Bacteriology PDF - Veterinary Microbiology

**GENERAL VETERINARY MICROBIOLOGY** **VMC-211 (1+1=2)** **DEPARTMENT OF VETERINARY MICROBIOLOGY** **VETERINARY COLLEGE, AAU, ANAND** **YEAR - 2017** **\ BACTERIOLOGY** **INTRODUCTION** **Microbiology:-**Branch of biology science that deals with study of microorganisms, which are tiny organism...

**GENERAL VETERINARY MICROBIOLOGY** **VMC-211 (1+1=2)** **DEPARTMENT OF VETERINARY MICROBIOLOGY** **VETERINARY COLLEGE, AAU, ANAND** **YEAR - 2017** **\ BACTERIOLOGY** **INTRODUCTION** **Microbiology:-**Branch of biology science that deals with study of microorganisms, which are tiny organism that live around us and inside our body. It is derived from three greek words, Micro-small, bios-living, logos-to study Microbiology word given by Lois Pasteur, The Word microbes was given by Sedillot. **Microorganism/Microbes:**- is a simple, very small organism that cannot seen with naked/unaided eye but visible using microscope. Microorganism can be one cell or cluster of cells **Organism:**-Organism is a living thing that ingest and breakdown food for energy and nutrients, excretes food as waste and capable of reproduction **Types of microorganism** - **Pathogenic microorganism:-**Disease causing microorganism that infect human, animals and plant - **Non pathogenicmicroorganism:-**many microorganisms help to maintain homeostasis in our bodies and used in the production of food and commercial products **Branch of microorganism** - Microorganisms are organised into five fields or various branch of microbiology are Bacteriology, Virology, Mycology, protozoology and phycology - Bacteriology:- Study of bacteria - Bacteria are unicellular prokaryotes organisms, cell multiply by binary fission - Prokaryotic organism does not have true nucleus - - - - - - ***Smallest to largest.....*** - *Prions* - *Viruses* - *Bacteria* - *Fungi* **Veterinary Microbiology** Veterinary microbiology is concerned with the etiology (causation)pathogenesis (mechanism of attack on body), laboratory diagnosis and treatment of infection in the individual and with the epizootiology (study of mass disease among animals) and control or prevention of infection in the community. **Difference between prokaryotes and eukaryotes cells** +-----------------+-----------------+-----------------+-----------------+ | | | Prokaryotic | Eukaryotic | | | | cell | cells | +=================+=================+=================+=================+ | **1** | **Nucleus** | | | +-----------------+-----------------+-----------------+-----------------+ | | Nuclear | Absent | Present | | | membrane | | | +-----------------+-----------------+-----------------+-----------------+ | | Nucleolus | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Chromosome | Single circular | Many | | | | chromosome | | +-----------------+-----------------+-----------------+-----------------+ | | Nuclear | Binary fission | Mitosis | | | division | | | +-----------------+-----------------+-----------------+-----------------+ | **2** | **Cytoplasm** | | | +-----------------+-----------------+-----------------+-----------------+ | | Mitochondria | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Golgi apparatus | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Endoplasmic | Absent | Present | | | Reticulum | | | +-----------------+-----------------+-----------------+-----------------+ | | Lysosomes | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Pinocytosis | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | **3** | **Chemical | | | | | structure** | | | +-----------------+-----------------+-----------------+-----------------+ | | Plasma membrane | No | Contain | | | | carbohydrates, | carbohydrates, | | | | No sterols | | | | | | Contain sterols | +-----------------+-----------------+-----------------+-----------------+ | | Muramic acid | Present | Absent | +-----------------+-----------------+-----------------+-----------------+ | **4** | **Structures** | | | +-----------------+-----------------+-----------------+-----------------+ | | Cells wall | Include | Chemically | | | | peptidoglycan | simple | | | | | | | | | Chemically | | | | | complex | | +-----------------+-----------------+-----------------+-----------------+ ***Bacteria - what comes to mind?