sequences of DNA from two separate sources
are integrated
▪ Induces an unexpected inheritable change due to the introduction of new
genetic material from a different cell
▪ New genetic material may be introduced by:
− Conjugation
− Transduction
− Transformation
o Transposons:
▪ Transposable genetic elements: segments of DNA that are capable to move
from one location to another
− Random movement
− Cannot self-replicate
− Transposition mediated by site-specific recombination
− May be accompanied by duplication
▪ Genetic elements that can move as a single unit from one replicon
(chromosome, plasmid, or bacteriophage) to another
▪ They do not possess an origin of replication and only replicate as the host
replicon into which they are inserted replicates

2. Describe the possible causes and results of bacterial mutations.

o Bacterium carrying a mutation= mutant
o Mutations arising in bacterial population can either be:
▪ Spontaneous
▪ Induced

TYPES OF MUTATIONS
o Base-substitution or point mutation producing
▪ Silent mutations
− Code for the same amino acid
▪ Mis-sense mutations
− Code for a different amino-acid
▪ Nonsense mutations
− Code for a stop resulting in a truncated protein
o Micro insertions or microdeletions of base pairs
▪ Frameshift mutations
 − +1 or -1 frameshifts
o Reversions
− Back mutations reversing point mutations (base-pair substituents)
o Deletions of multiple base pairs
o insertions during recombination resulting in errors
o Translocation of DNA segments within the genome
o Inversions
− Inverted orientation of a segment of DNA within the chromosome

3. Describe the different methods of bacterial recombination

o Genetic variation may occur due to:
▪ Mutation (where change occurs in the nucleotide sequence of a gene)
▪ Recombination (where new groups of genes are introduced into the
genome)

TYPES OF RECOMBINATION
o Conjugation
▪ Gene transfer from a donor to a recipient by direct physical contact between
cells
▪ Male (donor) bacteria: F+
− Source of donor DNA, the fertility (F) plasmid/ factor
− Form F pilus, through which genetic material can be transferred
▪ Female (recipient) bacteria: F-
− Lacks F factor
▪ Both participate
o Transduction
▪ DNA acquired either from
− the original bacteria chromosome
− or plasmid in in a previously infected bacterial cell
▪ can be in incorporated into phage nucleic acid and transferred by progeny of
the phage to susceptible recipient cells in a process called transduction
o Transformation
▪ Involves the transfer of free or naked DNA
▪ containing genes on a segment of chromosomal or plasmid DNA from a
lysed donor bacterium to a competent recipient

Characteristic TRANSFORMATION CONJUGATION TRANSDUCTION

Method of DNA Across cell wall and Through a By a virus
transfer cell membrane of conjugation bridge
recipient between two cells
Plasmid transfer Yes Yes Not likely
Chromosome No Sometimes No
transfer
Antibiotic Yes Yes Sometimes
resistance acquired
Theme C

C1: Prerequisites for pathogenicity

1. Define disease, infectivity, bacteraemia, pathogen, pathogenicity, opportunistic pathogen, septicaemia,

septic shock, toxaemia, toxigenicity, virulence. May have infectious/non-infectious etiology
• Disease: state of the body by which functions of part/whole body are disturbed.
• Infectivity: capacity of the organism to become established in tissues of the host → Infection ≠ always
disease
Ability to colonize, penetrate the tissues,
survive the host’s defenses and multiply • Bacteraemia: the presence of bacteria in the bloodstream
and disseminate within the host. Frank pathogens: pathogens
that can cause disease in some
• Pathogen: an organism with the potential to produce a disease. individuals with a healthy
• Pathogenicity: the capacity of an organism to produce a disease. immune system
Variation in this • Opportunistic pathogen: normally
capacity is described harmless commensals their normal
Usually only cause disease in
in terms of virulence. habitats → cause disease when they gain
immune-compromised host
access to other sites/tissues.
• Septicaemia: presence of bacteria and their toxic product in the bloodstream.
• Septic shock: characterised by inadequate tissue perfusion, following bacteraemia most freq. with Gram-
negative enteric bacilli.
• Toxaemia: presence of bacterial toxins in the bloodstream.
• Toxigenicity: capacity of certain organisms to produce toxins
• Virulence: Measure of the degree of pathogenicity
2. Apply “Koch’s postulates” in the understanding of the disease process.
• Koch’s postulates: Criteria that must be met to identify the causative agent of an infectious disease
beyond doubt.
• It stipulates that the pathogen must be:
o present in every case of the disease (+ absent in healthy individuals)
o isolated from diseased host + grown in pure culture
o The specific disease must be reproduced when pure culture of pathogen inoculated into healthy
susceptible host.
o recoverable from the experimentally infected host.
3. Explain how bacteria and mycoplasmas interact with the host and the environment to cause disease.
• Animals infected with bacteria soon after birth + remain infected their whole lives
• Infections don’t always cause disease
• Host-pathogen relationship kept in fine balance
o Disease results only when infectious agent cannot be kept in check
o + combination of damage done to host
o + host’s adaptive mechanisms result in infectious disease
• Environment, influences both, host, and pathogen
o + therefore, also plays role in both infection and disease outcome
• For a bacterium to produce disease after transmission, it must be able to:
o Colonise/adhere to host
o Invade the host
o Be able to multiply and leave the host
o Evade/overcome the host’s defences
o Cause essential tissue damage
 Bacteria • Infective
• Virulence factors
• Evasion of immune response
• Tenacity
• Immunity
• Genetic factors
• Management factors • Age
• Pollutants • Co-infections
• Climate • Gender
• Nutrition Environment Host • Anatomy

4. List the sources of potentially pathogenic bacteria.

• Carrier animals
o Most common source of pathogenic bacteria
o Clinically affected animals, those incubating the disease, animals carrying the agent but are
clinically unaffected and animals recovering from a disease for a period.
o Animals that carry bacterial infections may either be:
➢ Active carriers – those actively shedding the agent
➢ Passive carriers – those through which the agent just passes
➢ Latent carriers – organism being harboured but not shed (can become active carriers in
certain circumstances)
• Environment
o Bacteria may also originate from an environmental source
o i.e. drinking water/ feed
• Normal microflora (resident microbiota)
o Bacteria originating from the host
o Bacteria on body surfaces, s.a. skin, nasal + oral cavities, gastrointestinal, lower urogenital and
upper respiratory tracts.
o Viruses, bacteria, and fungi that live in/upon the normal animal without producing a disease
o Normal flora has potential/opportunistic pathogens
o Relationship between host and microflora in healthy individuals = mutualistic
o Some bacteria can cause disease if they gain deep tissues by trauma, surgery/other underlying
primary factors
o One organism can cause severe/fatal disease in young animals but can have no effect on healthy
older animals (eg. Rhodococcus equi.)
5. Describe how bacteria colonise, invade, and exit the body.
• Portals of entry include:
o Inhalation and infection via respiratory tract
o Ingestion and infection via alimentary tract
o Inoculation/infection via skin/mucous membranes
o Via genital tract as result of coitus/iatrogenic
o Transplacental infection
o Via umbilicus
o Via udder
• Colonisation
o Before colonisation → contact with host cell
o Eg. In respiratory tract bacteria would penetrate the mucous layer with aid of chemotaxis +
flagella/breakdown mucous by production of enzymes
o Other factors involved in docking (not adhesion):
➢ Hydrophobic interactions
➢ Electrostatic attractions
➢ Atomic + molecular vibrations resulting from fluctuating dipoles of similar freq.
 ➢ Brownian movement
➢ Recruitment + trapping by biofilm polymers interacting with bacterial glycocalyx (capsule)
o Once in contact → make use of adhesions to bind specifically to receptors on the host’s cell =
“anchoring”
o Need to adhere to prevent washout, invade the host/to produce exotoxins
o Microbial adhesions include:
➢ Fimbriae
➢ Membrane structures → outer membrane proteins normally aid specific bacteria-host cell
adhesions
➢ Capsules (glycocalyx) and slime
o Factors that may interfere with cell adherence of anchoring include:
➢ Isolated adhesin/receptor molecules
➢ Adhesin/receptor analogs
➢ Enzymes + chemicals that specifically destroy adhesins/receptors
➢ Antibodies directed against surface components, esp. adhesions, of bacteria
• Tissue invasion
o Bacterial entry into the host can either be directly by cell destruction/by phagocytosis
o Not all bacteria/fungi need colonise/invade the body to affect the body processes
o Some organisms grow outside of the body/within the body cavities with toxin penetration
o May be aided by extracellular enzymes produced by bacteria
➢ Results in cell destruction/
➢ By “ invasins” that allow cell membrane actin rearrangement and thus stimulating
engulfment of the bacteria.
o Bacterial proteins that may be involved in cell invasion:
➢ Hyaluronidase
▪ produced by streptococci, staphylococci, + clostridia
▪ attacks interstitial cement (“ground substance”) of connective tissue by
depolymerizing hyaluronic acid.
➢ Collagenase
▪ Produced by clostridium
▪ Breaks down collagen, framework of muscles,
▪ which facilitates gas gangrene due to these organisms
➢ Neuraminidase
▪ Breaks down neuraminic acid, an intercellular cement of epithelial cells of the
intestinal mucosa
➢ Streptokinase + staphylokinase
▪ Produced by streptococci + staphylococci, respectively
▪ Kinase enzymes convert inactive plasminogen to plasmin that digests fibrin and
prevents clotting of the blood.
▪ The relative absence of fibrin in spreading bacterial lesions allows more rapid
diffusion of the infectious bacteria
➢ Bacterial enzymes that result in cell lysis
▪ Usually act on the animal cell membrane by insertion into the membrane (forming
pore that results in cell lysis)
▪ / by enzymatic attack on phospholipids that destabilizes the membrane
▪ May be referred to as lecithinases/phospholipases
▪ And if they lyse red blood cells = hemolysins
➢ Bacterial protein enzymes/toxins with adenylate cyclase activity
▪ Increases levels of cyclic AMP
▪ Resulting in disruption of cell permeability
▪ E.g. oedema factor of bacillus anthracis
o Mycoplasmas = parasitic in nature + can attach to specific cells without killing the cells + thus
their infection process + progress can go undetected.
 o Mycoplasmas can grow in tissue fluids + can grow inside any living tissue cell without killing the
cells
• Transport and localisation of bacteria within the host
o Direct spread
➢ Localised spread of microbes from site of infection depends on host’s immune response.
➢ Radial spread = as organism multiplies
➢ Bacteria tend to produce spreading factors s.a. collagenase, elastase, hyaluronidase +
protease.
➢ Bacteria can move by gravitation (e.g. from upper respiratory tract to lungs)
➢ Presence of fluid (e.g. inflammatory oedemas) may also allow movement of microbes
➢ Scratching, licking, and biting will also spread organisms
➢ Basement membrane of epithelia acts as filter for microbes but can be broken down by
inflammation/epithelial cell damage allowing subepithelial invasion.
➢ Once microbes have penetrated barrier, they are exposed to host defences incl. tissue fluids,
lymph, and phagocytic cells.
o Spread via lymphatics
➢ Most bacteria that penetrate epithelial barriers will come into contact with + transported in
lymph to lymph nodes
➢ Where they are filtered and under normal circumstances destroyed
➢ Much of immune stimulation occurs in lymph nodes
➢ Filtering by the lymph nodes decreased when:
▪ Lymph flow is high
▪ Large numbers of infective particles + phagocytes fail to ingest infective particles
➢ Factors will allow systemic spread of micro-organisms
➢ Some bacteria will even divide in the lymph nodes
➢ Inflammation tends to impede lymph flow, containing infections with abscesses being noted.
o Spread via the blood (haematogenous
➢ Bacteria enter the blood either directly from the site of entry (e.g. lungs/from lymphatics)
➢ Bacteria and/or toxins are carried in the blood either freely in the plasma/leucocyte
associated
➢ Carriage rarely by the erythrocytes/platelets
➢ Blood microbe transport allows bacteria to establish themselves elsewhere in the body
o Spread via other systems
➢ Cerebral spinal fluid
▪ Once blood-brain barrier has been breached, movement of organisms is with the
flow of cerebral spinal fluid
▪ Movement across the blood-brain barrier requires its damage through eg.
Inflammation
▪ Young animals = thinner membrane barrier → meningitis + encephalitis is common
➢ Pleural and peritoneal cavities
▪ If organisms enter these cavities, spread is rapid
▪ Spread is held in check by phagocytes in omentum, peritoneum + pleura
▪ Rich lymphatic drainage from these areas
➢ Nerves
▪ Not common route of spread for bacteria and fungi
➢ Blood-foetal junction
▪ Between foetus + dam there is blood-foetal barrier that prevents transmission of
MOS from dam to foetus.
▪ Direct passage of organisms to foetus is rare
▪ In late pregnancy = placenta thins and little loss of maternal blood to foetus
▪ If dam is bacteraemic = thin placenta may allow bacteria to gain entry to the foetus
▪ Most MOS enter foetus in either 2 mechanisms:
 Some bacteria localize in ❖ Localisation in maternal blood vessels in placenta, where it multiplies and
placenta and then produce produce toxins which locally interrupt integrity of junction and thus infect
toxins that affect foetus fetus.
❖ Localisation in placenta and growth across placental junction
▪ Bacteria that induce high fever can indirectly cause abortions
➢ Tissue localisation
▪ Occurs when microbes are taken in the RE system of an organ/
▪ Are able to transverse capillary endothelium
▪ Crossing endothelial barriers aided by previous organ damage
❖ Eg. Endocarditis/by bacterial/fungal toxins and/or enzymes
▪ Common sites for mc localisation = brain, joints, kidney, testes, udder, skin, placenta
▪ Bacterial growth in these areas is often due to:
❖ Specific bacterial nutritional requirements
❖ Oxygen tension of tissue
❖ Specific host receptors
❖ Route of infection
❖ Blood perfusion

