ANIMAL MICROBIOLOGY AND PARASITOLOGY (WAP115) COURSE OUTLINE

1. INTRODUCTION

Diversity of micro-organisms, Microbes and human welfare

Beneficial and Harmful effects of micro-organisms

2. BACTERIOLOGY: General characteristics and classification

Morphology (cell structure, cell shape and arrangement, bacterial spores)

Bacterial Nutrition. Gene transfer in bacteria

3. MYCOLOGY: General characteristics and classification

Reproduction (asexual and sexual), General Life Cycles

Morphology (molds and yeasts), Dimorphism in fungi and nutritional adaptations

4. VIROLOGY: Characteristics and definition

Structure and composition

Virus multiplication and classification

Introduction: vaccines, immunity, diagnostic procedures

5. PARASITOLOGY

Internal and external parasites

Classification (Nematodes, Trematodes, Cestodes, Protozoa)

Control, treatment and prevention of parasites

6. Control of Microbial Growth

The growth curve, Control of Microbial growth (Physical and chemical methods),

Introduction to Food and industrial microbiology, Food spoilage

ASSIGNMENT 1

State and explain the chemical and physical methods that are used to prevent food spoilage in the food industries. HIGHLIGHT the effects of these methods on bacterial growth. {20}

ASSIGNMENT 2

Outline the general mechanisms of bacterial pathogenecity. (15)

BACTERIOLOGY - This is the study of bacteria -

General characteristics of bacteria- Bacterium means little rod. This is a typical example of a prokaryote.- Simplest of all microbial cells and are single celled organisms (unicellular)

and microscopic measuring (0,3-1,4nm) - Ubiquitous in distribution. Extremely diverse and numerous in soils and water - Contain naked DNA in the cytoplasm in a region called nucleoid. - Rapid growth and cell division (binary fission) under favorable conditions - Adapt quickly to various environments (capable of colonizing most

environments) - Release nutrients by decomposing organic matter. They fix atmospheric

nitrogen in the soil making it available for use by plant. Also carry out processes that produces Vitamin K Facilitate the degradation of food in the bowel

- Bacteria play an important role in digestion of cellulose in ruminants and animals.

- Survive as commensals e.g. E.coli , Streptococcus pneumoniae- Harmful bacteria cause disease and food spoilage - The overall form of a basic bacterial cell is that of a complex cell envelope

that encloses the cell protoplasm. Cell appendages protrude from the envelope into the environment surrounding the cell.

Constituents of bacterial cell components Cell Appendages: Flagella, Pili, Fimbriae Cell Envelope: Glycocalyx, cell wall, cell membrane, Periplasma Cell protoplasm: Ribosome, Mesosomes, granules, Nucleoid, Plasmid.

1. CELL APPENDAGES In general external structure are composed of polysaccharides, proteins or a mixture of both, General functions are as follows:

a. Prevent desiccation or nutrient loss b. Aid adherence to surface hence enhance bacterial pathogenecity c. Prevent the action of antibodies and delay phagocytosis d. Act as bacterial surface receptors

a.Glycocalyx A fibrous network of polysaccharides and or protein (polypeptides) – glycoprotein. It is made up of extracellular polysaccharides (EPS). It is formed inside the cell and extruded outside. It is a viscous (sticky) and gelatin substance that can prevent dehydration and its viscous nature may inhibit movement of nutrients from the cell. It is either referred to as capsule/ slime layer. Capsule – This is a dense layer of highly organized and cross -linked rigid polysaccharides (and sometimes proteins) that are firmly attached to the cell e.g in Streptococcus pneumonia. Capsules can be a source of energy and nutrients to microbes.-Protects the bacterial cells from phagocytosis by other micro=-organisms and the cells of host organisms (white blood cells).Often associated with pathogenic bacteria that are not easily identified by the phagocytic cells. Hence a capsule is associated with virulence. In artificial culture capsulated bacteria appear as mucoid colonies. -Polysaccharide from capsules can be used in vaccine development since they can be targets of protective immune response. Slime layer –this is when the glycocalyx is loosely attached to the cell.

2. Flagella – (sing flagellum) hollow hair –like structures that extend from the cell surface. They are composed of the protein (Flagellin)Bacterial flagella consist of a filament and hook which pieces the cell wall and attaches to the base of ring – like structures (motor) that are located just under the cytoplasmic membrane. The flagella rotate and move the bacterium in a fashion similar to a propeller , Grain positive have one set of ring (2 rings) and Grain negative have two set of rings ( 4 rings) -Primary function of flagella is motility. They allow bacteria cells to move away from toxins and towards nutrients. (+ve “taxis” movement) -towards favorable environment (-ve taxis) movement away from un favorable conditions. In culture some bacteria with flagella show a rapid swarming type of growth on solid media. Proteus species Basic arrangement of flagella 1. Monotrichous – a single flagellum at one pole of the cell 2. Amphitrichous – one or more flagella at both ends of the cell. 3. Peritrichous – flagella around the entire cell e.g. E. coli 4. Lophotrichous – a tuft of flagella at one pole of the cell. 5. Internal flagellum (endoflagellum) found inside the periplasmic space of gram positive bacteria e.g. spirochetes