*** - Diseases - Infections - Epidemics - Food Spoilage - Only 1% of all known bacteria cause human diseases - About 4% of all known bacteria cause plant diseases - 95% of known bacteria are non-pathogens ***Benefit Microbes*** 1. Bacteria are primary decomposers - recycle nutrients back into the environment (sewage treatment plants) 2. Microbes produce various food products - cheese, pickles, sauerkraut, green olives - yogurt, soy sauce, vinegar, bread - Beer, Wine, Alcohol 3. Microbes are used to produce Antibiotics 4. Bacteria synthesize chemicals that our body needs, but cannot synthesize - B vitamins - for metabolism - Vitamin K - blood clotting 5. Biochemistry and Metabolism - Very simple structure - rapid rate of reproduction - provides "instant" data 6. Microbial Antagonism - Our normal microbial flora prevents potential pathogens from gaining access to our body 7. Insect Pest Control - Using bacteria to control the growth of insectsEg.*Bacillus thuringiensis* - bollworms - corn borers 8. Bioremediation - Using microbes to clean up pollutants and toxic wastes 9. Recombinant DNA Technology\ Gene Therapy\ Genetic Engineering - Bacteria can be manipulated to produce enzymes and proteins they normally would not produce - Insulin - Human Growth Hormone - Interferon 10. Microbes used in vaccine preparation 11. Microbes form the basis of the food chain - Marine and fresh water microorganisms **Branches of microbiology** - Veterinary Microbiology - Medical Microbiology - Air Microbiology - Aquatic Microbiology - Dairy Microbiology - Soil Microbiology - Industrial Microbiology \*\*\* **HISTORY OF MICROBIOLOGY** 1. **Roger Bacon (1220-1292) --** Disease is produced by invisible living creatures. 2. **Girolamo Fracastoro (1483-1553) --** Theory that invisible seeds caused disease. 3. **Antony Van Leeuwenhoek (1677) -**Observed Animalcule means \"little animals\".FIRST TO SEE MICROORGANISM. First to discovered microscope. 4.  [**Francesco Redi**](http://www.britannica.com/eb/article?eu=64582) (**1688**)-Disproved the idea of spontaneous generation 5. **[Edward Jenner](http://www.sc.edu/library/spcoll/nathist/jenner.html) (1796)-**Small pox vaccine Previous infection with cowpox could offer protection against smallpox. He collected scrapings of cowpox lesions from the fingers of Sarah Nelmes, a young milkmaid and injected it into James Phipps, an 8-year-old boy. James got mild fever and typical cow pox lesions.  A few weeks after recovery, Jenner injected James with the live smallpox virus and found that the boy was protected from the disease. Jenner invented the word \"Vaccination\" for his treatment (from Latin *vacca*, a cow). Pasteur adopted this word for immunization against any disease. 6. **Spallanzani -** no organism survive on boiling of broth for one hour and concluded air is essential for life. "Disapproval of spontaneous generation" 7. **[Louis Pasteur](http://www.accessexcellence.org/AB/BC/Louis_Pasteur.html) (1861)**-**Disproved spontaneous generation and given Germ theory of disease.** - [**Louis Pasteur**](http://www.accessexcellence.org/AB/BC/Louis_Pasteur.html)repeated series of experiments of Spallanzaniusing a swan neck sterilized flask and showed that contamination of nutrient broth when exposed to air resulted from microorganisms in dust particles setting on the fluid. - Pasteur also showed that microorganisms caused fermentation - a process used in baking and brewing. Pasteur solved the problem of rancid wines in France\'s vinyards. He **recommended sterile technique and *Pasteurization***  of milk and other food products - Pasteur studies on chicken cholera and discovered one could **attenuate (decrease virulence) cultures** and produce artificial vaccines. First to use word vaccine. - Developed **vaccines for fowl cholera, anthrax and rabies** (Joseph Meister a boy -- injected rabies vaccine). 8. [**Joseph Lister**](http://www.sjsu.edu/depts/Museum/lis.html)-**Antiseptic surgery** (1867). By spraying carbolic acid on surgical instruments, wounds and dressings, he reduced surgical mortality due to bacterial infection considerably. Lister used antiseptics on wounds and during surgery. He showed that the healing was faster with the antiseptic treatment. He was also the **first to isolate a pure culture by serial dilution:** *Bacterium lactis.* 9. **Robert Koch (1876)-**First proof of **Germ Theory of Disease**using *B. anthracis*organism First to cultivate anthrax bacteria outside the body using blood serum at body temperature 10. **[Robert Koch](http://nobelprize.org/medicine/laureates/1905/) (1881)-Growth of Bacteria on solid media**, The plate technique is used to isolate pure cultures of bacteria from colonies growing on the surface of the plate. 