C2: Mechanisms of tissue damage by bacteria

1. Describe how bacteria and mycoplasmas cause direct damage to tissues.

• Some microbes will directly cause death to cell within which they multiply
o E.g. by cell lysis/by toxin production
• Damage also caused by mechanical damage
o i.e. a vasculitis will result in ischaemia + tissue necrosis of tissues supplied by blood vessel.
2. Explain the effects of endotoxins on the host animal.
• Endotoxins form part of outer layer of bacterial cell of Gram neg. bacteria
• Small amounts released during bacterial growth, but for the most part they remain associated with cell
wall until death + disintegration of bacteria.
• Endotoxins = responsible for most of toxic effects of Gram neg. → other cell wall components may also
cause disease
• Gram neg. may also produce very potent
exotoxins
• Properties:
o LPS consisting of 3 portions:
➢ Lipid A – embedded in bacterial cell
wall and is responsible for most of
toxigenic effects and is usually
found in all Gram neg. bacteria
➢ Core oligosaccharide – specific to certain genera
➢ Long chain oligosaccharide – which confers smoothness to bacteria, is antigenic + bacterium
specific.
▪ It also causes complement activation
▪ Called the O-antigen
▪ Used to type members of the Enterobacteriaceae in lab.
o Heat stable + does not form a toxoid → can be destroyed by oxidizing agents
o Generally, less potent than exotoxins, req. higher doses to be toxic
o Potency of toxins vary between Gram-neg. bacteria
o Induces different pharmacologic + immunological changes at low and high concentrations.
• Role of endotoxin for bacteria:
o Permeability barrier that only allows penetration of low molecular weight, hydrophilic molecules
o It impedes destruction of the bacterial cells by serum components + phagocytic cells
o Plays important role as surface structure in interaction of pathogen with its host.
Eg. LPS may be involved in adherence, / resistance
to phagocytosis, / antigenic shifts that determine
the course + outcome of an infection.
• Adaptations to endotoxin:
o Normally present at high levels within normal GIT.
o There they are contained by intestinal mucosal barrier
o If they do happen to invade body, they are either removed by the mononuclear-macrophage
system/by specific antibodies
o Always small amounts of endotoxin that penetrate mucosal barrier, which stimulate host’s
immune system + thus help host keep Gram-neg. bacteria in check.
o Only when endotoxins are in very high levels within body that endotoxaemia occurs.
➢ Either as sequelae of Gram-neg. bacterial infection/ by increased permeability of intestinal
mucosa
o Other sources = Gram-neg. infections of mammary gland, urogenital + respiratory tracts.
o Certain vaccines may be high in endotoxin + thus may cause transient decrease in animal prod.
o Endotoxin level = increased as direct result of antimicrobial actions of antimicrobial agents
o Spp. susceptibility to endotoxin varies with equi., bov., + rabbits being most susceptible
➢ Endotoxins target different organs in different animal spp.
➢ E.g. In horse it is intestinal tract + in humans + dogs it is the lungs + kidneys
• Pathogenesis:
o Regardless of bacterial source, all endotoxins produce same range of biological effects in animal
host.
➢ Lipid A in bloodstream → lipopolysaccharide binding protein (LBP) → produced by
hepatocytes as part of acute phase response → forms complex → cells of IS produce variety
of pro-inflammatory mediators → incl. cytokinesis, tumour necrosis factor (TNF), Interleukin
1 (IL-1) etc.
➢ In cells containing phospholipase A (i.e. Neutrophils, monocytes, platelets) platelet
activating factor (PAF) also released → increased vascular permeability
➢ Activation of blood-clotting cascade → coagulation, thrombosis, acute disseminated
intravascular coagulation → depletes platelets + various clotting factors → internal bleeding
➢ Plasmin activation → fibrinolysis + haemorrhaging
➢ Activation of complement alternative pathway + kinin activation → releases bradykinins +
other vasoactive peptides → hypotension
➢ Net effect → induce inflammation, intravascular coagulation, haemorrhage + shock
➢ Other effects → stimulation of stress hormones, induction of acute phase proteins,
endogenous opioids, alterations in carbohydrate, amino acid + lipid homeostasis + changes
in certain metal ions.
➢ LPS → B cell mitogen → stimulate polyclonal differentiation + multiplication of B-cells +
secretion of immunoglobulins, esp. IgG + IgM.
• Pathophysiology:
o Fever
o Depression of reticuloendothelial system
o Neutropoenia
o Lymphocytes
o Haemodynamic effects
o Haemostasis
o Glucose haemostasis
o Reduced blood supply to tissues
o Cardiac failure
o Endotoxin induced abortions.
o Gastrointestinal tract
o Swartzmann reaction
o Death => pulmonary oedema, generalised anoxia secondary to respiratory insufficiency, cardiac
arrythymias, DIC, cerebral anoxia/combination factors
3. Explain the effects of exotoxins on the host animal.
• Soluble proteins secreted by mainly Gram-pos. bacteria but also some Gram-neg. bacteria, which have
toxic effects on animal
• Properties:
o Produced in log phase of growth of bacteria (extracellular toxins)/on bacterial lysis (protoplasmic
toxins)
o Potency varies, but generally more potent than endotoxins
o Mode of action varies greatly dependent on type of toxin
o Heat labile + can be inactivated by formalin to form toxoids
o Very antigenic
o Mode of action like that of enzymes
• Cell-to-cell signalling systems:
o Why large numbers of bacteria are more likely to express virulence factors than small numbers
o Many bacteria have ability to sense population density
o + communicate with each other using polypeptides in case of Gram-pos. bacteria
o + small molecules known as autoinducers in case of Gram-neg. bacteria
o The more bacteria present, the more of these molecules are produced until they reach critical
concentration,
o Where they are able to increase the expression of certain genes,
o Some of these being virulence factors (e.g. Exotoxin A in case of Pseudomonas aeruginosa)
• Exotoxins acting locally
o Many will exert effect where they are produced
o Known as cytotoxins (cell destructive)/ haemolysins
• Enterotoxins
o Exotoxins that specifically affect small intestine
o Causing changes in intestinal permeability that lead to diarrhoea
o Certain enteric bacteria produce enterotoxin which able to affect electrolyte + fluid transport of
enteric cells
➢ Results in osmotic diarrhoea + electrolyte imbalances
o Other enteric bacteria will penetrate intestinal epithelium
➢ Produce cytotoxin which will cause cellular destruction resulting in diarrhoea
• Systemic effects of exotoxins
o Nerve toxins e.g. tetanus + botulinum toxins
o Toxins that lyse cells e.g. lecthinase, haemolysin + leukotoxin
o Toxins that block protein synthesis e.g. exotoxin A of Pseudomonas aeruginosa
o Toxins that stimulate immune system
➢ Anthrax lethal toxin stimulates macrophages to produce cytokines (IL-1 + TNF)
➢ + oxidizing radicles that induce systemic shock and death
• Clinical uses of toxins
o Toxoid vaccines
o Toxins that bind to membranes can be used as medicine carriers
4. Explain the general effects on the host by enzymes produced by bacteria.
• Degrade connective tissue barriers and promote spread of infection
• Produce substances that are not directly toxic to host,
• But aid in all aspects of virulence of bacteria (e.g. coagulase, neuramidase, DNAse lipase, etc.)
5. Describe how bacteria and mycoplasmas can cause indirect damage of the host animal by induction of
inflammatory responses.
• Acute inflammation attracts and activates polymorphonuclear leucocytes + macrophages
• Normal host cells = secondarily injured by proteolytic enzymes + free radicals released by phagocytes
• Abscesses freq. result
• Small number of bacteria survive and replicate inside host cells
• Incl. Mycobacteria, Rickettsiae, Chlamydiae, Listeria monocytogenes, Brucella + certain Mycoplasma
• Most of these do not produce toxins/directly damage cells themselves
• Injury results from host immune responses which injure tissues secondarily
o as they attempt to eliminate microbial invaders
o takes form of chronic inflammation
• Mycoplasmas can reside extra-cellularly bit also enter + grow inside any living tissue cell without killing
the cells
o Reason why Mycoplasma infection can remain + progress undetected for extended periods of
time
o Once attached to host cell, they begin to compete for nutrients inside the host cells.
o As nutrients deplete = host cells may malfunction/even change normal functioning of cell
o Can even cause RNA and DNA mutations of host cells + have been linked to certain cancers
o Can also invade host cells which evade immune system, esp. white blood cells
o Inside white blood cells, they can travel throughout the body and even cross the blood-brain
barrier, + into central nervous system and spinal fluid.
C3: Microbial interactions with other microorganisms

1. Define viral-bacterial synergism, and using various examples describe how it plays a role in the
pathogenesis of disease in animals.
• Many bacteria on their own cannot cause primary disease + not only require susceptible host
• , but other microbes may interact with bacteria to cause infectious disease
• Bacterial infections of RT = common after primary viral infection
• E.g. “shipping fever” (respiratory disease of cattle)
o Respiratory virus infections, augmented by pathophysiologic effects of stress, alter susceptibility
of cattle to Pasteurella spp. that are normally present in URT
• Viruses aid bacterial virulence by number of mechanisms
o Immunosuppressive/can damage macrophage + neutrophil functions in RT
➢ Not only will this allow invasion by external bacteria,
➢ But could also activate pre-existing chronic infections
o Induce inflammatory responses which damage RT epithelium
o Alteration of surface properties of cells, allowing bacterial adhesin with growth of bacterial
microcolonies
o Release of iron, which enhances bacterial growth + colonisation
o Conversely, proteases secreted by bacteria may activate influenza infectivity by proteolytic
cleavage of haemagglutinin
2. Describe how bacteria interact with other microorganisms to produce disease.
• Many Gram-neg. anaerobic infections = polymicrobial
o e.g. ovine footrot
➢ constant wetting of claws of sheep allow invasion by diphtheroid + cocci in interdigital
epidermis
➢ bacteria generate favourable conditions for Fusobacterium necrophorum from soil to invade
➢ Growth factors produced by these bacteria + tissue damage produced, allows Dichelobacter
nodosus to invade + cause extensive tissue damage by protease production.
• Other microbial spp. such as protozoa, fungi + helminths may act synergistically with bacteria
o High parasitic loads lower host resistance to viruses and bacteria
o Damage to intestinal mucosa by intestinal protozoa + worms can allow either absorption of
endotoxins/ invasion of body by Gram-pos./Gram-neg. bacteria.
3. Describe polymicrobial infections by means of appropriate examples.
• Anaerobic infections = typically endogenous in origin
• Once mucosal + skin barriers have been broken, anaerobic bacteria likely to localise in sites of low
oxygen tension.
• Common sites of anaerobic infections include:
o Lung,
o Deep sited wounds,
o Abscesses,
o Necrotic tissue,
o Abdominal organs,
o Areas with poor blood supply.
• Most anaerobic infections = polymicrobial
o Containing both obligate + facultative anaerobes
o Because often develop following tissue contamination + aerobic bacteria use up oxygen in
tissues allowing anaerobes to multiply.
o Some bacteria act as helpers for anaerobes
• Are able to produce potent toxins causing severe disease + also can allow other bacteria to survive
• Immunosuppression/immunodeficiencies can predispose to anaerobic infections
• Obligate anaerobes incl. spp: Bacteriodes, Clostridia, Dichelobacter, Fusobacterium, Peptoniphilus +
Prevotella.
Theme D: Rickettsiales

D1: Introduction, terminology, staining and visualization

1. Define the terminology used to describe different life cycle stages.

• Reproduction stages takes place in both ixodid ticks (vectors) + vertebrate animals
• Vector: organism which contributes to spreading of pathogen in population of hosts.
• Ehrlichia spp. + Anaplasma spp. – able to cause persistent infection in vertebrate hosts which allows
them to be reservoirs of infection
• Ixodid ticks able to transmit Ehrlichia spp. + Anaplasma spp. transstadially rather than transovarially
• Transstadially: from larvae to nymphs and from nymphs to adults
• Transovarially: from eggs to new generation of ticks
• Uninfected ticks become infected y feeding on previously infected animals
• + at next stage can transmit infectious agent to other mammals.
• When ticks feed on hosts:
o Pathogens first enter the tick midgut epithelium, where their primary replication takes place
o Then pathogens move to tick salivary glands to invade epithelial cells
o In salivary epithelial cells, bacteria undergo second replication cycle + enter salivary gland
secretion when tick feeds on next vertebrate host.
• Basic epidemiology
o Members of Rickettsiales = labile outside host cells
o Animal hosts + arthropod vectors = reservoirs for most organisms in order
o In certain spp. silent cycle involving ticks + small wild mammals = possible source of infection for
domestic animals.
o Some organisms produce latent infections.
o In arthropods, rickettsiae replicate in epithelial cells of gut before spreading to other organs,
incl. salivary glands + ovaries
➢ Where further replication may occur.
o Organisms (Rickettsia rickettsii) maintained in tick population by transovarial transmission
o Transmission of E. canis + A. phagocytophilum occurs in ticks by transstadial routes
o Majority of members of Rickettsiales transmitted by Arthropods.
2. Describe staining of blood and organ smears for visualization of Ehrlichia and Anaplasma.
• Anaplasma spp.
o Stain bluish-purple with Romanowsky methods
o Romanowsky method: prototypical staining technique that was forerunner of several distinct
but similar methods, incl. Giemsa, Jenner, Wright, Field + Leishman stains
➢ Used to differentiate cells in pathologic specimens
➢ Group of eosin-methylene blue stains generally used for blood smears, protozoa + bacteria.
o Best stained with Romanowsky stains
➢ Organisms develop dark blue appearance
➢ As opposed to violet colour of eukaryotic cell nucleus
o Stain poorly Gimenez method but easily with acridine orange
• Ehrlichia spp.
o Best stained with Romanowsky stains
➢ Organisms develop dark blue appearance
➢ As opposed to violet colour of host cell nucleus
o Stain poorly with Gimenez method, but easily with acridine orange
• Blood/ tissue smears stained by Giemsa technique
o Used to demonstrate the morphology of members of family Anaplasmataceae.
o Giemsa technique: used in cytogenetics + for histopathological diagnosis of malaria + some other
spirochete + protozoan blood parasites.
➢ Classical blood film stain for peripheral blood smears + bone marrow specimens
 ▪ Erythrocytes stain pink, platelets show light pale pink, lymphocyte cytoplasm stains
sky blue, monocyte cytoplasm stains pale blue, + leukocyte nuclear chromatin stains
magenta
➢ Also, differential stain
▪ Can be used to study adherence of pathogenic bacteria to human cells
▪ Differentially stains human + bacterial cells purple and pink respectively
➢ Also used to visualize chromosomes
▪ Specific for phosphate groups of DNA
▪ + attaches itself to regions of DNA where there are high amounts of adenine-
thymine bonding
▪ Used in Giemsa banding, commonly called G-banding, to stain chromosomes
▪ + often used to create an ideogram
▪ Can identify chromosomal aberrations such as translocations + rearrangements
• Macchiavello stains: differential staining procedure for richettsiae,
o In which fixed smear is stained with basic fuchsin, differentiated with 0.5% solution of citric acid
o + counterstained with 1% solution of methylene blue,
o Rickettsiae stain red, and tissue cells stain blue.
• Gimenez staining technique: uses biological stains to detect + identify bacterial infections in tissue
samples
o Valuable for detecting certain slow growing/fastidious bacteria
o Basic fuchsin stain in aqueous solution with phenol + ethanol colours many bacteria (Gram-pos.
+ Gram-neg.) red, magenta/pink.
o Malachite green counterstain gives blue-green background cast to surrounding tissue.

D2: Morphology

1. Describe the morphology of Ehrlichia and Anaplasma spp.

• Order Rickettsiales
o Order of Alphaproteobacteria containing families Rickettsiaceae + Anaplasmataceae
o Small, obligate intracellular, Gram-neg. coccobacillary bacteria
o Parasitizing host cells as source of ATP
o May be pathogenic for humans + other animals
o Generally, replicate within erythrocytes, leucocytes/endothelial cells.
o Small, non-motile + pleomorphic
• Family Rickettsiaceae
o Obligate intracellular bacteria
o Grow freely in cytoplasm of eukaryotic cells
o Cell walls like that of Gram-neg. bacteria
o Cell walls contain peptidoglycan
o Cultured in specific cell lines/fertile eggs
o Tropism or vascular endothelium
o Vector-borne bacteria that incl. emerging infectious agents a.w.a. old pathogens that are re-
emerging among homeless people in urban areas
o No flagella/endospores
o Can be parasitic in man + other vertebrates, causing diseases
o Some confined to invertebrate host as pathogens/symbionts.
• Family Anaplasmatceae
o E.g. Anaplasma + Ehrlichia
o Vector-borne parasitic bacteria
o Transmitted by ixodid ticks
o Obligate intracellular parasites
o Found exclusively within membrane-bound vacuoles in host cell cytoplasm
o Outer membranes similar to Gram-neg. bacteria → lack obvious peptidoglycan layer
 o Almost all organisms multiply in both vertebrates + invertebrates
o No cell walls
o Difficult to culture in vitro
o Tropism for cells of haemopoietic origin
o Anaplasma
➢ occur in membrane-bound vacuoles in cytoplasm of cells of hematopoietic origin
➢ forming inclusions that contain variable numbers of organisms
➢ infect predominantly erythrocytes, neutrophils, / mononuclear phagocytes in vivo
➢ approx. 0.3 um in diameter but may vary considerably in size + shape
➢ inclusions (morulae) range in size up to 4 um in diameter
➢ weakly gram-neg.
o Ehrlichia
➢ same as anaplasma
➢ but individual organisms = approx. 0.5 um in diameter, but size may vary

D3: Genetics of Ehrlichia and Anaplasma spp.

1. Define the basic methods of replication in Ehrlichia and Anaplasma spp.

• Replication of family Rickettsiaceae
o Rickettsia bacteria = obligate intracellular pathogens
o Dependent on entry, growth, + replication within cytoplasm of eukaryotic host cell
o Host cells lyse + releases rickettsial progeny to initiate new infection cycle
o Infection generally does not result in complete shutdown of host machinery
o Preferentially infect endothelial cells lining small blood vessels by parasite-induced phagocytosis
o Once in host cell → bacteria lyse phagosome membrane with phospholipase
o + get into cytoplasm where they replicate
o Mode of exit from host cell may be cell lysis, budding/extrusion depending on species
o Normally multiply by transverse binary fission
o Under poor nutritional conditions → cease dividing + grow into long filamentous forms
o Which subsequently undergo rapid + multiple division into typical short rod forms
➢ When fresh nutrients is added
o 6 – 10 daughter cells will form within host cell before cell ruptures + releases them.
 • Replication of Anaplasma + Ehrlichia
o Preferentially infect leukocytes
o Enter cell by phagocytosis +
o Once in host cell they inhibit phagolysosome fusion
o Organisms grow within membrane-bound phagosome +
o Released by lysis of cell
o Inclusion body containing organisms = morulae

D4: Systematic division of Ehrlichia and Anaplasma spp.