3. Fimbriae and Pili – short fine hair like structures These originate from the cytoplasmic membrane of gram negative bacteria. -Act as adhesions and allow bacteria to colonize cells especially eukaryotic cell sometimes causing diseases-Can detect chemical signals and are important in bacterial cell communication.-Act as receptors for viruses that infect bacteria (bacteriophages) -In some streptococcus species fimbriae act as an important virulent factor by adhering to host cells and resisting phagocytosis. -Bacteria are constantly losing and reforming pili as they grow in the body and may switch the adhesive tips of the pili in order to adhere to different surface and evade immune defenses. -Sex pili / F pili :these serve to join two cells together so that they can exchange genetic information (allow transfer of DNA from one cell to another -bacterial conjugation.

Cell Envelope Cell wall Functions of bacterial cell wall

1. Lends characteristic shape to the cell and maintains it 2. Act as a barrier protecting the cell contents from external environment3. Maintains cell integrity i. e protection against osmotic lysis 4. Serves as a primer for new cell wall synthesis 5. Provides a receptor site for viral and flagella attachment 6. Some cell wall components are endotoxins7. Contributes to sensitivity to certain anti-microbial agents and the immune

system( antibodies , phagocytes)

GRAM STAINING – MORPHOLOGY AND STAINING PROPERTIES

The most common staining procedure for bacteria. (Differential staining) 1) Apply methyl violent / gentian violet (primary stain) 2) Iodine (Mordant) 3) Apply ethyl acetone (alcohol) –decolourizer 4) Neutral red /dilute carbol fuchsin /safranin – counter stain.

Alcohol dehydrates the peptidoglycan resulting in a decrease in porosity of the cell wall.Gram (positive) : violet / blue in color and resist decolourazation because of the peptidoglycan in their cell walls . (PURPLE) Gram (negative): is decolorized –violet / blue color and take up the counter stain which is red. (PINK)This results in retention of the crystal violet iodine complex. Gram positive bacteria have a large peptidoglycan layer 60-90%. This appears as a broad, dense wall 20-80nm thick consisting of numerous interconnecting layers of peptidoglycan. Peptidoglycan murein / mucopeptide are the principle component. Peptidoglycan is made up of N-acetyglucosamine (NAG) and N-acetylmuramic acid (NAM) with a polypeptide coming off the NAM (amino acids and amino sugars) Peptidoglycan is laid down in cables of several cross linked glycan strands.Interwoven in the wall of grain position are teichoic acid and lipoteichoic acids. Teichoic acids extend through and beyond the rest of the cell wall and are polyalcohols composed of polymers of glycerol, phosphates , and the sugar alcohol ribitol and are covalently bound to the peptidoglycan . Teichoic acids covalently bound to the cytoplasmic membrane lipids are called lipoteichoic acids. The outer surface of the peptidoglycan is studded with surface proteins that differ with the strain and species of the bacterium. Functions of the Gram –Positive cell wall components

1. The peptidoglycan in the gram positive cell wall prevents osmotic lysis 2. The teichoic acid probably helps to make the cell wall stronger. 3. The surface protein in the bacterial peptidoglycan depending on the strain

and species , carry out a variety of activities including : - Functioning as enzymes Services as adhesins. Adhesins enable the bacterium to adhere intimately to host cells and other surfaces in order to colonize and resist flushing - Function as invasions –enable some bacteria to penetrate host cells - Aiding certain bacteria in resisting phagocytic destruction - Gram positive cell wall components are associated with Bacterial

Pathogenecity

Gram –Negative Cell Wall (Structure and composition)

1) A thin, inner wall composed of peptidoglycan that is generally 2-3 (nm) thick and contains just 2-3 layers of peptidoglyan. About 10-20% of the gram negative cell wall is peptidoglycan.

2) An outer membrane – appear as a lipid bilayer about 7 nm thick. It is composed of phospholipids, lipoproteins , lipopolysachrides ( LPS) 40% of the surface structure and protein Phospholipids are located mainly in the inner layer of the outer membrane , as are the lipoproteins that provides anchor for the outer membrane to the peptidoglycan.

- Lipopolyssaccharides that are located on the outer layer of the membrane, consist of a lipid portion called Lipid A embedded in the membrane and a polysaccharide portion extending from the bacterial surface.

- LPS also known as endotoxin.- There are pore -forming proteins called porins that span the outer membrane.

The porin function as a channel for the entry and exit of solutes through the outer membrane of Gram negative cell wall.

- The outer membrane is studded with surface proteins that differ with the strain and species of the bacterium.

Functions of the Gram – Negative Cell Wall Components 1) The peptidoglycan prevents osmotic lysis 2) The outer membrane is semi-permeable and acts as a coarse molecular

sieve, helps retain certain enzymes and prevent some toxic substances such as penicillin G and lysozyme from entering (protective role), protects the cell from phagocytosis.