11. **Robert Koch(1882)-Koch's postulates using***Bacillus anthracis*organism Koch's postulates... i. The pathogenic microorganism must be present in every case of the disease but absent from healthy animals. ii. The suspected microorganism must be must be isolated and grown in pure culture. iii. The same disease must be reproduced when a pure culture of the agent is inoculated into a susceptible healthy animal. iv. The microorganism must be isolated again from the experimentally infected animal.  12. **John Tyndal (1876)-**Developed fractional sterilization (**Tyndallizatio**n) 13. **Christian Gram (1884):**develops the Gram stain. Bacteria which retain the violet dye are classified as gram-positive. The distinction in staining is later correlated with other biochemical and morphological differences. 14. **Fanny Hesse (1850-1934):-**Suggested use of agar as a solidifying materials for microbiological media. 15. **Robert Koch -** Agar as solidifying agent in bacteriology media. This suggestion was given by **Frau Hess.** 16. **Schroeder and Dusch (1854)** Introduce the use of cotton plug in microbiology 17. **[Paul Ehrlich](http://www.chemheritage.org/EducationalServices/chemach/ppb/pe.html)(1882)**-Developed acid-fast staining technique, Modern concept of chemotherapy. 18. **Ziehl (859--1926)--Neelsen (1854--1898):-** Modified the acid fast staining and named as the acid-fast stain used to identify acid-fast organisms, mainly Mycobacteria 19. **ElieMetchinikoff (1883)-**Discovered phagocytosis 20. **Christian Gram (1884)-** Developed Gram Staining technique to differentiate bacteria into Gram positive and Gram negative 21. **[R.J. Petri](http://www.hoslink.com/history2.htm#petrie) (1887)- **Invented Petri Dish 22. **Dmitri Ivanovski(1892)-** Discovery of firs viruses (Tobbaco mosaic virus) 23. **Loeffler and Frosch (1898) -** The first animal virus discovered Foot-and-Mouth-Disease virus 24. **[Alexander Fleming](http://www.pbs.org/wgbh/aso/databank/entries/bmflem.html) (1929)- **Discovery of Penicillin 25. **[Knoll and Ruska](http://www.britannica.com/eb/article?eu=66125)(1931)-**First electron microscope 26. Golden age of microbiology -- 1880-1900 1. Father of Microbiology - **Antony Van Leeuwenhoek** 2. Father of Modern Microbiology -[**Louis Pasteur**](http://www.accessexcellence.org/AB/BC/Louis_Pasteur.html) 3. Father of Bacteriology & Medical Microbiology- **Robert Koch** 4. Father of Immunology-- **Edward Jenner** 5. Father of Virology - **W. M. Stanley** 6. Father of antiseptic surgery - **Joseph Lister** **\*\*\*\*\*** **CLASSIFICATION OF MICROORGANISMS** - **Classification:-**Orderly arrangement of unit under study into groups of larger units. - **Taxonomy:**Classification of living organisms into groups - **Phylogenetic Classification System:**Groups reflect genetic similarity and evolutionary relatedness - **Phylogeny versus Taxonomy** - Phylogeny and taxonomy are not synonyms - Taxonomy -- classification, systematics of extant species - Phylogeny -- the history of evolution since the origin of species - One should not contradict the two with each other - - Classification of organisms - To establish relationship - It provides a common reference - Act as universal language of communication. **Two kingdom** **Carlluslinnoeus (1707-1778)-** First used classification system. Two kingdom, Plant and animals. **Three kingdom** **Haeckel (1866):-** Suggested third kingdom -- Protista which included unicellular microorganism (bacteria, fungi, algae & protozoa) that are neither plant nor animals. **Five Kingdoms System** **Living organisms are classified in five kingdoms(Whittaker,1969):**Based on nutrition requirement. i. **Monera:** Bacteria, originally classified as plants because they have cell walls. - **Bacteria** and cyanobacteria classified into kingdom **Monera.** ii. **Protista:** Algae, slime molds, protozoa (have a cell wall, not cellulose). iii. **Fungi:**Fungi, yeast, mold, mushrooms. In past were classified as plants because they\ have cell walls and are 'rooted' in ground. Multinucleated higher fungi. iv. **Plantae:** Complex multicellulareukaryotes, obtain nutrients by\ photosynthesis v. **Animalia:** Complex multicellular eukaryotes, obtain food by ingestion - Later on based on electron microscopy study cells are divided into two kingdom, Prokaryotic cells (Bacteria) and Eukaryotic cells (Fungi, algae and protozoa). **Difference between prokaryotes and eukaryotes cells** +-----------------+-----------------+-----------------+-----------------+ | | | **Prokaryotic | **Eukaryotic | | | | cell** | cells** | +=================+=================+=================+=================+ | **1** | **Nucleus** | | | +-----------------+-----------------+-----------------+-----------------+ | | Nuclear | Absent | Present | | | membrane | | | +-----------------+-----------------+-----------------+-----------------+ | | Nucleolus | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Chromosome | Single circular | Many | | | | chromosome | | +-----------------+-----------------+-----------------+-----------------+ | | Nuclear | Binary fission | Mitosis | | | division | | | +-----------------+-----------------+-----------------+-----------------+ | **2** | **Cytoplasm** | | | +-----------------+-----------------+-----------------+-----------------+ | | Mitochondria | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Golgi apparatus | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Endoplasmic | Absent | Present | | | Reticulum | | | +-----------------+-----------------+-----------------+-----------------+ | | Lysosomes | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | | Pinocytosis | Absent | Present | +-----------------+-----------------+-----------------+-----------------+ | **3** | **Chemical | | | | | structure** | | | +-----------------+-----------------+-----------------+-----------------+ | | Plasma membrane | No | Contain | | | | carbohydrates, | carbohydrates, | | | | No sterols | | | | | | Contain sterols | +-----------------+-----------------+-----------------+-----------------+ | | Muramic acid | Present | Absent | +-----------------+-----------------+-----------------+-----------------+ | **4** | **Structures** | | | +-----------------+-----------------+-----------------+-----------------+ | | Cells wall | Include | Chemically | | | | peptidoglycan | simple | | | | | | | | | Chemically | | | | | complex | | +-----------------+-----------------+-----------------+-----------------+ | | Eg | Bacteria | Fungi, algae | | | | | and protozoa | +-----------------+-----------------+-----------------+-----------------+ **The difference between a domain and a kingdom** - A domain is a taxonomic category above the kingdom level. The three domains are: Bacteria, Archaea, and Eukarya, which are the major categories of life. Essentially, domains are superkingdoms. - A kingdom is a taxonomic group that contains one or more phyla. The six kingdoms system arePlantae, Archaebacteria, Eubacteria, Protista, Animalia, and Fungi. - **THREE DOMAIN SYSTEM OF CLASSIFICATION** - The Three Domain system,proposed by **Woese** and others in 1978 is an evolutionary model of classification based on differences in the sequences of **nucleotides in the cell\'s ribosomal RNAs (rRNA),** as well as the cell\'s membrane lipid structure and its sensitivity to antibiotics. - **The three Domains are (i) Eubacteria/Bacteria (ii) Archaea (iii) Eucarya** - The domain **Bacteria** include all of the pathogenic and non pathogenic prokaryotes found in soil and water. - The domain **Archaea** include prokaryotes that do not have peptidoglycan in their cell walls and can live in extreme conditions. - They include **methanogens**; strict anaerobes that produce methane from carbon dioxide and hydrogen, extreme **halophiles**; which require high concentration of salt for survival and **hyperthermophiles**, which normally grow in hot environments - The domain **Eukarya** includeProtozoa, Fungi, Animals and Plants. - **Classification of Bacteria according to Bergey's manual** - 1927-Bergey's published **Bergey's Manual of Determinative of Bacteriology** - The manual grouped bacteria into phonetic groups - It is now its 9^th^ edition in 1984 - Taxonomic classification schemes of bacteria identified using Bergey'sManual - Bergey'sManual -- 1984, bacteriabelongs to kingdom prokaryotes has four division - **Criteria for characterization and classification of microorganisms as per Bergey's Manual** i. Morphology -- Shape, size, spore ii. Cultural characters - colony characters iii. Staining characters -- Gram positive and Gram negative iv. Metabolism -- Biochemical reaction v. Oxygen requirement -- aerobic and anaerobic vi. Growth characteristics -- Colony size, shape, colour vii. Serological reaction -- Antigen -- Antibody reaction viii. Phage typing -- Using bacteriophage to identify bacterial strains ix. Genetic characterization -- Analysis of DNA components. DNA base is a constant character of a particular species and express as mole % G+C, range 23-75% x. DNA hybridization -- closely