1. Give the family, genus, and species names for Ehrlichia and Anaplasma spp. of veterinary importance.
Theme F: Chlamydiales

F1: Staining and visualization

1. Explain the methods used to visualize and identify chlamydial species in the laboratory.
• Direct microscopy
o Suitable for detection of organisms in smears/tissue sections containing moderate numbers of
organisms
o Suitable chemical staining procedures incl.
➢ Ziehl-Neelsen
➢ Geimsa
➢ Modified Machiavello
➢ Casteneda methods
o Methylene blue-stained smears can be examined by dark-field microscopy
o Immunofluorescent staining improves sensitivity of detection of chlamydial EBs in smears
➢ But usually does not identify spp. involved
• Serological procedures
o Several are available for detection of antibodies to chlamydiae
 o Incl. complement fixation, ELISA + indirect immunofluorescence
o Although complement fixation test is most widely recognized serological test,
➢ it is time consuming + only moderately sensitive
• Chlamydia can be isolated
o Either in embryonated eggs, following inoculation into yolk sac,
o /in number of continuous cell lines
o Cell lines vary in susceptibility to infection by different chlamydial spp.
o Sensitivity of isolation procedure increased by use of non-replicating cells
• PCR techniques
o Have been developed for detection of chlamydial DNA in samples
o Real-time PCR + micro-array also available for both detection + spp. identification

F2: Morphology

1. Describe the morphology of chlamydial species.

• Contain DNA, RNA + ribosomes + make their own proteins + nucleic acids
• Now considered to be true bacteria
• Possess inner + outer membrane similar to Gram-neg. bacteria
• LPS, but no peptidoglycan layer
• Although they synthesize most of metabolic intermediates → unable to make own ATP = “energy
parasites”
• Roughly spherical + measures between 0.3 + 1 um in diameter,
• According to stage of development
• Both small + large cell types contain complete cell walls
o Similar to cell walls of Gram-neg. bacteria
• Under cell wall lies separate cytoplasmic membrane made up of large amounts of lipid
• DNA occurs as irregular mass in cytoplasm
• No nuclear membrane
• Ribosomes can be seen throughout cytoplasm
• Cells contain no capsule/flagella

F3: Genetics of chlamydial species

1. Define the terminology, (elementary body, reticulated form) and basic strategies used for replication by
chlamydial species.
• Elementary body (EB)
o Small (0.3 – o.4 um)
o Infectious form
o Rigid outer membrane → extensively cross-linked by disulphide bonds
➢ Resistant to harsh environmental conditions when chlamydia outside their eukaryotic host
cells.
o Bind to receptors on host cells + initiate infection
o Most infect columnar epithelial cells but soe can also infect macrophages
o Form contains DNA genome with molecular weight of 66 x 107
o In add. contains RNA polymerase responsible for transcription of DNA genome
➢ After entry into host cell cytoplasm + initiation of growth cycle
o Ribosomes + ribosomal subunits found in these bodies
• Reticulate body (RB)
o Non-infectious
o Intracellular form
o Metabolically active replicating form
o Possesses fragile membrane lacking extensive disulphide bonds
o Basically, structure in which
➢ chlamydial genome transcribed into RNA, proteins synthesized, + DNA replicated
 o divides by binary fission to form particles which develop into new infectious EB progeny
➢ after synthesis of outer cell wall

• Basic strategies used for replication

o Undergo unique biphasic developmental cycle
o Characterized by 2 morphological forms
➢ EB
➢ RB
➢ Also, intermediate forms
o Starts with endocytosis of EBs by eukaryotic cells
➢ Residing within small intracytoplasmic inclusions
o EBs transformed into larger RBs + replication then occurs by binary fission
o As RBs multiply inclusion rapidly fills + expands in size
o After 24-48h → RBs transform back into metabolically inactive, infectious EBs
➢ Which are then released following host cell rupture + go on to invade neighbouring cells
o Central replication = similar to growth cycle of other organotrophic bacteria
➢ Chlamydiae growth and cell division restricted to intracellular environment
o Beginning + end of cycle = make chlamydiae unique
o EB characterized by resistance to physical + chem. factors in EC environm.
➢ + lack of metabolic activity
➢ Resistance consequence of rigidity of cell envelope = osmotically stable + poorly permeable
➢ Also, consequence of greatly reduced surface area of EB vs RB
➢ EB adapted for prolonged EC survival
▪ Which in case of animal pathogens may mean many months outside natural host.
Some features of rickettsiae and chlamydiae:

• Rickettsiae and chlamydiae are classified as bacteria.

o They can be seen with the light microscope. All contain DNA and RNA and are susceptible to various
antibiotics.
• They are small, pleomorphic coccobacillary forms existing as obligate intracellular parasites.
• They have cell walls, including two membrane systems that resemble those of Gram-negative bacteria.
o They stain reasonably well with Giemsa, Castaneda, Giménez, and Macchiavello stains, but poorly with
Gram’s stain.
• They possess many of the metabolic functions of bacteria but require exogenous cofactors from animal cells.
o Rickettsiae can generate their own energy, but they also depend on their host for some energy,
o whereas chlamydiae are more dependent on energy from their host cells.
o Rickettsiae can exchange their internal ADP for ATP from the host cells.
o They can also lose their viability in storage due to the loss of their intracellular ATP pool and several
coenzymes.
• Most rickettsiae and chlamydiae grow readily in the yolk sac of embryonated eggs and in cell cultures.

There are some fundamental differences between the rickettsiae and chlamydiae:

• Rickettsiae have cytochromes and their metabolic reactions are aerobic. Chlamydiae do not have cytochromes
and their metabolic reactions are essentially anaerobic.
• Rickettsiae multiply by simple binary fission, whereas chlamydiae have a singular development cycle.

Theme I

I1: Fungal invasion of tissues

1. Differentiate between disease in animals as a result of fungal invasion to that caused by mycotoxins.
• Mycosis = fungal infection
• Mycotoxicosis = disease caused by intake of fungal toxins
• Mycoses caused by either true fungal pathogen that cause primary infections/opportunistic fungal
pathogens => secondary infections
• Opportunistic fungal pathogens = part of normal flora
• Primary fungal pathogen
o A true primary fungal pathogen can invade and grow in a healthy, non-compromised host.
o They usually come from and environmental reservoir and infect animals that have been exposed
to a large dose of spores or that are immunologically naïve to the fungus.
o For example, the dermatophytes.
➢ The dermatophytes most encountered are the Trichophyton species and Microsporum
species.
o potential host more susceptible to fungal invasion when natural physical barriers broken down,
➢ for example skin abrasions open the way for invasion of dermatophytes and development of
dermatophytosis.
➢ Dermatophytes also produce keratolytic substances that break down keratinocytes.
o Suppression or deficiency in the cell mediated immune response (CMI) leads to increased
susceptibility to fungal invasion.
o Increased progesterone levels increase the susceptibility of female animals to candidiasis (the
disease caused by Candida species).
o Candida species are yeasts (single celled fungi) that often form part of the normal flora on
mucosal surfaces.
• Opportunistic (Secondary) fungal infections
o Opportunistic fungal pathogens take advantage of hosts that are debilitated or
immunocompromised due to a primary condition to cause infection.
 o
These fungi may be from an environmental reservoir for example Cryptococcus or Aspergillus.
o
They may also be commensals (normal flora) in healthy hosts for example Candida species or
Malassezia species.
o However, when it comes to fungi
➢ the distinction between primary pathogens and opportunists is a grey area.
➢ Sometimes opportunists appear to cause disease in healthy hosts.
• Predisposing factors
o A very important predisposing factor for opportunistic fungal infections is antibiotic therapy.
o The overuse of broad-spectrum antimicrobials kills off the bacterial normal flora that keep
yeasts under control and so pave the way for yeast overgrowth.
o A good example is otitis externa in dogs due to Malassezia pachydermatis.
o Long term corticosteroid treatment is another factor as it suppresses the immune system.
o Diabetes also suppresses the immune system. Catheters can also carry fungi into the blood
stream.
2. Define dermatophytosis and subcutaneous and systemic mycoses.
• Mycoses can be divided into 3 types:
o Cutaneous/superficial
➢ Fungi that only grow at room temperature = superficial mycoses
➢ Cannot survive at body temperature
➢ Dermatophytosis = superficial mycosis of skin involving keratinised layers of epidermis

• Primary mycoses tend to be cutaneous and superficial. Fungal spores on blankets,

halters, brushes or saddles contaminate the skin surface or hair follicles.
• The spores germinate and hyphae penetrate the keratinised layers of the skin.
• An example is dermatophytosis (ringworm)
o which is an infection of the superficial layers of the skin by fungi of the
genera Microsporum, Trichophyton or Epidermophyton.

o Subcutaneous
➢ Involves deeper layers of skin + other subcutaneous structures
➢ E.g. dermis + lymphatics
• Subcutaneous infection results when fungal spores are inoculated into the deeper layers
of tissue below the skin.
• Lymphocutaneous sporotrichosis (Sporothrix schenckii) develops when the spores are
inoculated into the skin by a thorn or wood splinter.
• Opportunistic fungi can also sometimes cause such infections.
o Systemic
➢ In warm-blooded animals → involves internal organs
➢ + caused by fungi that can grow at host’s body temp.
➢ Grow at 37 ⁰C + higher

• The respiratory tract can also be a portal of entry as some spores are so small that they can be inhaled and
settle in the lungs or airsacs.
• The spores attach to and are taken up by epithelial cells and phagocytes.
• A good example is aspergillosis in birds. The intestine can also serve as a portal of entry as spores can be
ingested with mouldy feed such as hay, peanuts and parrot seeds.
• Once again aspergillosis is a good example.
• The spores germinate inside the phagosome and escape into the cytosol of the host cell.
• Such infections can develop from localised to systemic when fungi erode blood vessels to gain access to the
blood stream.
I2: Mechanisms of tissue damage by fungi

1. Describe how fungi cause damage to the host’s tissue.

• Fungi that cause mycoses can damage tissues in several ways.
• They can produce enzymes (proteins) such as
o keratinases,
o elastases
o and collagenases
o that digest keratinocytes, collagen, or blood vessel endothelium.
• The primary purpose is often to free the nutrients inside the cells for absorption by the fungus.
• Fungal growth is invasive producing masses or balls of hyphae that lead to physical obstruction of
bronchi and arteries as well as subsequent necrosis of tissues.
• These fungal masses can also displace or destroy vital structures.
• The infection elicits an inflammatory response that also causes tissue damage.
• The response is pyogranulomatous.
• Hypersensitivity can develop to particular fungal spores.
• Virulence factors
o Fungi produce virulence factors that enhance their ability to cause disease and the severity of
disease they can cause.
o A fungus can have a number of virulence factors that work in combination.
o These virulence factors evolved to
➢ aid the survival of fungi in the environment,
➢ to resist ingestion by amoebae or to survive inside amoebae,
➢ but many are also useful when the fungus has to survive in an animal.
o Virulence factors are proteins, polysaccharides, phospholipases, and structures (capsules) that
help the fungus to colonise and survive the immune system attacks of the host.
o Some of these factors are:
➢ The ability to adhere to host cells by means of cell wall glycoproteins,
➢ capsules that resist phagocytosis or aid in survival inside the host cell.
o Fungi are primarily saprophytes that digest their substrate by secreting enzymes such as
➢ keratinase,
➢ elastase
➢ and collagenase.
o They then absorb the nutrients that are released from damaged cells.
o The ability to secrete these enzymes is a virulence factor
➢ enables dermatophytes to colonise and survive on skin.
o Another virulence factor is the ability to grow at higher temperatures including the fever range.
➢ Opportunistic fungi can acquire this virulence factor.
➢ Non-pathogenic fungi and fungi that cause superficial infections do not have this factor and
so cannot survive in a warm-blooded animal.
➢ The ability to exhibit thermal dimorphism improves the chances of survival of a fungus in a
warm-blooded host.
➢ A fungus is thermally dimorphic when it can grow as a mould at 20 to 30°C, and as a yeast at
37°C.
➢ The yeast form can survive and reproduce at temperatures up to 40°C, low oxygen tension
and low nutrient availability.
➢ It more resistant to killing by phagocytes as well.
2. Describe how fungal toxins cause tissue damage in the host animal.
• Some filamentous fungi produce mycotoxins at certain developmental stages in response to
environmental stressors as they grow on a substrate such as peanuts or maize.
• Mycotoxins serve various purposes in the environment such as suppressing competing microorganisms.
• However, they can cause mycotoxicosis in animals.
• The portal of entry for these toxins is ingestion.
 • When ingested in large amounts, they can have cytotoxic effects in animals such as
o cellular disorders,
o oxidative stress,
o inhibition of translation of mRNA,
o DNA damage
o and apoptosis.
• Many are carcinogenic.
• The most well-known of these toxins is aflatoxin, produced by Aspergillus flavus, that causes liver
damage. Other producers of mycotoxins are Fusarium and Penicillium.

Theme M

Normal Flora

1. Explain the presence and role of normal flora in different body tissues (Nose, ear, conjunctiva, oral cavity,
upper respiratory tract, gastrointestinal tract, urogenital tract, and skin) in relation to disease diagnosis.
• In utero all newborn mammals are free of microorganisms.
• acquire normal flora from
o their mothers, starting during the birth process,
o other members of the family and social grouping,
o as well as from the environment.
• Normal flora are found on all the body surfaces exposed to the external environment including the
o skin
o conjunctiva
o outer ear canal
o nose
o oral cavity
o upper respiratory tract
o lower urethra
o intestine, especially in the large intestine
• Normal Flora of the skin
o The density and composition of the normal flora of the skin vary with anatomical locale.
o The majority of skin microorganisms are found in the most superficial layers of the epidermis
and the upper parts of the hair follicles.
o Skin bacteria or their metabolites may protect against colonization by dermatophytic fungi.
o Qualitatively, the bacteria on the skin near any body orifice may be similar to those in the orifice.
• Normal Flora of the respiratory tract.
o The nostrils are always heavily colonized.
o The healthy sinuses, in contrast are sterile.
o A large number of bacterial species colonize the upper respiratory tract (nasopharynx).
o The lower respiratory tract (trachea, bronchi, and pulmonary tissues)
➢ virtually free of microorganisms,
➢ mainly because of the efficient cleansing action of the ciliated epithelium which lines the
tract.
➢ Any bacteria reaching the lower respiratory tract are swept upward by the action of the
mucociliary blanket that lines the bronchi, to be removed subsequently by coughing,
sneezing, swallowing, etc.
➢ If the respiratory tract epithelium becomes damaged, as in bronchitis or viral pneumonia,
▪ the individual may become susceptible to infection by pathogens descending from
the nasopharynx.
• Normal Flora of the oral cavity.
o The presence of nutrients, epithelial debris, and secretions makes the mouth a favorable habitat
for a great variety of bacteria.
 o Oral bacteria include streptococci, lactobacilli, staphylococci and corynebacteria, with a great
number of anaerobes, especially bacteroides.
• Normal Flora of the conjunctiva.
o A variety of bacteria may be cultivated from the normal conjunctiva but the number of
organisms is usually small.
o The conjunctiva is kept moist and healthy by the continuous secretions from the lachrymal
glands.
o Blinking wipes the conjunctiva every few seconds mechanically washing away foreign objects
including bacteria.
o Lachrymal secretions (tears) also contain bactericidal substances including lysozyme.
o There is little or no opportunity for microorganisms to colonize the conjunctiva
➢ without special mechanisms to attach to the epithelial surfaces
➢ and some ability to withstand attack by lysozyme.
• Normal flora of the urogenital tract
o Urine is normally sterile, and since the urinary tract is flushed with urine every few hours
➢ microorganisms have problems gaining access and becoming established.
o The flora of the anterior urethra, as indicated principally by urine cultures,
➢ suggests that the area may be inhabited by a relatively limited, consistent normal flora
consisting of staphylococci,
o Enterococcus faecalis, and some alpha-hemolytic streptococci.
o In addition, some enteric bacteria (e.g. E. coli, Proteus) and corynebacteria, may occasionally be
found at the anterior urethra.
➢ which are probably contaminants from the skin, vulva, or rectum
• Normal Flora of the gastrointestinal tract.
o The bacterial flora of the GI tract of animals has been studied more extensively than that of any
other site.
o The composition of the GI flora differs between various animal species, and within individual
animal species.
2. Explain the potential of normal flora to become pathogenic organisms.
• BENEFICIAL EFFECTS OF THE NORMAL FLORA
o The normal flora synthesizes and excrete vitamins more than their own needs,
➢ which can be absorbed as nutrients by their host.
➢ For example, in humans, enteric bacteria secrete Vitamin K and Vitamin B12, and lactic acid
bacteria produce certain B-vitamins.
o The normal flora prevents colonization by pathogens by competing for attachment sites or for
essential nutrients.
➢ This is thought to be their most important beneficial effect,
➢ which has been demonstrated in the oral cavity, the intestine, the skin, and the vaginal
epithelium.
➢ In some experiments, germ-free animals can be infected by 10 Salmonella bacteria,
➢ while the infectious dose for conventional animals is near 106 cells.
o The normal flora may antagonize other bacteria through the production of substances which
inhibit or kill non-indigenous species.
➢ The intestinal bacteria produce a variety of substances ranging from relatively nonspecific
fatty acids
➢ and peroxides to highly specific bacteriocins, which inhibit or kill other bacteria.
o The normal flora stimulates the development of certain tissues,
➢ i.e., the caecum and certain lymphatic tissues (Peyer's patches) in the GI tract.
o The normal flora stimulates the production of natural antibodies.
➢ Since the normal flora behave as antigens in an animal,
▪ they induce an immunological response, in particular, an antibody-mediated
immune (AMI) response.
 ▪ Low levels of antibodies produced against components of the normal flora are
known to cross react with certain related pathogens,
▪ and thereby prevent infection or invasion.
• HARMFUL EFFECTS OF THE NORMAL FLORA
o 1. Bacterial synergism between a member of the normal flora and a potential pathogen.
➢ This means that one organism is helping another to grow or survive.
➢ There are examples of a member of the normal flora supplying a vitamin or some other
growth factor that a pathogen needs in order to grow.
➢ This is called cross-feeding between microbes.
➢ Another example of synergism occurs during treatment of "staph-protected infections"
▪ when a penicillin-resistant staphylococcus that is a component of the normal flora
shares its drug resistance with pathogens that are otherwise susceptible to the drug.
o Competition for nutrients.
➢ Bacteria in the gastrointestinal tract may get to some of our utilizable nutrients before we
are able to absorb them.
➢ Germ-free animals grow more rapidly and efficiently than conventional animals.
➢ The explanation and rationale for incorporating antibiotics into the food of swine, cows and
poultry is that they grow faster (and thereby get to market earlier).
o The normal flora may be agents of disease.
➢ Members of the normal flora may cause endogenous disease if they reach a site or
tissue where they cannot be restricted or tolerated by the host defences.
➢ Many of the normal flora are opportunistic pathogens,
➢ and if they gain access to a compromised tissue from which they can invade, disease
may result.