3) The LPS adds strengths to the outer membrane 4) The outer membrane may also form vesicles that contain signaling

molecules , enzymes. This also allows for communication between Gram negative bacteria.

5) LPS cause a net negative charge on the surface of the cell 6) The surface proteins function as enzymes, adhesins , invasins (penetration of

host cells) and aid bacteria in resisting phagocytic destruction (side chains of LPS are capable of variation – allowing Gram negative bacteria to evade the immune response)

7) The LPS contribute to Bacterial Pathogenecity

Acid fast stain /Ziehl Neelsen Stain - Stain with hot carbol fuchsin for 5 minutes and then decolourise with

sulphuric acid and 95% ethyl alcohol. - Counter stain with methylene blue / malachite green which stain micro-

organisms that have not retained carbol fuchsin and also host cells and other structures providing a suitable background against which to see the red mycobacterium. E.g. Mycobacterium spp. M. tuberculosis, M. bovis

Acid –fast bacteria - The cell wall contains peptidoglycan in their cell walls and tends to stain

weakly gram positive. - Contain 60% mycolic acid, thick waxy material. - The microbes grow slowly as lipids present a barrier to nutrients into cells . - In addition to peptidoglycan these contain a large amount of glycolipids

(especially mycolic acids). The peptidoglycan layer is linked to arabinogalacton) arabinose and galactose) which is then linked to higher molecular weight mycolic acids. The mycolic acid layer is overlaid with a layer polypeptides and mycolic acids consisting of free lipids, glycolipids and peptidoglycolipids.

- Porins are also present and these are required to transport small hydrophilic molecules through the outer membrane of the acid –fast cell wall.

- The outer surface of the acid –fast cell walls studded with surface proteins that differ with the train and species of the bacterium.

Functions of the Acid –Fast Cell Wall Components - The peptidoglycan prevents osmotic lysis - The mycolic acids and other glycolipids also impede the entry of chemicals

causing the organisms to grow slowly and be more resistant to chemical agents and lysosomal components of phagocytes than most bacteria . There are far fewer porins in the acid -fast wall compared to the gram-negative cell wall and the pores are much longer . This is thought to contribute significantly to the lower permeability of acid-fast bacteria.

- The surface proteins depend on the strain and species including - A. functioning as enzymes - Serving as adhesions

Acid- fast cell wall contribute to bacterial pathogenecity Periplasma - gelatinous material between cell wall peptidoglycan and cytoplasmic membrane

- Contains hydrolytic enzymes for nutrient breakdown as well as periplsmic binding proteins to facilitate the transfer of nutrients across the cytoplasmic membrane. This is about 15nm wide .Periplasmic binding proteins facilitate the transfer of nutrients across the cytoplasmic membrane.

Plasmalemma (inner membrane / cell membrane) - Composed of lipid and protein - Phospholipids bilayer with polar head on either sides of the membrane -

selective permeable barrier -fluid in nature. - Substances are limited by pore sizes and the hydrophobic nature of the

membrane - Integral (transmembrane) proteins form channels and act as carriers.

Peripheral proteins can act as receptors and as enzymes for metabolic reaction such as ATP production ad photosynthesis

Endospores - These are non- reproductive structures that develop intracellularly, dense

Round oval in shape. - The ability to produce spores is a type of cell differentiation (one cell give

rise to one spore) - In culture spores are produced at the end of the logarithmic growth

phase ,when nutrients become limited and bacterial waste products have accumulated.

- Spore formation (sporulation) sporogenesis - The cell membrane invaginates and a copy of the DNA is pinched off into a

separate part of the cell called the forespore.- Across wall called a septum separates the spore from the rest of the cell.

- The spore is eventually protected with a heat resistant calculated spore coat which is surrounded by inner and outer spore, membranes. (plasma membrane and core wall)

- Beyond the core wall lies the spore cortex ( the major component is peptidoglycan)

- Outside the cortex lies the spore coat (composed of protein and inorganic phosphates)

- Inner coat is alkali , sensitive , the outer coat is resistant to alkali - A thin exosporium encloses the whole structure (composed of protein ,

carbohydrate and lipid) - Spores maybe located in the middle of bacterial cell (central

spores ) ,towards one end (sub-terminal spores ) or at the end of the cell terminal spores)

- Spores contain DNA , small amount of RNA and little free water. - There is reduced metabolic activity - Have thick walls and resistant to adverse conditions. They are resistant to

heat (can withstand boiling for an hour) desiccation, UV radiation, chemical disinfectants and toxic substances.

- The resistance of the spore increase the pathogenecity of bacteria - When conditions are favorable there is a rapid shift in metabolic activity and

a return to vegetative form of the cell. Examples of bacterial genera that have endospores, Bacillus, Clostridium, Sporosarcina Sporolactobacillus, Desulfotomaculum, Oscillospira and Thermoactinomyces