Definitions:

• The mixture of micro-organisms regularly found at any anatomical site in healthy animals is referred to as the
“Normal Flora” or “Indigenous Microbiota” or simply as commensals.
o Bacteria are by far the most numerous microbial components of the normal flora, along with a few
fungal species and protozoa.
o Normal flora are extremely abundant in terms of absolute numbers and may comprise more (but
much smaller) cells than the host organism itself.
• Symbiosis: “living together”.
o Normal flora are symbionts,
➢ meaning they maintain a long-term interaction with the host, whereby at least one (or both) of
them is benefitting from the interaction
o Depending on the kind and degree of interaction the following types of symbioses are differentiated:
➢ Commensalism: The microbes neither hurt, harm, nor benefit the carrying host.
➢ Mutualism: Interactions between the microbe and the carrying host in which both organisms’
benefit.
➢ Parasitism: Interaction in which the micro-organism benefits and the host is harmed.
• Resident microbiota: Are a part of the normal microbiota throughout life
• Transient microbiota: Members of the normal flora that are present for only a few days, weeks, or months
before disappearing
• Microbial antagonism: A process by which pathogenic microorganisms are inhibited by normal flora from
colonizing healthy organisms
SAMPLING TECNIQUES

GOOD SAMPLING TECHNIQUES

• Labs are the practitioners link to accurate diagnosis and they facilitate cost-effective
treatment and control of diseases in livestock and companion animals
• Accurate diagnosis is required if a zoonosis (e.g. rabies) is suspected
o To ensure safety of pet owners and vets
• Most important aspects of specimen collection for subsequent laboratory examination are:
o Correct selection
o Methods of collection and submission
o Requesting appropriate tests
• 3 components contributing to meaningful diagnosis of disease:
I. The clinician: Clinician
▪ Collects specimen
▪ Interprets result
II. The diagnostic assay use:
▪ Choice and suitability
III. Lab technologist:
▪ Quality control of assay
▪ Interprets the test result

Interaction and communication between Lab technologist Diagnostic assay

all components are vital

SPECIMEN SELECTION

1. Selecting the animal

The best animal for diagnostic purposes is an untreated, acutely ill animal
• Live animals:
− Samples:
▪ Blood
▪ Faeces
▪ Urine
− If clinical signs seen in more than one animal of a group:
▪ Take specimens from all/ most of the clinically affected animals
▪ As well as some healthy animals in contact
− If the groups are large
▪ Sufficient to take samples from 10% of the clinically ill animals
− Many labs will handle multiple specimens as one case and will not charge
extra or offer discount
• Dead animals:
− Good quality samples:
▪ If case allows it and owner permits it, it is preferrable to sacrifice an
animal in extremis At the point of death
▪ Second best: recently dead animal
 − Post-mortem invasion of tissues by intestinal and other bacteria can be
rapid, particularly in warm weather
− Ruminants, obese animals, long-haired animals:
▪ Tend to cool slower
▪ So autolysis (especially of abdominal viscera) will proceed quickly
− This hampers with:
▪ identifying the causative agent
▪ histopathology where cell cultures will be lost

2. Choosing tissues for submission

• Live animals
− Specimens should be taken from easily accessible affected sites as early as
possible after onset of clinical signs
− Examples:
▪ Blood in anticoagulant for virus
▪ Protozoa and bacteria isolation
▪ Serum for serology
▪ Faeces if diarrhoea is present
▪ Urine if cystitis is suspected
− If it is difficult to pinpoint where the lesions are, in that case use:
▪ Haematological tests
▪ Clinicopathological tests
− Note that specimens required to detect carrier animals may be different
from those for clinically ill animals. Example:
▪ Foot-and-mouth disease carriers- pharyngeal scrapings
▪ Clinically affected animals- vesicle fluid and epithelium covering the
vesicles

• Dead animals
− Samples from tissues or organs with lesions are preferred
− Best to collect specimens from the edge of lesions, including some healthy
tissue. Why?
▪ Usually the site of active replication
− If there are no apparent gross legions: select tissues from organ systems to
which the clinical signs are referred
− If a particular disease is suspected: choose tissues targeted by this disease
− If there are no gross lesions and the clinical signs do not narrow the disease
process to a particular organ system: take tissue samples from several
organs. Appropriate specimens:
▪ Lung
▪ Liver
▪ Spleen
▪ Brain
▪ Kidney
▪ Heart
▪ Duodenum
▪ Jejunum
 ▪ Ileum
▪ Colon
▪ Body fluids
− The bone marrow can be sent if no other suitable samples are available
Putrefaction: decay of organic matter by since it is less accessible to putrefactive organisms
the action of microorganisms ▪ Send 50-100mm od rib stripped of muscle and periosteum in a
sterile well-sealed container
− In the case of aborted foetesus, neonatal animals/birds/small pets (chickens,
rats, canaries): the whole body can be sent to the lab
− If uncertain as to which specimens and how to take them:
▪ Contact the VDL
▪ Find out if the lab can preform the relevant test

TISSUE HANDLING AND PACKAGING

• Many diagnostic tests are able to detect non-viable microorganisms: the preservation of
specimens for these tests are a lot less stringent than for culturing methods
• Specimens should be collected as aseptically as possible: prevents aetiological agents from being
overgrown by contaminating bacteria
• Handle specimens gentle: rough tissue handling exacerbates tissue artefacts and autolytic
changes
1. Fixed specimens for micro-pathology and immunocytochemistry
− Take thin tissue samples: 5mm thick
▪ So that formalin can penetrate the tissue
− Place in 10% buffered formalin: the formalin to tissue ratio should be 4:1
− Do not freeze
▪ May result in artefact formation Artefact: something that is not typical of the actual
substance but as a result of processing
2. Fresh tissues for microbial examination
− Individual tissues in separate sterile containers for microbial isolation
− Do not allow specimens for culture to come into contact with disinfectants or
antiseptics (formalin)
− For microbiological examination as generous amount of sample as possible should
be taken (within reason) and submitted. E.g.:
▪ Blocks of tissue: 4cm3
▪ Biopsy material
▪ Several millilitres of pus, exudate or faeces
− When possible submit together with microbiological specimen:
▪ A few unstained air-dried smears of
o Exudates
o Urine sediments
o Tissue impressions
▪ These provide a picture of the disease process in the body
▪ Could aid in rapid presumptive diagnosis
▪ If indicated these smears can be used for a direct fluorescent antibody test
− Appropriate containers for organ specimens:
o Sterile screw top jars If glass jars used: should be well
o “Whirl-pak” plastic bags packed to avoid breakage
 o Plastic bags that are well sealed
▪ NOT:
o Zipper seal bags: tend to leak
− If swabs are used:
▪ Should preferably be placed in appropriate, non-nutritive transport medium
to prevent desiccation
▪ Commercially available are swabs with transport medium which are suitable
for:
o Aerobic
o Mycoplasma
o Fungal culture
− Do not allow specimens to dry out
▪ If sample size is small: place in appropriate transport medium
o Different transport media are available for aerobic and anaerobic
bacteria, viral and mycoplasma isolation
o Can usually be obtained from VDL
o Viral transport medium usually contain antibiotics so they cannot be
used for bacterial culture
o May need specialised transport medium for culture of
a. Fastidious microorganisms (like Campylobacter spp.)
b. Obligate anaerobes
− To slow autolysis and prevent unchecked proliferation of contaminants: fresh tissue
samples should be submitted in a container containing:
To maintain them at a cold temperature
▪ Adequate refrigerant for the entire shipping process
▪ Sufficient insulation
o Usually use a small polystyrene container with at least 2 frozen ice
packs that are well wrapped
o If transportation is for longer than 24h:
• Packs substituted with dry ice
o Because cold air is heavier than warm air, samples should be kept at
bottom of container
− Most specimens should not be frozen
− Fixed and fresh specimens should be separated:
▪ Formalin leaking from fixed specimens will render fresh samples unusable
− Ensure that packages sent to lab are:
▪ Watertight
▪ Correctly labelled
− As complete a history of the animal as possible should be provided
▪ Why?
o Lab personnel did not examine the animal and are essentially
“blind”
o All info given will aid the lab personnel in interpreting the results as
well as preforming any extra relevant tests
▪ What should be included?
o Animal species
o Sex
o Age
 o Numbers affected
o Any relevant clinical and management info
These can affect o Recent treatment with antimicrobials
interpretation of results o Vaccination history
o If the samples are taken at necropsy the necropsy findings should be
included
▪ Forms containing the relevant info on the specimen should be placed in a
separate zipper sealed plastic bag

SPECIMENS REQUIRING SPECIALISED TREATMENT

1. Anaerobic specimens for bacterial culture

− Strict anaerobes survive only for short period in air so strict care must be taken
when collecting for anaerobe examination
− Specimens should be taken aseptically to prevent contamination with normal
mucosal micro-flora (of which anaerobes form part)
− Samples from animals that have been dead for longer than 4h:
▪ Unsuitable
▪ Because of rapid post-mortem invasion of the body by anaerobes from the
intestinal tract
− If the samples are sufficiently large and collected aseptically: the centre of the tissue
remains anaerobic without taking special precautions
▪ In this case sample should reach lab in 2-3h
− In the case of fluid exudates:
▪ the sample can be placed in disposable syringe
▪ air is expelled from syringe and it is plugged
▪ alternatively: fluids can be injected through the cap into red-capped
vacutainer
▪ preferable not to send these specimens on ice because oxygen dissolves
better in fluids at low temperatures
− swabs must be placed in an anaerobic transport medium immediately after
collection

2. Blood specimens
− For bacterial isolation
▪ If bacteraemia or septicaemia is suspected: blood should be cultured
▪ Best to collect blood during fever peak
▪ Strict aseptic procedures should be taken when collecting blood
▪ Area over venipuncture site
1. must be shaved
2. cleaned thoroughly with detergent
3. dried
4. 70% alcohol applied to skin and allowed to act for at least 30
seconds
▪ Bacteraemia can be intermittent so take 3-4 blood cultures within a 24h
period
 ▪ Blood culture bottles are usually available from most VDLs or medical
diagnostic laboratories

− For viral isolation

▪ In the case of viruses known to cause viraemia: blood can be submitted for
either:
Green cap vacutainer (heparin)
o Viral isolation
o Fluorescent antibody test Lilac capped (EDTA)
▪ 10ml of blood collected
▪ Specimens should be collected during the primary fever peak

− For microfilarial isolation:

▪ Blood can be taken if:
o In a dog
o Cardiopulmonary signs or itchiness
o Aetiology of signs cannot be determined
o Or dog has been to area known to be endemic to heartworm
▪ 10ml of blood collected
▪ Either in heparin or EDTA for identification of microfilaria

2. Specimens for serology

− Serological tests are used in the diagnosis of many infectious and immune-mediated
diseases
− These test can detect antibody responses in the animal
▪ Not as definitive as isolation of causative agent
− Advantages:
▪ Sampling is easy
▪ Many animals can be tested
▪ Used when the causative agent cannot be identified
▪ Used to assess the prevalence and distribution of an infectious agent
▪ Used in the monitoring of control programmes and disease therapy
− Method of sampling:
▪ Blood collected in 10ml red capped vacutainer
▪ Allowed to clot
▪ Sample is sent on ice @4C to VDL
▪ Sample should be uncontaminated and non-haemolysed for best results
▪ If blood sample cannot reach lab in 24h:
o Place tube on its head and leave until the blood has clotted
Then discard the clot attached to the cap and
submit the serum (remaining fluid)
o Centrifuge sample
and pipette the serum into sterile tubes or red-capped vacutainers

o Serum should be stored at -18C or at least in a refrigerator if

transport is delayed
o paired serum samples should be taken
 a. one in acute phase of disease and the other in the next 2-3
weeks
b. a four-fold rise in antibody titre is indicative of a recent
infection
c. Unless measuring lgM (immunoglobulin M)

TIME CONSTRAINTS IN LABORATORY

− Lab endevours to get the results to you as soon as possible but there are constraints
including:
1. Time specimens take to reach the lab
2. Time required for specimen processing
3. If culture is required:
▪ Can easily take long
▪ Dependant on:
• speed of growth of organism
• presence of contaminants
• number of tests required

TEST TIME REQUIREMENT For results to reach clinician

Bacterial culture
− aerobic 3 days
− anaerobic 7-10 days
− Campylobacter and Brucella 5-15 days
− Mycoplasma 2 weeks
− Mycobacterium paratuberculosis 13 weeks
− Listeria spp. 2 months
Fungal culture More than 2 weeks
Virology culture 1-3 weeks
Serology
− IFA, ELISA 1 day
− CFT 2 days
− Serum neutralisation 5 days
Molecular assays 1-2 days
 THEME J1: INTRODUCTION, TERMINOLOGY AND VISUALISATION OF VIRUSES

INTRODUCTION:

− Viruses infect every living thing on earth

− 8% of our own genome is viral (we eat, breath billions of virions)
− We have co-evolved and co-existed with viruses since the beginning
− Viruses play NB role in different ecosystems
− Virus’s strategy for success: make large number of copies of itself
− Viruses are the most abundant biological entities on earth
− Bacteriophages: viruses that parasitize bacteria by infecting them and replicating inside
them
− 94% of all particles in ocean are viral
− Viruses play important role in carbon cycle as it is released from infected bacteria and
protists (source of carbon)
− At any given time we all have about a dozen viruses in us
− Herpes viruses:
o We are all infected with at least two
o Can’t get rid of them
− Our immune systems can deal very effectively with most viral infections
− Good viruses?
o Viruses infecting humans and animals only represent minute fraction of all viruses
o Play an NB role in our microbiome and participate in the normal development of our
immune system
o Grass: Dichanthelium langinosum, grows at soil temperatures of 50C, requires
thermal endophyte and this thermal endophyte (Curvularia protuberata) requires a
virus (Curvularia thermal tolerance virus (CThTV)) to enable thermotolerance effect

NB WHAT IS A VIRUS

An infectious, obligate intracellular parasite, comprising genetic material (DNA or RNA) surrounded
by a protein coat and/or envelope derived from the host cell membrane

→ Viruses are infectious, i.e. can be transmitted between hosts and cells
→ Obligate intracellular parasites (meaning they need something from host), they have to get
into a cell in order to replicate. Otherwise don’t do anything
→ They are parasite: dependent on the host
→ DNA or RNA as genetic material:

DNA genome RNA genome

herpesvirus Influenza
African horse sickness
Coronavirus
Only things that can have RNA as genome (all
other life forms depend on DNA as genome)

→ The genetic material (nucleic acid) is surrounded by a protective protein shell in majority of
viruses (all viruses infecting vertebrates)
 → Protein shell= virus capsid: comprised of 3D morphological subunits called capsomeres
→ Some viruses have the capsid enclosed in an envelope that
▪ contains proteins and lipids
▪ resemble the host cell membranes
▪ acquired from the host cell during the release of virus from the cell
▪ viruses with envelopes are susceptible to lipid solvents (e.g. coronavirus)
→ Peplomers → surface projections of varying lengths spaced at regular intervals on viral
envelope

DEFINE THE CHARACTERISTICS SHARED BY ALL VIRUSES

CHARACTERISTICS OF VIRUSES

 small infectious agents

 are all obligate intracellular parasite needs to be inside host cell cannot survive outside of cell
 genetic material is either DNA or RNA
 genetic material surrounded by protective protein shell: capsid
o in some viruses this shell is enclosed in an envelope made of proteins and lipids
 viral genetic material programs the infected cell to synthesize viral genetic material
 all viruses must make mRNA that can be translated into proteins by the host ribosomes
 viral genome is also multiplied
 assembly of these building blocks makes new virions in the host cell
 have no functional ribosomes or organelles: they utilize the host cell’s energy and ribosomes
 viruses do not replicate by binary fission;
o have an eclipse (lag) phase in which the individual building blocks are made before
assembly (use host cell’s machinery)
▪ building blocks will incl. structural + non-structural proteins a.w.a. genomic
material
▪ materials are then assembled during eclipse phase to produce many virions
• that are released during burst phase
o Bacteria does replicate through binary fission → each daughter cell gets it’s own
copy of the DNA
 the new virions produced in the host cell then transport the viral genetic material to another
host cell/ organism to carry out another round of infection
 from this we get the definition of a virus

ARE VIRUSES ALIVE?

• It has two phases (living + non-living phase):

o It is not alive while it is sitting in a test tube or if it sits on a surface
▪ It is alive when it infects and takes over the host cell’s machinery.
▪ The cell together with the virus becomes a living organism.
▪ Some viruses have the enzyme; reverse transcriptase polymerase
 which can make a DNA template from their own RNA
 and then incorporate this DNA into the host cell genome
 and remain there for the rest of the cell’s life.
 When activated they can reproduce and infect other cells
VIRUS TERMINOLOGY:

Capsid: the symmetric protein shell which encloses the nucleic acid genome
Capsomere: morphological units, seen under the electron microscope, they are substructures of
virus particles.
• They are composed of aggregates of polypeptide chains that interact to form the basic
structural units of the capsid
Envelope: the outercoat acquired by some viruses as they penetrate or bud from the nucleus or
cytoplasmic membranes of the host cells (DNA or RNA viruses).
• This surrounds the capsid and is made of proteins and lipids
Virion: a complete infective virus particle that exists freely outside the host cell.
• A virus that has lost its envelope is not referred to as a virion anymore, although these
viruses are still infective and virulent
Peplomere: surface projections of varying lengths spaced at regular intervals on the viral envelope.
• They consist of viral glycoproteins.
• Look like spikes.
• Several functions.
o 2 examples of peplomers:
I. Haemagglutinin:
− acts as the receptor on the influenza virus through which it binds to the host
cell before entry.
− also responsible for haemagglutination (binding to red blood cells)
− is an antigenic determinant Epitope: antigenic determinant, is the part of antibody
II. Neuroaminidase: that is recognised by the immune system
− Enzymatic activity
− Responsible for the release of influenza viruses from the host cell
Pseudovirion: are synthetic viruses used to inject genetic material (including DNA and RNA) with
specific desired traits into bacterial and eukaryotic cells.
• Pseudovirions are closely related to viruses in structure and behaviour but lack many
characteristics of true viruses, like capability to replicate
Defective interfering viruses: spontaneously generated virus mutants in which a critical portion of
the particle’s genome is lost due to defective replication
Nucleic acid: viruses contain only one nucleic acid type with respect to transmitting genetic material
RNA viruses or DNA viruses
 double stranded segmented

RNA viruses Resembles mRNA, can directly interact with

+ sense strand cells ribosomes to translate into various viral
proteins
single stranded
- anti-sense Must first produce + sense strand before it can
strand be translated to viral proteins. Uses enzyme:
genomes RNA-dependant RNA polymerase (one of non-
structural proteins of the virus)
circular
double stranded
(dsDNA)
linear

DNA viruses

circular
single stranded
(ssDNA)
linear

Viroid: smallest known pathogens, they are naked circular ssRNA molecules,
• that do not encode proteins yet replicate autonomously when introduced into host plants.
• No proof of animal diseases they infect plants.
Prions: misfolded proteins with the ability to transmit their misfolded shape onto normal variants of
the same protein. Neither bacterial, fungal, viral.
• Can trigger normal proteins in brain to fold abnormally
• Contains no genetic material. Responsible for many degenerative brain diseases like,
o Bovine Spongiform Encephalopathy (BSE, mad cow disease), scrapie in sheep and
Creutzfeldt-Jakob disease in humans.
• Non-degradable “bad” prions are virtually indestructible and will induce the disease if
consumed

CLASSIFICATION OF VIRUSES:

• Traditionally described and classified by:

1. Dimensions of virion and capsid
2. Symmetry of protein shell (capsid)
3. Presence or absence of lipid membrane (enveloped or naked)
4. Nature of nucleic acid in the virion
• Now we largely rely on genetic sequencing to discriminate between taxa

METHODS USED TO VISUALIZE VIRUSES:

• All animal viruses are sub-microscopic physical particles.

− Viruses are much smaller than bacteria
− Cannot be observed with light microscope
− This adaption allows viruses to infect these larger cells
− Virion size can range from 20nm- 900nm (for typical large viruses)
− Parvovirus, 20nm (smallest virus of veterinary importance)
• Some found in amoeba (like mimivirus) is large enough to be seen with a light microscope
 Direct methods:

Electron microscope:

• Dimensions of viruses are below the resolving power of visible light

• Visualization requires the shorter wavelengths available with EM
• For EM magnetic fields must be used to focus electrons
• Higher energy of electrons accelerated through high voltage produces very short
wavelengths with resulting high resolving power
• Types of electron microscopes
1. Transmission Electron Microscope (TEM)
▪ Uses an electron beam from above the specimen
▪ Focused using a magnetic lens
▪ Projected through the specimen onto a detector
− To go through the specimen must be extremely thin (20-
Requirements of specimen for electron beam
100nm thick)
to be projected through
− and it must be in a vacuum
▪ Detector captures the image
▪ Produces a 2D image
▪ Magnification power of 500 000x
▪ The image is produced because of varying opacity in various parts of
the specimen
− Opacity can be enhanced by staining
2. Scanning Electron Microscope (SEM)
• Methods used to stain the virus:
1. Shadow casting
▪ Involves the deposition of an electron-dense material (heavy metal)
− Tungsten or platinum metal vapour
− Filament of metal (as above) is vaporised by passing an
electric current through it in a vacuum
▪ At an oblique angle
− Angled disposition leaves an area on the support film free of
electron dense material and as an electron image this will
resemble a shadow of the particle being studied on a
photographic negative
− Virus becomes dark on one side and bright on the other
▪ Onto surface containing 3D specimens
2. Negative staining
▪ A concentrated suspension of virus particles is deposited on a grid
This supplies an electron transparent ▪ Grid is coated with a transparent plastic film
support to particles
▪ The particles settle onto the surface of the plastic film
▪ A droplet of negative stain is applied to the surface of the grid
leaving a thin solid glossy film
− E.g. heavy metal salt phosphotungstic acid or urinyl acetate
▪ The stain surrounds the viral particles
▪ And to a limited degree penetrate the exposed irregularities of the
viral surface
▪ Image: background will be dark and virus bright
 3. Positive staining
▪ Certain viral components selectively absorb certain electron dense
salts
▪ To study virus particles in cells or centrifuged pellets:
▪ Thin sections are cut from the concentrated pellet
▪ Stained with a positive stain
▪ E.g. uranyl acetate

Indirect methods:

Host system

• Detect the presence of a virus by looking at the effects they have on the host system
1. Tissue culture
2. Lab animals
3. Embryonated chicken eggs

Serological tests

• To demonstrate the presence or absence of a virus

1. Haemagglutination
▪ Some viruses have the natural ability to bind to red blood cells and
then cause agglutination of the cells
THEME J2: MORPHOLOGY OF VIRUSES

What causes the differences in viral shapes?

• Shape of their capsids

• Determined by the nature and composition of the capsid proteins (capsomeres) that
interlock to form the closely packed capsid
• The nature of the capsomeres can be such that they self-assemble in test tubes to form
empty capsids

DESCRIBE THE DIFFERENT MORPHOLOGICAL FORMS OF VIRUSES:

I. Helical
o The capsid is cylindrical or rod shaped with the genome fitting just inside the length
of the capsid
Enveloped Naked Folded to form a spherical
virion
Rabies (bullet shaped) Tobacco mosaic virus Coronavirus

II. Icosahedral
o Extremely common among viruses
o Consists of 20 triangular faces delimited by 12 fivefold vertexes
o Consists of 60 asymmetric units
o Resemble a soccer ball
Enveloped Naked
Coronavirus Bluetongue virus

o Can be 5-fold, 3-fold, 2-fold (5:3:2 symmetry)

III. Complex
o these viruses can take on different shapes
Brick shaped Binary (both helical and polyhedral)
Poxvirus: T4 bacteriophage
Intricate surface characteristics
not seen in other categories of
capsids
THEME J3: QUANTIFICATION OF VIRUSES

Virus quantification:

− Involves counting the number of viruses or virus components in a specific volume to get the
# 𝑜𝑓 𝑣𝑖𝑟𝑢𝑠𝑒𝑠
virus concentration [𝑣𝑖𝑟𝑢𝑠] = 𝑣𝑜𝑙𝑢𝑚𝑒
− If you want to know how many infectious viruses in a sample you need to determine its
infectivity
o But some virus particle can be defective and not necessarily infectious
o So we need to use different techniques to determine this (e.g. virus plaque assay)

Briefly describe the methods of quantification of viruses:

1. Total particle count

a. Direct counting
• TEM
• Images can show individual virus particles
• with a reference particle like latex spheres in known concentrations as a
control
• quantitative image analysis can be used to determine virus concentrations
• cannot distinguish between infectious and non-infectious virus particles

b. Haemagglutination
• Indirect method
• Relies on fact that haemagglutinin (surface protein) binds to and
agglutinates red blood cells
• E.g. influenza virus
• In this assay dilutions of influenza sample are incubated with a 1%
erythrocyte solution
• The virus dilution at which agglutination first occurs is visually determined
• Assay results are recorded as haemagglutination units or haemagglutination
titre
• Since non-infective viruses will still agglutinate red blood cells it does not
measure number of infective viruses

c. Flow cytometry
• Samples are stained using 2 dyes
− One specific for proteins (in the virus envelope or capsid)
− Other specific for nucleic acids
• A virus counter quantifies the number of virus particles or components
thereof by using laser beams o detect different colours of fluorescence after
staining
• This determines total number of particles not number of infectious particles
 2. Infective particle count
• These methods quantify infectious units so a host system for the growth of viruses
must be used.
o The cytopathic effect of the infectious particles on the host will be used to as
indicator of the presence of the virus
• Both methods based on preparation of serial dilutions and thereafter determining the
or or
limit of endpoint of some specific evidence of infection
• Keep in mind:
→ Dilution factor: calculate dilution factor into final answers
→ Environmental conditions: temperature, light, pH, stabilizers. All play role in growth
of virus and thus the outcome
→ Host system used:
o Volume of inoculum
o Route of inoculation
o Effect on host
o Factors influencing suceptibillity
• Methods used include:
a. Plaque/pock/focus formation
ALIQUOT: a portion of a larger whole,
i. Plaque assay: plaque forming units especially a sample taken for chemical
• To preform plaque assay analysis or other treatment.
• 10fold dilutions of a virus stock are prepared
• 0.1ml aliquots inoculated onto susceptible cell monolayers
• Monolayers are susceptible single layer tissue culture cells that
grows on and cover the bottom of the flask
• The layer is confluent when it covers the whole surface of the flask
What is the purpose of adding a semi- • We add the different virus dilutions (aliquots)
solid agar overlay on the monolayer of • Incubation period, to allow virus to attach to cells 45-60min
infected cells? • Monolayers are covered with a nutrient medium containing a
To restrict viral diffusion after lysis of
substance (agar) that causes the formation of a gel
infected cells • When the plates are incubated the original infected cells release
viral progeny
The damage caused by the virus seen • The rapid spread of the new virus particles to neighbouring cells is
as plaque or focus in the cell restricted by the gel
monolayer
• Consequently, each infectious particle produces a circular zone of
infected cells called a plaque
• Eventually plaques become large enough to be visible to naked eye
• Dyes that stain living cells often used to enhance the contrast
between the plaques and the cells
− Stains: neutral red/Crystal violet
− Plaques do not stain and form white hole in the monolayer
• Titre of a virus stock can be calculated in Plaque-Forming Units per
millilitre (PFU/ml)
− Each plaque or lesion in the monolayer is assumed to be
formed by a single virus particle
− i.e. each plaque originates from one genetically similar virus
− can be used to purify viruses by selecting a plaque of a
genetically pure strain of a virus
 ii. Specific lesion count

b. Determination of infectious units

i. Endpoint dilution assay: (infective dose 50) TCID50
• Used to measure virus titre before development of plaque assay
• Still used for viruses that do not form plaques
• Serial dilutions of a virus stock are prepared
• Inoculated onto replicate cell cultures (often in multi-well formats)
• 1:100, 1:1000, 1:10 000 dilutions are prepared and added to
individual wells of each row of the multi-well plate
• Incubation period
• The number of cell cultures in the individual wells that are infected
is then determined for each virus dilution by looking at cytopathic
effect
• Wells scored + if they display cytopathic effect
• At high dilutions: none of the cell cultures display cytopathic effect
because none of the particles are present
• At low dilutions: all of the wells infected
• Where half of the cell cultures show cytopathic effect: endpoint
− Dilution of the virus at which 50% of the cell cultures are
infected
− Number calculated from data and expressed as 50%
Infectious Dose (ID50) per millilitre
• Endpoint dilution method can also be used to determine virulence
of a virus in animals
− Same approach is used
− Serial dilutions are made and inoculated into different test
animals
− Infection of the animal can be determined by death or
clinical symptoms
− Results expressed as 50% Lethal Dose (LD50) per millilitre or
as 50% Paralytic Dose (PD50) per millilitre when death or
paralysis is used as endpoints
• The 50% point of infectivity indicates the concentration of a virus at
which 50% of the host system will be affected
• @50% area of response a very small change in dose is reflected by
large difference in % of host affected The host will show much more severe affects after 50% infection is reached
when you reach the 50% infected mark a small increase in infection will lead to a bigger change in the affects on the host
• At extremes of the curve large changes in dose must be made to
see
affet % of host affected
• Thus the point of maximum sensitivity of the assay can be
establsihed at the 50% level
• Endpoint method: this is a statistical unit which indicates the
dilution of the virus that will damage 50% of the host cells or
organsims
• The terms of conditions must always be defined
− Volume of inoculant
− Route of infection
− Host system
THEME J4: LABORATORY CULTURE OF VIRUSES

IN VIVO: when we grow viruses within a lab animal

IN VITRO: when we grow viruses within cells in an artificial environment (e.g. cell culture flask)

1. USE OF LABORATORY ANIMALS

• Currently used for
− Limited to only viruses that cannot be adapted to grow in cell cultures
− Used as a model for the study of pathogenesis, immunity and pathology of a
disease where it is not possible or ethical to use domestic animals or
humans
o Use of animals strictly controlled by:
o research ethics guidelines and committees
o good justification in absence of alternatives needed
− Used as a model for the study of pathological changes, tissue tropism and
cellular reactions
− To evaluate cross protection
o Antibodies of one virus protect animal against infection with
another virus
− When other hosts are not available
− Infectivity titration
− In vivo serological tests (titre)
− To prepare antisera for that can be used as diagnostic reagents for the
detection of viruses or their specific antibodies in patients

• Hampered by several factors

− Variation in susceptibility:
o Same spp. Can have diff. susceptibility to the same virus
− Previous infection and immunity:
o Immune status of animals not known
− Practicality:
o Large animals availability and accommodation problematic
− Unnatural hosts
− Prior adaption to a virus:
o Therefor not always susceptible to the virus

• Disadvantages
 $$$ (facilities, cages, equipment, management) D
 Problems with capture and transport of free-living primates (stress, disease, N
D
unscrupulous dealers)
S
 Problems with establishing breeding units in captivity I
 Cross infection in close confinement T
 Dangers of human infections C
 Latent infections complicating test results
 Previous exposure and resulting immunity of animals
 • Signs of infection
− Death
− Paralysis
− Specific internal and external lesions
− Febrile reaction

2. USE OF EMBRYONATED CHICKEN EGGS

• Used for
− Identification and diagnosis of pathogenic viruses in clinical specimens
− Production of vaccines (e.g. influenza virus)
− Basic research studies

• How does this work

− Embryo serves as incubator for viral replication
− Provides specialized organ system
− and extra-embryonic membranes
− Fast dividing cells
− Protected by shell from other bacterial and fungal pathogens
− Many avian viruses replicate more readily like this than in cell culture
− Allows for culture of viruses with different tissue tropism
o Target sites:
a. amniotic cavity 10-14 days
▪ influenza virus
▪ mumps
b. allantoic cavity 10-12 days
▪ influenza virus
▪ Newcastle virus
c. yolk sac 6-8 days
▪ gives systemic infection
▪ vaccine production
Routes of infection: different viruses d. chorioallantoic membrane 10-12 days
require different routes of infection to ▪ pox
rapidly grow and present an effect ▪ herpesvirus
e. intravenously into vessels of shell membrane and embryo
(rarely)
▪ bluetongue virus
f. intracerebral infection

• Advantages
✓ Readily available, cheap and easy to maintain
✓ Relatively free form contaminants
✓ Easy to handle and to infect
✓ Absence of non-specific defence mechanisms
✓ Specialised systems and cells available, so can use different routes of
infection for different viruses
✓ Extra-embryonic membranes
✓ Rapidly dividing cells (21 days)
 ✓ Protection provided by shell

• Disadvantages
 Immune status of donor hens not known
 Susceptibility to bacterial contaminants
 Cost of Specific Pathogen Free (SPF) poultry breeding unit

• Evidence of infection
− Embryo death
− Haemorrhages
− Dwarfing and malformation: viruses that disrupt embryo growth
− Oedema and haemorrhages or chorioallatoic membrane
− Pocks lesions (on chorioallantoic membranes) caused by pox virus
− Microscopic lesions

3. USE OF CELL CULTURES

• Primary cell culture
Primary culture: first in vitro − Freshly prepared from animal tissues or organs
outgrowth, cells obtained − Cells extracted from tissues via
from embryos or foetuses o Mechanical scraping or mincing
o Enzymatic method using trypsin or collagenase to break up tissue
and release single cells into suspension
− Anchorage dependence requirement: primary cell cultures require a liquid
culture medium in a petri dish or tissue-culture flask so cells have a solid
medium surface (glass/ plastic) for attachment and growth
− Usually have limited life span
o Cells undergo mitosis and a sufficient density of cells are produced
o Cell-to-cell contact
o Mitosis triggered to stop
o Called contact inhibition
o It prevents the density of cells from becoming too high

• Secondary cell culture

− To prevent contact inhibition: cells from the primary cell culture must be
transferred to another vessel with fresh growth medium
− to maintain cell growth:
o Periodically cell density must be reduced by pouring off some cells
o and adding fresh medium to provide space and nutrients

• Continuous cell lines

− Derived from transformed cells or tumours
Cell line: when a primary cell − Can be sub-cultured many times
culture has been sub-cultured
− Or grow indefinitely: immortal
approx. 70 times. Cells
become transformed or
o E.g. HeLa cell line
changed so that they will not − Why does continuous cell lines grow in piles or lumps resembling tumours
loose their ability to divide o Not anchorage-dependant and will grow in suspension
Diploid cell line: 75%
retention of karyotype
(number and structure of
chromosomes)

o Lost their contact inhibition

• Requirements for in vitro cultivation of cells

1. Source of cells
o Embryonic tissue
▪ Most ideal
o Adult tissue with high concentration of epithelial cells and no
contaminants; lung heart kidney
o Buffy coat (leucocytes)
▪ Very difficult and not commonly used
o Macrophages
▪ Well established, very difficult and not commonly used
o Tumours; HeLa cells
▪ Grow well but dangerous for virologist
o Tissue of infected animal
▪ A way of identifying viruses by growing the infected material
2. Environment
o Temperature (28, 30, 37C)
▪ Some cells specifically infected with virus prefer certain
temperatures to grow, e.g. insect cells grow at 28
o Isotonicity (buffer system)
o pH (physiological pH)
o matrix or substrate
▪ glass or plastic, normally in closed environment
▪ e.g. petri-dishes, bottles, microtiter plates etc.
3. Nutrients
o Serum (foetal calf serum, bovine serum): provides cells with
proteins, amino acids and other growth factors
o Partial substitutes like:
▪ lactalbumin
▪ defined amino acids and vitamins
▪ tissue extracts
▪ yeast extracts

• Evidence of virus replication in monolayer cultures

1. Cytopathic effect (CPE)
o Definition: distinct observable cell abnormalities due to viral
infection. It is the degeneration and death of cells
o Demonstrated by: D
a. General degeneration N
b. Necrosis
c. Detachment of cells from surface D
d. Syncytium formation: S
▪ fusion of the cell membranes to form one large
multinucleated cell called the syncytium
I
e. Inclusion bodies T
Canine morbillivirus
▪ Small aggregates of viral proteins found either in C
(distemper) and pox virus
produce eosinophilic
nucleus or cytoplasm
S
intracytoplasmic inclusions V
 ▪ Can be visualised with fixing and staining the cell
monolayer
f. Transformation
▪ Integration of viral nucleic acid into cells of the
monolayer
▪ The characteristics of the cell monolayer (karyotype)
change and can eb demonstrated with specialized
techniques
g. Changes in cell shape
▪ From round to flat
h. Shrinkage of the nucleus
i. Vacuoles in the cytoplasm
2. Non- or partial cytopathic effect
o The presence of the virus must be demonstrated by the use of a
second laboratory test
a. Metabolic inhibition
b. Interference:
▪ When viruses of the same group or subgroup can
prevent the entrance of one another or
▪ When the viruses have penetrated the one inhibits
the replication of the other
c. Fluorescent antibody
▪ Use a labelled antibody to detect the antigen within
the cell monolayer
d. Haemagglutination
▪ The binding or agglutination of red blood cells to the
virus
▪ Different dilutions of the virus are reacted with a
constant 1% red blood cell suspension
▪ Viruses with haemagglutination properties will bind
to the red blood cells and cause them to stick
together
▪ Possible outcomes:
Virus with Haemagglutination, Red colour
haemagglutinin forms a lattice throughout
structure well
Virus without Red blood cells do Red dot in
haemagglutinin not bind and sink to centre of
bottom of well well

e. Hemadsorption
▪ Red blood cells will bind to infected cells in cell
culture
f. Electron microscopy

• Disadvantages
 Contamination of the cell culture medium by bacteria and yeast
 Cytotoxic factors: toxins in water used to produce medium
  Time consuming and expensive: filtration and sterilisation of the medium
 Interferon production: specifically when primary cell cultures used

• Advantages
✓ High virus yield
o 1 host cell: 3-4 (or more) viruses produced
✓ Low tissue content
o Small number of cells used to isolate virus
✓ Versatile application
o Use in serology as antigens
o Antigens for vaccines
o Preparation of antigens for research etc.
✓ No immune interference
o No antibody production
✓ First stage in establishing the characteristics of a virus
THEME J5: CHEMICAL COMPOSITION OF VIRUSES

I. Viral nucleic acid

• DNA/ RNA
• Encodes genetic information necessary for replication of the virus and viral
proteins
• Single stranded or double stranded
• Proportion of genetic material varies for different viruses
− Influenza: 1%
− Bacteriophages: ±50%
• The amount of nucleic acid is an index of genetic complexity
II. Viral protein
• Studied by separating them though the action of detergents
• Then dissociation
• Then separation by electrophoresis or chromatography

a. Structural proteins of viruses

• Functions: (PAISE)
− Protect viral genome against inactivation by nucleases
− Capsid proteins are highly resistant to proteolytic enzymes (e.g.
trypsin)
− Assist in attachment of virus particles to a susceptible host cell
− Act as immunological antigen
− Give structural symmetry
− Responsible for electrophoretic mobility
b. Internal proteins
• Preform specialised tasks in the virion
• They produce:
− Enzymes for replication cycle
▪ Like reverse transcriptase: revert RNA to DNA,
o important in transformation of cells by tumour
viruses.
o The DNA of these viruses usually integrates into the
host cell chromosomal material
− Proteins for attachment to cells
▪ Like haemagglutinin
III. Viral enzymes
• Viral enzymes are internal proteins
− Neuraminidase: assist with release of virus from infected cell
• Larger viruses contain enzymes
− DNA transcriptase
− RNA transcriptase
Can transcribe both RNA and − DNA dependant RNA polymerases
DNA into DNA − RNA dependant RNA polymerases
− RNA dependant DNA polymerases
− Reverse transcriptase
IV. Viral lipids
• Found in viruses that have an envelope
• Lipid content in viral envelope causes these viruses to be sensitive to
− Ether and
− Chloroform
• This characteristic is used to divide viruses into 2 major groups:
a. Enveloped
• lipids are added to the viral envelope as it matures or buds through the cell
or nuclear membrane
• therefor the viral envelope contains the lipid and protein constituents from
the membrane it is derived
• also adds virus specific glycoproteins (spikes or peplomer) to the envelope
b. naked

V. Viral carbohydrates
• Viral envelopes contain a significant amount of carbohydrates in the form of
glycoproteins
• NB components of antigenic determinants
• Glycoprotein synthesis:
− Partly controlled by virus
− Also determined by host cell genome
THEME J6: VIRAL REPLICATION

Viruses are obligate intracellular parasites

Meaning they require living cells to provide:

• Energy and
• Enzymes for metabolic activity

Virus multiplication was first studied in bacteriophages because:

1. System is easy to prepare

2. Growth cycle is short
3. Assay of bacteriophage replication is very simple
• Because we can easily observe the development of plaques

5 stages in the bacteriophage lytic cycle

1. Attachment
→ Phage interacts with specific bacterial surface receptors
2. Penetration
→ Entry
3. Biosynthesis of new viral components
have to make -DNA that will be template for +mRNA
-DNA also template for +DNA
→ Hijacks the host cell to replicate, transcribe and translate the necessary viral
components for the assembly of new viruses
4. Maturation
→ New virions are created
5. Lysis
→ Release
→ Mature viruses burst out of host cell (lysis) and progeny viruses released into
environment to infect new cells

BACTERIAL REPLICATION

− Binary fission
− Sigmoidal curve lag, log, stationary and death phases
− Double the number of cells with every division

VIRAL REPLICATION

− Essential to synchronise (as far as possible) viral and cellular activity

o So that the same events take place in all the cells at the same time
o Achieved by: infecting all the cells with at least one viral particle and eliminating all
the unabsorbed viruses
− Diversity of mechanisms by which viruses ensure their proteins are made, reflected in but
not deduced from their genomic structure
− Vary in number of genes they contain
− But all viruses encode proteins that can facilitate a minimum of 3 functions:
i. Replication of the viral genome
ii. Package the genome into virions (assembly)
 iii. Alter the structure/ function of the infected cell formed
− Ability to remain latent in host: function of specific proteins and enzymes for by the genome
of some viruses
− Does not follow a sigmoidal curve rather a one step growth curve

One step growth curve: Synchronise the infection by using one phage per bacterial cell

Burst: host cells release many

Represents number of phages detectable in supernatant
LOGARITHM OF NUMBER OF INFECTIOUS VIRIONS:

viral particles Burst size: number of virions

released per bacterium
Inoculation:
inoculum of
(outside of cells)

virus binds to
cells

Eclipse: virions penetrate

the cells

HOURS:

Represents the time during the phage replication

cycle

1) INOCULATION:
▪ Inoculum of viruses attach to host cells
2) ECLIPSE
▪ Virions penetrates cell (so there will be no virions in medium)
▪ Virus gains control of host cell synthetic machinery to produce the produce the
individual components required to assemble into new virions
▪ Phase during which virion has entered the cell and before progeny is made
▪ NO INFECTIOUS VIRUS PRESENT
▪ Definition: period between the start of infection to the first appearance of
intracellular infectious virus

LATENT PHASE:

Includes the eclipse phase and is from the start of infection to the appearance of infectious virus in the surroundings

Some animals don’t have a classic burst but rather release viruses slowly over a long period of time during this time it is
possible to find large internal pools of viruses
 3) BURST
▪ Host cell releases many viral particles
▪ Different to bacterial growth curve in that all the virions are released form the lysed
host cell at once: called a burst
4) BURST SIZE (virus yield or total amount of virus)
▪ Number of virions released per bacterium
▪ Steep rise in viral titre= (number of virions per unit volume)

If no viable host remains the virus particles begin to degrade during the decline of the culture

ESSENTIAL STEPS IN VIRUS REPLICATION:

I. Attachment
− Specific virus protein binds to a cell surface receptor
− Examples:
Humans are susceptible to • Haemagglutinin of influenza
COVID 19 because of • Complex viruses such as pox can bind more than one receptor
mutations that increase • Angiotensin-Converting Enzyme 2 (ACE2) serves as entry point for
binding affinity between spike coronavirus, including SARS-CoV-2 (COVID 19). The spike protein in the
protein and ACE2 cell coronavirus envelope binds to the ACE2 receptor on the cell membrane
receptors
− Cellular receptors are mostly glycoproteins
− Initial attachment of a virus to a cell receptor is due to electrostatic forces:
• Will only occur if specific affinity exists between the cell and the virus
• This is the reason for TISSUE TROPISM: the type of cell in which the virus is
able to replicate
o Expression or absence of receptors on the surface of cells
determines the TROPISM
o NB factor in pathogenicity
o Absence of these factors why certain spp. Are refractory to infection
with a particular virus
− This adsorption occurs at specific sites distributed over the cell and virus
• These sites are called receptors
− Interaction of the cellular receptor and its corresponding ligand on the protein is
dependant on correct:
• Temperature
• pH
• molarity
− initial bond between cell membrane and virus can be:
i. reversible
o only loose contact between cell membrane and virus
o dependant on electrostatic forces induced by specific electrolyte
concentrations Fun facts:
o temperature independent
Poliovirus only attaches to human cells
ii. irreversible
o late stage of binding process Viruses can be adapted to attach to new cell types, when they
o multiple point attachments form grow in vitro for some time (vaccine production)
Some viruses can grow in wide range of cells

Receptors may only be present for short time during growth

Different viruses may share the same receptor sites in host

cells
 o virus now protected from antibodies
o temperature dependant

II. Penetration (viropexis)

− Energy dependant step
− Occurs quickly
− Several ways in which this occurs:
• Endocytosis of the entire particle
o Resulting in accumulation of virus particles inside a cytoplasmic
vesicle
o E.g. dengue virus
• Fusion of the cellular membrane with the virion envelope
o Direct release of nucleocapsid into the cytoplasm
o E.g. paramyxovirus, herpes, HIV

III. Uncoating
− General term applied to everything after uncoating which allows the virus to express
its genome
− Different viruses uncoat in different ways
− Process brought about by lysosomes from the host
− Protein coats (if more than one is present) is systemically removed
• Some viruses may have their own uncoating enzymes
− Nucleic acid then set free in the cytoplasm
− Beginning of the eclipse phase

IV. Biosynthesis (eclipse phase)

− Transcription: the principle for both RNA and DNA genomes is that a specific mRNA
must be transcribed from the viral genome before further expression and
duplication
• Transcription of mRNA
• Translation of mRNA
• Replication of viral nucleic acid
• Transcription of further RNA from progeny and parental nucleic acid
• Translation of late mRNA into structural protein
V. Assembly
VI. Release of new virus

REPLICATION EXPRESSION STRATEGIES OF VIRUSES

− A gene is expressed via process of transcription and translation

− Transcription:
o DNA as template to produce mRNA
o Enzyme: RNA polymerase
− Translation:
o mRNA translated to build polypeptide encoded for on the original DNA
o mRNA used as template for protein synthesis= sense strand or + strand
 − during the replication stage the virus must replicate its own genome, produce the building
blocks for progeny viruses and produce the necessary machinery to assist its own replication
− All viruses must direct the synthesis of mRNA to produce proteins
− No viral genome encodes a complete system for translating proteins
o Therefor all viral protein synthesis completely dependent on the translational
machinery of the host cell
− Retrotranscribing viruses:
o Viruses that have reverse transcriptase enzymes
o That will make a DNA copy from the viral genome
− Viral genomes:
DNA viruses RNA viruses Retro-transcribing viruses
dsDNA ssDNA dsRNA ssRNA(+) ssRNA(-) ssRNA (RT) dsDNA(RT)

− Baltimore classification of viruses:

o Based on:
▪ Central role of translational machinery
▪ Importance of mRNAs in programming viral protein synthesis
o He placed mRNA in the middle
o And described the 7 pathways to mRNA from DNA and RNA genomes
have to make -DNA that will be template for +mRNA
-DNA also template for +DNA that will be genome of viral progeny

+ DNA

6. 2.
7. ±DNA
+RNA template for rt polymerase that creates -DNA that is incorporated into host genome
Host then carries code for viral mRNA and viral proteins
dsDNA is gaped
needs RNA intermediate with rt polymerase to replicate the genome
+RNA -DNA ±DNA from the +RNA they make dsDNA again with rt polymerase and then they can transcribe +mRNA

1. +mRNA produced from anti-sense DNA strand

3.
+RNA -RNA mRNA (+) ±RNA

genome RNA = mRNA segmented genomes

For viral replication +mRNA must make -RNA that is template for +RNA for new viral genome + each RNA segment is transcribed separately into mRNA

4.

-RNA Complement of +mRNA

must first be copied to make complementary +RNA to make proteins

5.

1. Double stranded DNA viruses

• Have sense and anti-sense DNA strand
• +mRNA is produced from the already present -DNA strand
• E.g. adeno and pox viruses

2. Single stranded (+) sense DNA viruses

 • Have to make -DNA strand that will serve as a template for the +RNA strand
• The -DNA also serves as a template for making +DNA which will then be used as the
genome of the progeny virus

3. Double stranded RNA viruses

• These usually have segmented genomes
• Each genome segment is transcribed separately to produce mRNAs
• Reovirus like bluetongue or African horse sickness

4. Single stranded (+) sense RNA viruses

• Genome RNA is the same as mRNA so it can be translated directly to protein
• RNA alone is infectious
• To replicate the genome the +mRNA must first make -RNA as template for the +
genomic RNA
• E.g. coronavirus

5. Single stranded (-) sense RNA viruses

• The virion RNA is antisense (complementary to mRNA)
• Must be copied to make the complementary + sense RNA to make protein
• E.g. rabies virus

6. Single stranded (+) sense RNA viruses with a DNA intermediate in life cycle
• The RNA genome is a + strand but it does not serve as mRNA (unique among viruses)
• Serves as a template for reverse transcriptase
• So it first makes an intermediate DNA
• Seen in Retroviruses
• They encode RNA- dependent DNA polymerase (reverse transcriptase, within capsid)
to make the DNA provirus
• This DNA is then incorporated into the host cells DNA from where it can make viral
mRNA
• E.g. human immunodeficiency virus

7. Double stranded DNA viruses with RNA intermediate

• These viruses have gaped dsDNA genome
• Require an RNA intermediate with reverse transcriptase to replicate their genome

REPLICATION CYCLES OF VIRUSES

→ In all cases the first step of the eclipse phase, after uncoating, is the synthesis of mRNA
(always sense or + polarity)
→ Then the translation of proteins
o For DNA viruses
▪ Enzymes first: enzymes may assist in duplicating original viral genome to
produce progeny viral genomes
▪ Then further transcription will result in more mRNA that will be translated to
structural proteins
 oFor RNA viruses
▪ Posttranslational cleavage will simultaneously produce both enzymes and
structural proteins
→ Synthesis of nucleic acid
→ Assembly and release

Replication cycle of a positive sense RNA virus

• The RNA of (+) sense RNA viruses can act as template to be directly
• translated into viral protein.
• In many cases this is a large complex protein that can be cleaved into several separate
enzymes that play a role in duplicating the original viral genome.
• The enzymes are RNA-dependent RNA polymerases that first make a negative template from
the original positive RNA genome and from there they produce the positive RNA progeny
virus genomes.
• Structural proteins are also produced when the original complex protein is cleaved.
• The structural proteins are then assembled to encapsulate the progeny viral genome before
release.
Replication cycle of a negative sense RNA virus

• RNA from (-) sense RNA viruses must first synthesize (+) sense mRNA
• that can be translated into viral proteins.
• To replicate the original negative sense genome an intermediate positive strand must first
be made to act as template for the negative progeny RNA genomes.
• The structural proteins will then encapsulate the progeny RNA before release of the virus

Replication cycle of a retrovirus

• Here is the example of HIV which is a +ssRNA virus belonging to Group 6 of the Baltimore
classification.
o HIV fuses to the cell surface
o The viral RNA, and the revers transcriptase enzyme, enters into the cell.
o The reverse transcriptase synthesizes a complementary ssDNA (cDNA) copy using
the +ssRNA genome as a template.
o The ssDNA is then made into dsDNA, which can integrate into the host chromosome
and become a permanent part of the host.
o From here mRNA can produce viral proteins that can encapsulate viral mRNA to
form new virus particles.

ASSEMBLY AND RELEASE OF A VIRUS

o MOLECULAR CHAPERONES: Some viruses make morphogenetic factors that are not
structurally part of the virus but whose presence is required for normal assembly
o Cellular chaperones may also take part in process
o Other viruses SELF-ASSEMBLE
o TMV(Tobacco Mosaic Virus) and RNA will assemble and make infectious viruses in
the test tube
o Suggests that TMV structure is a minimum free energy state for its constituents
o SYMMETRY is NB in viral assembly
o It reduces ambiguities in the assembly process
Unenveloped viruses

o Capsids are formed by self-assembly of monomers:

o Structural proteins produced in cytoplasm
o Into structural capsomeres
o Into complete capsids
o Nucleic acid not required for process
o Shown in presence of empty capsids in cytoplasm
o Some viruses concentrate at the site of production to form intracellular crystals or inclusion
bodies
o RNA viruses are usually released very rapidly accompanied by destruction of the cell
o Icosahedral DNA viruses: released slowly after they have matured in nucleus and their
release is accompanied by autolysis

Enveloped viruses

o After the nucleocapsid is formed, envelopes are formed around the nucleocapsid by budding
of the cell
o Either at surface of cell
o Or into cytoplasmic vacuoles
o Some viruses (herpesvirus) assemble in the nucleus
o Acquire envelope as they pass through the inner nuclear membrane
o Then accumulate between inner and outer lamella of the nucleus
o From here pass in vesicle to the cell surface
o They are protected from the cytoplasm
o Budding process is invariable cytolytic
o Some viruses acquire their lpid bilayer from the plasma membrane
o Capsid assembly can take place in cytoplasm
o Or (HIV) in the plasma membrane
o Budding can also take place in the vacuole
o Release of the virus then takes place after fusion of the vacuole with the membrane
o Some viruses derive an envelope from the Golgi body
o E.g. vaccinia
Viruses may be released from the
cell, by cell lysis or if enveloped
may bud from the cell. Budding
does not necessarily kill the cell
THEME J7: VIRAL INTERFERENCE AND GENETICS

1. CONDITIONAL LETHAL MUTANT

• These mutants can ontly replicate under specific physiological environmental
conditions
• Most NB group: Temperature Sensitive mutants (TS):
− Can replicate under normal physiological temperatures
− When temp rises they stop their replication
− Are used to study the replication of viruses in infected cells
− After replication at 37C a rise in temp to 40C will stop the replication
process and its viral replication stage can be studied

2. ATTENUATION AND ATTENUATED MUTANTS

• Attenuated or weakened virus mutants have lost the ability to cause disease in
humans/animals
• Can still infect cells
− Used as live vaccines to elicit an active immune response
• PATHOGENICITY IS LOST NOT ANTIGENICITY
• Attenuation occurs when a virus is grown/passaged in vitro in an unnatural host
(embryonated chicken eggs or cell culture for many passages)

3. DEFECTIVE VIRUSES
• Viruses not always productive, i.e. don’t produce live active progeny
• This type of infection: abortive infection
• Reasons
− Host cell defective
▪ Does not have a certain enzyme for viral replication
− Virus itself defective
▪ Cannot replicate
• Possible reason for chronic and latent infection where the release of viruses are slow
or sometimes never
• If cells are infected with a suspension containing a large concentration of defective
viruses the defective viruses can block adhesion receptors on cells: called DEFECTIVE
ADHESION PARTICLES

4. GENETIC INTERACTION BETWEEN VIRUSES

• Recombination
− Physical interaction of viral genomes in co-infected cells
− Remarkable feature of retroviruses
− Defined as when two parent viruses exchange genetic material in one host
cell
• Gene reassortment
− Viruses with segmented genomes can exchange segments (e.g. influenza
virus)
− Antigenic shift
▪ Avian or swine fu virus can replicate in the same cell with a human
flu strain
 ▪ A viable reassortment can result in a new human flu strain that
carries a glycoprotein from the animal virus
− Antigenic drift
▪ Spontaneous point mutations that can lead to minor but
serologically detectable differences
• It is the envelope proteins that determines the serological properties of the virus
and thus the severity of the disease but these can be subjected to sudden changes
− Infection with flu virus does not (in contrast to most other viruses ) lead to
long lasting or reliable immunity
THEME K: PATHOGENESIS OF VIRAL INFECTIONS

Pathogenesis: mechanism by which disease is caused

Virulence: ability of an infectious microbe to cause disease

• Virulence of a virus is a measure of the severity of the disease it is capable of causing

• Measured by: LD50 (lethal dose) among other things

MECHANISMS OF VIRAL INFECTION AND VIRUS DISSEMINATION

To be able to cause a disease a virus must follow these steps:

1. Entry into the host and primary virus replication

To get entry
o Must evade hosts natural protective and cleansing mechanisms
o @ cellular level: must take over host-cell functions for its own replication
process
2. Local or general spread in the host (defined by tissue tropism) with secondary virus
replication
To get established
o Evade host defences in the form of innate immune and inflammatory
responses and natural barriers
o Damage to the host may occur at this and later stages
3. Shedding from the host
Must be able to exit
o At specific site
o At sufficient [] to ensure infection of the next host
4. Clearance from the host
o Mediated by the adaptive immune response
o Not always complete: some viruses may persist and contribute to long-term
shedding and chronic disease

Viral infection is not synonymous Death

with disease as many viral
infections are subclinical Severe disease
(asymptomatic or inapparent) Moderate disease
whereas others result in disease of
varying severity that is typically Mild disease
accompanied by characteristic
Subclinical infection
clinical signs in the affected host
Exposure without infection

OUTCOME OF THE VIRUS-HOST ENCOUNTER

Product of the virulence of the virus and the susceptibility of the host
Virulence determinants:
➢ Multigenic
➢ Tropism
Susceptibility/ resistance determinants:
➢ Multifactorial
➢ Host factors
➢ Environmental factors
− Initiating an infection:
o Sufficient virions must be available
▪ Theoretically: a single virion should be able to initiate an infection
▪ Practically: not realistic
• Infection= series of stochastic events (random event)
• Failure of one event will lead to a failure of infection
o Examples of failed infections:
▪ Interaction of virus with debris, extracellular materials or surface antibodies
▪ Delivery of virus to lysosome upon entry of cytoplasm
▪ Defective virus particles
• Not all viruses are capable of replication because of mistakes/
mutations during virus production

− Attachment and bypass of host defences

o To initiate an infection a virus must overcome host defences
o Defences may be overcome passively:
▪ By overwhelming inoculum of virus particles, swamping the host defences
▪ Some only take place through abrasions or cuts or needle sticks in skin
▪ Some viruses infect cells with a low exposure to immune system (skin)
o Attachment/adsorption of viruses to mucous membranes:
▪ Facilitated by virion attachment sites
▪ Cell surface receptors on the host cell
o After attachment the virus is not affected by presence of antibodies
o Enveloped viruses:
▪ Require intact envelope for attachment
▪ Envelope fulfils attachment function, not protective function
o Non-enveloped viruses:
▪ Attachment facilitated by capsid proteins
o Penetration of cells for most viruses takes place by means of receptor-mediated
endocytosis (phagocytosis)
▪ Exception: viruses that contain F (fusion) proteins in envelope
▪ These peplomers facilitate fusion of the host cell plasma membrane and the
viral envelope

− Routes of virus entry

I. Respiratory tract
• Most common route of viral entry
• Numerous host defence mechanisms against viral entry like:
o Mechanical barriers:
▪ Muco-ciliary blanket: from nasal passages to the airways in
the lung
Consists of layer of mucous that is kept in continuous flow
by the coordinated beating of cilia
 Particle size Up to where does is reach
>10 m Trapped on nasal mucosa
5-10 m Carried to trachea and
bronchioles: trapped on
mucous blanket
<5 m Reach alveoli by means of
inhalation

o Cellular and Humoral immune responses

• Virus examples:
o Rhinovirus
o Influenza virus
o Foot-and-mouth disease
o Canine distemper virus

II. Digestive system

• Ingestion of contaminated food or drink
• Extremely hostile environment for virions:
o Stomach is acidic
o Intestines alkaline
o Lumen contains digestive enzyme and bile acids
o Surface epithelium lined by mucous, antibodies and phagocytic cells
• So virions must at least be:
Example: poliovirus is acid-resistant and establishes an infection in
o Resistant to extremes in pH
the upper intestine
o To proteases
o To bile But rhinovirus (who is from the same family) is acid-labile
(destroyed in acid environment) and cannot infect the intestine only
the respiratory tract

• Hostile environment is sometimes used by viruses to facilitate infection

o Rheoviruses are converted by proteases in the intestinal lumen into
infectious subviral particles: which are the form that infects
epithelial cells
• Mechanism used by some enteric viruses to cross the epithelial barrier
explained:
o Lymphoid follicles (Peyer’s patches) in the intestinal tract are
covered on the luminal side by M cells (microfold epithelial cells)
o M cell cytoplasm is very thin:
▪ Creates a membrane like bridge between the lumen and the
subepithelial space
o Material taken up by M cell on luminal side transverses the
cytoplasm and is delivered to the underlying lymphoid tissue,
virtually intact: called TRANSCYTOSIS
▪ Some enteric viruses use this mechanism to cross the
epithelial barrier (Rheovirus)
▪ Others replicate in the M cells and do not spread to the
underlying tissues (coronavirus)
• Examples:
o Poliovirus
 o Rheovirus
o Parvovirus
o Rotavirus
o Coronavirus
o Bovine viral diarrhoea virus

III. Urogenital tract

• Via genital tract common since many viruses may be present In semen
• Protected by:
o Mucous
o Low pH (vagina)
• Microtears during sex can provide an entry point for viruses
• =venereal transmission

• Placenta: viruses transmitted vertically from mother to foetus

o Viruses can cross the placental barrier by growing over the placental
junction by means of:
▪ cell-to-cell spread
▪ injury to cells and leakage
▪ carried in migrating cells
o transplacental infection of foetus and outcome thereof depends on:
▪ stage of pregnancy
▪ tropism of virus for foetal cells
▪ duration and extent of maternal viraemia
o foetal infection of certain viruses may lead to: IMMUNE TOLERANCE
▪ during early pregnancy the foetus will not recognise the
foreign virus
▪ so antiviral antibodies are not produced
▪ animal is born persistently viraemic without ever developing
a detectable immune response
▪ e.g. bovine diarrhoea virus
• examples:
o bovine/ equine herpesvirus
o papillomavirus
o equine arteritis virus

IV. Epithelial barriers

• Viruses can cross the epithelial barrier by means of
o mechanical injury
▪ papillomavirus
o iatrogenic
▪ needle injections
o bite of animals
▪ rabies virus
o insect bites
▪ insect vectors are either
i. biological vectors
 • arthropods that support the replication and
amplification of viruses in their tissues
before transmission
• midges and bluetongue virus
ii. mechanical vectors
• become contaminated during the feeding
process on Infect hosts and then inoculate
host
the virus into a new virus by subsequent
feeding
• biting insects and lumpy skin disease
V. conjunctiva
• much less resistant to viral invasion than skin
• it is constantly cleased by:
o flow of tears
o mechanical wiping of eyelids
• e.g.
o adenoviruses
o enteroviruses
o substantial number of viruses can be experimentally entered via this
route

− Virus spread
o After replication at site of entry:
▪ Viruses can remain localised
▪ Or spread to other tissue
o Local spread: may be the result of cell lysis and release of viral particles although
lysis is not needed for release of viral particles
o Fusion of host cells makes it possible for viruses to move directly into new host cells
o Some viruses bud from cellular membranes without destroying the cell
o Systemic spread of viruses:
▪ Facilitated by drainage to the regional lymph nodes via lymphatic vessels
▪ Lymphatic capillaries are considerably more permeable than circulatory
system capillaries
▪ In the regional lymph node:
• Virions may be taken up by leucocytes and inactivated
• Other viruses may replicate in these cells:
o Retrovirus
o Canine distemper virus
▪ After replication in the regional lymph nodes spread to other organs take
place through the bloodstream. Either:
• Free or
• Within leucocytes
o Viraemia: describes the presence of infectious virus in the blood
▪ Passive viraemia
• Virus present in the blood without viral replication at the site of
entry (inoculation)
▪ Active viraemia
 • Virus present in the blood as a result of replication
▪ Primary viraemia
• Virus released into blood after initial replication at the site of entry
• Clinically silent
▪ Secondary viraemia:
• Subsequent systemic infection of target organs results in a much
higher concentration of virus in the blood

− Organ invasion
o Once virus is in the blood it needs to invade new cells
o TROPISM: Restrictions of the cell or tissue type the virus can infect
▪ On the cell surface the receptor population determines whether the cell can
be infected by a specific virus
o There are 3 main blood vessel-tissue junctions that serves as routes of tissue
invasion:
I. Sinusoids
▪ Where are sinusoids present?
• Liver, spleen, bone marrow, adrenal glands
• Characterised by presence of sinusoids lined by macrophages of the
mononuclear -phagocyte system
o Known as Kupffer cells in the liver
o Virions may infect the Kupffer or endothelial cells
themselves
o Or may be transcytosed across these cells to infect the
underlying hepatocytes

II. Fenestrated endothelium

▪ Where is this found
• Choroid plexus
• Villi of the intestine
• Renal glomerulus
• Pancreas
• Endocrine glands
▪ To enter tissues that lack sinusoids: virions must first adhere to cell
receptors on endothelial cells where blood flow is the slowest
• Some viruses cross the endothelium while being carried by infected
leucocytes, called: DIAPEDESIS
• Or can be transcytosed across endothelial cells

III. Continuous endothelium

▪ Where is this found
• Central nervous system
• Connective tissue
• Skeletal and cardiac muscle
• Skin
• Lungs
▪ The endothelium is continuous and backed by a dense basement membrane
 ▪ In CNS:
• Basement membrane is the foundation of the blood-brain barrier
• But in certain areas like the choroid plexus the basement membrane
is sparse and the endothelium is fenestrated
o Providing entry point for some viruses
o Like: mumps virus, togavirus
• Some viruses find their way into the CNS by:
o Trans-endothelial migration of infected leukocytes or
o Injury to endothelial cells with resultant leakage
• Deposition into astrocytes possible by means of pinocytotic vesicles
• Virus migration along long nerve trunks= well know mechanism of
migration of certain viruses
▪ Neurotropic virus: can infect neurons and can be: or
• Neuroinvasive: spread within nervous system
• Neurovirulent: cause disease of nervous tissue
o E.g. herpesvirus: neurovirulence but neuro-invasiveness
▪ Readily enters PNS but rarely CNS
▪ When it does consequences severe (fatal)
o Mumps: neurovirulence but  neuro-invasiveness
▪ Often infects CNS but neurological disease is mild
o Rabies: neurovirulence and neuro-invasiveness

VIRAL MECHANISMS TO EVADE HOST DEFENCES

− General mechanisms
I. Non-cytocidal infection
▪ Virus establishes chronic infection without killing cells within which they
replicate
▪ E.g.
• Arenavirus
• Hantavirus

II. Cell-to-cell spread

▪ Viruses cause adjacent cells to fuse together so that virus can spread
contiguously from cell to cell without being exposed to host’s immune
mediators
▪ E.g.
• Morbillivirus (canine distemper virus
• Herpesviruses

III. Infection of non-permissive, resting or undifferentiated cells

▪ Viruses may undergo productive replication in one cell type but non-
productive latent infection in another
▪ E.g.
• Herpesvirus assumes latent state in ganglionic neurons but
replicates productively in mucosal epithelial cells
 IV. Restricted gene expression
▪ Viral latency may be maintained by restricting expression of genes that have
the capacity to destroy the cell
▪ E.g.
• In latent infections herpesviruses only express a few genes to
maintain latency
• Reactivation from stress/ hormones causing whole genome to be
transcribed again

V. Evasion of neutralising bodies

▪ Viruses produce two surface proteins
• one forms part of the capsid
• other secreted extracellularly
o decoy
o appears to mop up neutralizing antibodies
• e.g. Ebolavirus
▪ proteins of some viruses are heavily glycosylated, and these carbohydrate
chains interfere with the ability of antibodies to neutralise the virus
• e.g. rift valley fever

VI. Induction of immune tolerance

▪ In prenatal viral infections the foetus may not recognise the virus as foreign
if the infection happens during the development of immune system, in later
life antiviral antibodies are not produced in response to natural infection
with the same virus
▪ E.g.
• Bovine viral diarrhoea virus

VII. Immunologically privileged tissues

▪ Infection of the CNS and epithelial cells confers some degree of protection
from the immune system to the virus
• Due to the blood-brain barrier (brain) and basement membrane
(epithelium)
▪ Cytomegaloviruses replicates in the
• Kidney
• Salivary glands
• Mammary glands
They are shed more or less continuously in the corresponding secretions
even when there are abundant neutralizing bodies and cytotoxic T-cells in
the surrounding tissues

VIII. Integration into the host cell genome

▪ Integration of retroviral proviral DNA into the host cell assures maintenance
of the virus

IX. Genetic/ antigenic drift

 ▪ Continuous mutation especially of RNA viruses enables the virus to evade
the immune response raised by previous variants
▪ E.g.
• Influenza virus

HOST MECHANISMS TO ELIMINATE VIRUSES

− Immune system
− RNA interference (RNAi)
I. Double stranded RNA viruses (e.g. African horse sickness virus (Reoviridae)) infects
cell
II. Early transcription of dsRNA genome occurs
▪ Where: inside the sub-viral particle (naked core)
▪ Enzyme: viral polymerase
▪ Why: so that the dsRNA is never exposed to the cytoplasm
III. Exposure to the cytoplasm would result in degradation of the RNA due to RNA
interference (RNAi)
IV. The RNAi pathway is found in many eukaryotes (including animals)
▪ Initiated by enzyme dicer which cleaves long dsRNA molecules into short
fragments

NECROSIS: a form of cell injury which

EFFECT OF VIRUS ON HOST CELLS
results in the premature death of cells in
− Cytopathic effect living tissue by autolysis
o Microscopically virus induced NECROSIS characterized by
APOPTOSIS: rapid, self-programmed
▪ Pyknosis
mechanism of cell death
▪ Karryorhexis
▪ Bursting of cell membrane
▪ Cells swell, rupture and release toxic cellular by-products
▪ Induce a strong inflammatory response
o Another form of cell death that is morphologically distinct from necrosis is
APOPTOSIS
▪ Cells gradually shrink in overall size
▪ Nuclear chromatin shown condensation and fragmentation
▪ Plasma membrane blebs
▪ Ultimately cell is fragmented into apoptic bodies
• These bodies are rapidly phagocytosed by surrounding cells
▪ Does not cause inflammatory response
o Vacuolisation
▪ In the cytoplasm of host and cavitations in tissues
▪ Easily seen by light microscopy
o Cell fusion
▪ Resultant syncytia formation
▪ Characteristic of many viruses
▪ Large amounts of virus can be produced in these giant cells
o Inclusion bodies
▪ Represent within host cells the sites of virus directed biosynthesis and
morphogenesis
 ▪ Detectable by means of most common cell staining procedures
▪ Can be seen with light microscopy in the:
• Cytoplasm
• Nucleus
• Or both

− Viral damage to tissues and organs

o The cytopathology caused by a virus in tissue culture does not necessarily relate to
the severity of the disease in vivo
▪ some viruses are cytocidal in tissue culture but do not produce any clinical
signs
• many enteroviruses
▪ others are non-cytocidal in vitro but cause lethal disease in animals
• retroviruses and rabies
o in the respiratory tract
▪ viral infections start off with the destruction of a few epithelial cells and the
protective layer of mucous
▪ infection spreads contiguously from cell-to-cell
• aided by beating cilia of epithelium: help by transporting viruses
along tract
▪ large areas may ne denuded of epithelia
• results in accumulation of
o transudate
May block the passages o necrotic debris
o inflammatory cells

o in the gastrointestinal tract

▪ rotaviruses infect cells at the tip of the villi
▪ causes:
• marked shortening and
• occasional fusion of the adjacent villi
• so that the absorptive surface of the intestine is reduced
o resulting in:
▪ fluid accumulation in the in the lumen of the gut
▪ diarrhoea
▪ parvovirus infect and destroy dividing crypt epithelium
▪ causes:
• removal of the source of the short-lived villus epithelial cell
o viral infections can predispose the host to secondary bacterial infections
Respiratory tract ▪ e.g. parainfluenza virus 3 may destroy the ciliated epithelium allowing
Pasteurella hemolytica and other bacteria to invade the lungs (shipping
fever)
Gastrointestinal tract ▪ rota and coronaviruses may lead to an increase in susceptibility to
enteropathogenic Escherichia coli

− Persistent infection and chronic damage to tissues and organs

o Persistent infections
 ▪ Occur where infectious virus is demonstrable continuously in the circulation
whether or not there is an ongoing disease
▪ Persistent viruses important for several reasons
• May be reactivated and cause a recrudescent episode of disease in
the host
• May lead to immunopathological disease
• May be associated with neoplasia
• May allow viral survival in vaccinated animals
• May be epidemiologically important
o Movement of infected animals
o Reintroductions
▪ Many different patterns of persistent viral infections:
• Acute
• Latent
• Chronic
• Acute occasionally chronic
• Slow

o Latent infections
▪ Infectious virus is not demonstrable except when reinfection occurs
▪ E.g. herpes virus
o A lot of viruses have chronic manifestation

o Viral tumorigenesis
▪ Viruses can induce transformation in cells leading to neoplasia or tumour
formation
▪ Transformation is presumed to be a deregulation of cell growth by an
interaction between gene or gene products of the infecting virus and the
infected cell
▪ Transformed cells have
• an altered morphology
• changed behaviour
• altered biochemistry
THEME H1: INTRODUCTION TO FUNGI

Aerial mycelium: hyphal elements growing above the agar surface

Anamorph: an asexual state of a fungus

Asexual reproduction: spores (reproductive bodies of a fungus) are formed directly from the
vegetative mycelium or from specialized hyphae

Bud: a young conidium, usually used to denote the young blastoconidium of a yeast

Budding: asexual multiplication by the production of a small outgrowth or bud from a parent cell

Conidiophore: specialised hypha on which conidia develop

Conidiogenous cell: a cell that forms conidia

Conidium: an asexual reproductive propagule formed in any manner that does not involve
cytoplasmic cleavage. Function as organs of dissemination

Biphasic (dimorphic): ability of some fungi to grow as either yeast or filamentous stages, depending
on conditions of growth

Eukaryotes: organisms possessing a true nucleus (such as fungi) as opposed to prokaryotes who do
not contain a nuclear membrane (such as bacteria)

Hypha: a single filament of a fungus

Mold: term generally referring to filamentous fungi

Macroconidium: the larger of two different types of conidia formed by fungi in the same manner

Microconidium: the smaller of two different types of conidia formed by fungi in the same manner

Mycelium: the mass of hyphae making up the thallus of a fungus

Nonseptate: without septa (cross-walls)

Pleomorphic: having more than one form

Rhizoids: root-like structures

Sexual reproduction: zygote/ spore formation follows the fusion of two haploid nuclei

Septum: a cross wall in a hypha

Spore: generally the reproductive body of a fungus, occasionally a resistant body for adverse
environment

Teleomorph: the sexual form of a fungus

Thallus: vegetative body of a fungus

Yeast: pasty of mucoid form of fungus growth, microscopically shows a predominance of budding
cells
FUNGI CHARACTERISTICS:

• Eukaryotic
• Non-photosynthetic (heterotrophs)
o Produce enzymes and obtain nutrients through absorption
• Kingdom: Fungi
• Widely distributed in environment
• Cell walls contain chitin and other polysaccharides
• Branching hyphae (molds) and unicellular yeasts are the two major forms
Molds Yeasts
− Grow as branching filaments called − Unicellular
hyphae − Oval or spherical
− 2-10m − 3-5m

• Reproduce both sexually and asexually through the production of spores

• Grow aerobically at 25C
o Some molds are strict aerobes
• Majority are saprophytes, some cause opportunistic infections
• Dermatophytes are pathogens that cause ring worm in animals and humans

HABITAT:

• Fungi are found worldwide in soil and on animals

• They colonize skin, nails, and hair

CULTIVATION

• Fungi grow slowly on media

• Media used: Sabouraud dextrose agar (pH 5.5)
• Fungi tolerate high osmotic pressures
• Prefer low pH values
• Resistant to antimicrobial drugs
• After cultivation → identification of pathogenic fungi through direct microscopic
examination of wet preparations using morphology
• Stains:
o Lactophenol cotton blue
▪ For wet preparation to visualize structures for microscopy
o Methylene blue
o Gram stain
▪ For yeast

IDENTIFICATION

• Based on morphological features from direct microscopic examination of wet preparation

(from sample or culture)
• Differentiation of fungal species
o Colonial characteristics of fungi on media
▪ Size and appearance after incubation time
o Vegetative hyphae
 ▪ Presence or absence of septa
▪ Hyaline (colourless) or dematiaceous (pigmented)
▪ Specific hyphal structures such as racquet-shaped and spiral hyphae
o Examination of sporing heads for spore arrangements and type of morphology of
the spores
▪ Presence of mature sporangium identifies fungus as a zygomycete
o Form of sexual stage (teleomorph) is used for assigning a fungus to a phylum
• Most fungi species are identified according to their morphology and sexual reproduction
• Molecular and biochemical tests can be used to classify fungi spp.

BASIC MORPHOLOGICAL FORMS OF FUNGI

• Asexual reproduction:
o Two main types of spores formed:
1. Conidia
• Formed on conidiophores
i. Macroconidia
Formed in dermatophytes
ii. Microconidia
2. Sporangiospores
• Formed within a sporangium (a sac-like structure borne on an aerial
hypha termed a sporangiophore)
• Formed only by fungi in phylum Zygomycota
o In most yeasts asexual division is by budding
• Sexual spores: (fusion of nuclei during sporulation)
1. Ascomycota
• Forms ascospores
• in a sac-like cell known as an ascus
2. Basidiomycota
• Forms basidiospores
• Produced on a specialized club-like structure called a basidium
3. Zygomycota
• Forms a zygospore
• Thick-walled spore
o Deuteromycetes (fungi imperfecti): has no known sexual stage
H2: FUNGAL GENETICS

DIFFERENT WAYS OF FUNGAL REPRODUCTION

• Remarkable in that they produce many kinds of reproductive structures in many ways
• Produce spores both sexually and asexually
• A single fungus produces a single type of sexual spore

ASEXUAL REPRODUCTION

• Ability to produce asexually is common in all fungi

1) Budding and binary fission
▪ Simplest form
▪ Onset of cellular events accompanied by nucleur events of mitosis
2) Fragmentation
▪ Any mycelium that is fragmented or disrupted can grow into a new colony
▪ Provided that:
o The fragment contains the equivalent of the peripheral growth zone
▪ Many are subcultured using this method
3) Sporulation (mitosis)
▪ Most NB
▪ Spore formation
▪ Asexual reproduction is important to fungi because:
o Produces a large number of spores throughout the year
▪ Spores are formed in anamorphic phase of fungal life-cycle
▪ There can be more than one anamorphic state for each spp. Of fungus
▪ And even for different species the anamorphic state can look very similar
▪ This contributes to the problem with creating a taxonomy for the fungi that
only possesses asexual states
Teleomorph state much more
SEXUAL REPRODUCTION stable and thus more reliable for
taxonomic purposes
• Sexual reproduction nuclei fuse to undergo meiosis
• This reduction division that potentially brings about variation in the progeny
• Events followed by formation of spores
o In most cases are resting spores that can withstand adverse conditions