related organisms xi. Ribosomal RNA hybridization -- Distantly related organisms **Naming of Microorganism** - Carlous Linnaeus (Carolus Linnaeus, also called Carl Linnaeus, Swedish Carl von Linné) - firs to gave "Binomial Nomenclature" (two words) - Two latine name to identify each organism - Principle of nomenclature as follows - Bacteria of each distinct kind designated as species - Species name has two words first name *Genus* and second *Species* - Genus -- Noun, always first letter capitalized, - Species -- Adjective, lower case - Both italicized or underline - The basic taxonomic group (taxon) is the species i.e. a collection of strains having similar characteristics. - Bacterial species consist of a special strain called **type strain**. - Genus is composed of a collection of similar species. Genus is composed of a collection of similar species Family:- A group of similar genera Order:- A group of similar families Class:- A group of similar orders Division:- A group of similar classes Kingdom:- A group of similar divisions **Hierarchical classification** - **Taxon:** A group or "level" of classification - **Hierarchical;** broad divisions are divided up into smaller divisions: - Kingdom (Not used by most bacteriologists) - Phylum (Called "Division" by botanists) - Class -- **siffix-- a** *Scotobacteria* - Order-- **siffix-- ales** *Spirochaetales* - Family-- **siffix--aceae** *Leptospiraceae* - Genus **(plural: Genera)** *Leptospira* - Species (Both singular & plural) *L. interrogans* \*\*\*\*\* **MORPHOLOGY** **SIZE OF BACTERIA** - Micrometry:- Accurately measure the size of bacterial cell - The size of bacteria is expressed in term of a 'micron (micrometer) µ' - 1µ = 1/1000mm and one thousands of millimeter - 1nanometer (millimeter) =1/1000µm, one thousandth of a micron or one millionth of a millimeter - The resolution power of unaided eye is about 200µ and that of an ordinary light microscope is 0.1µm - General size of bacteria pathogenic in nature is measure in range from 3-5µm (length) x 0.2-1.5µm (diameter) **SHAPE AND ARRANGEMENT OF BACTERIA** - Shape of bacteria is due to rigid cell wall - The type of cellular arrangement is determined by the plane through which binary fission takes place and tendency of the daughter cell to remains attached even after division - Depending of their shape, bacteria are classified into several varieties 1). Cocci/coccus--Coccus--Greek word kokkos, means a berry, are spherical or oval cells - On the basis of arrangement of individual organisms, they are given descriptive arrangement such as i. ii. iii. iv. v. 2\) Bacilli -- Bacillus meaning rod, Straight rods-bacilli (bacillus) *Latin word bacillus, means a stick* - On the basis of arrangement they further subdivided as vi. vii. viii. ix. 3\) Spirilla -- are rigid spiral forma 4\) Spirochetes -- Speira meaning coil and chaite meaning hair. It is flexuous spiral forms. Spiral shape having several coils. EgLeptosipra 5\) Actinomycetes -- acts meaning ray, mykes -- fungus. They are branching filamentous bacteria. They are termed such because of their resembling to sun rays when seen in tissue lesions. Eg. Actinomyces 6\) Pleamorphic -- Mycoplasma (cell wall deficient) lack in rigid cell wall. Mycoplasma are highly pleomorphicand of indefinite shape. They occur as round or oval bodies and interlacing filamentous. Straight rods-bacilli (bacillus) *Latin word bacillus, means a stick* Helically curved rods-spirilla (spirillum) 03\_20 **ARRANGEMENT** Bacteria are usually arranged in a manner characteristic of their species. **Cocci** It occurs in several characteristic arrangements depending on the plane of cellular division and whether daughter cells stay together following division. - Diplococci: cells divide in one plane and remain attached predominantly in pairs. - Streptococci: Cells divide in one plane and remain attached to form chains. - Tetracocci: Cells divide in two planes and characteristically form groups of four cells. - Staphylococci: Cells divide in three planes, in an irregular pattern, producing "bunches" of cocci - Sarcinae: Cells divide in three planes, in a regular pattern, producing cuboidal arrange ment of cells.   **Bacilli\ \ **They occur mostly singly or in pairs (diplobacilli). Some form chains (streptobacilli) (eg: *Bacillus subtilis*). - Some trichomes -- similar to chains but have a much higher area of contact between adjacent cells. (eg: *Saprospira species*). - Palisade arrangement -- cells are lined up side by side like match sticks and at angles to one another( corynebacterium) - Streptomycis species form long,branched,multinucleate filaments called hyphae. The hyphae collectively form a mycelium. - Curved rods are usually curved with a twist or turn. Bacteria with less than one complete twist or turn are called vibrioid (eg: vibrio). Those with one or more complete turns have a helical shape. - Spirilla : rigid helical bacteria. - Spirochetes: highly flexible. They can twist and contort their shape. BACTERIAL ARRANGEMENT **STRUCTURE/ANATOMY OF BACTERIAL CELL** - Bacteria are classified under kingdom Monera, fferences in cellular organization & biochemistry. - Bacteria and blue green algae are prokaryotes, while fungi, mould, protozoa and other algae are eukaryotes. The basic components of bacterial cells include: A. Structure that form the cell envelop enclosing cytoplasm: 1) cell wall, 2) cytoplasmic membrane B. The cytoplasmic constitutes: 1) DNA, 2) Ribosomes 3) Mesosomes, 4) storage granules C. External structures that project through or cover cell envelop:1) capsule 2) flagella 3) pilli **Essential structures:-** required for survival of organism Eg. Nuclear material, cell membrane, cell wall, certain cytoplasmic components **Accessory structures:-**required for survival and functioning but not essential for survival eg. Flagella, Pilli, Capsule & Spore A. Cell envelop: 1. Cell wall: Definition:-Cell wall is a tough and rigid structure, surrounding the bacterium like shell. - The rigid cell wall provides protection and imparts shape to the cell. - Cell wall is constitute 10-40% of dry weight of call wall. - The rigid part of cell wall is peptidoglycan(murein). - Peptidoglycan is a principle structural component of cell wall & is mixed polymer consist of alternating strand of N-acetylmuramic acid (NAM) & N-acetylglucosamine(NAG) which cross linked by peptide chain. - NAM & NAG linked to short peptides of identical chain of four amino acid ( L-alenine, D-alenine, D-glutamate & diamino acid.)   - **Cell wall of gram positive &gram negative bacteria:** - Both contain peptidoglycan 1. Gram positive bacteria: - It has simple but thick cell wall - Peptidoglycan layer is thicker - Most of gram positive bacteria contain special component like teichoic acid made up of glycerol/ribitol. - **Mycobacterium** have **high lipid** content in the cell wall called **mycolic acid** which gives acid fast character. - Due to high peptidoglycan content in the cell wall of Gram positive bacteria they are susceptible to lysozyme and penicillin. 2. Gram Negative bacteria: - Contain complex cell wall structure. - Cell wall thinner than gram positive, with less peptidoglycan & no teichoic acids. Outer membrane: - It has additional outer membrane that covers the cell wall - Outer membrane is thicker than the peptidoglycan layer & is composed of lipoprotein &lipopolysaccharide (LPS-endotoxin) attached to the peptidoglycan. - Periplasmic space is present in Gram negative bacteria. - The outer membrane contain "O" antigen & the endotoxin that is characteristic of Gram negative bacteria. - **Porins:-**Porins are small pores in outer membrane. **Differences between Gram positive and Gram negative** --------------------------------------------------------- --------------- --------------- Gram positive Gram negative Structure Simple Complex Thickness Thicker Thinner Variety of amino acids Few Several Lipid Absent/scant Present Teicoic acid Present Absent Periplasmic space Absent Present  - Removal of cell wall is result in cell lysis → Osmotic pressure - Protoplasts: complete removal of gram positive cell wall result in formation of protoplast, which lysis unless it is osmotically stabilized. - Spheroplasts: partial removal of cell wall of gram negative bacteria with damages cell wall called spheroplast. - L- forms: Lister Institute of London- wall less organisms. Cell wall deficient form of bacteria. Because of lake of cell wall- L-form do not have regular size & shape & it is pleomorphic e.g.:**Mycoplasma (naturally lack peptidoglycan)** - Cytoplasmic membrane/cell membrane: Cytoplasmic/ plasma membrane is a thin, elastic, semipermeable layer & lies beneath the cell wall separating it from the cell cytoplasm. a. Cytoplasmic membrane exhibits well defined selective permeability. b. Electron transport system, the principle energy system located on cytoplasmic membrane. c. Mesosomes are complex invaginations of the cytoplasmic membrane are seen in many but not all bacteria. d. Anchor to bind a pull apart daughter chromosomes during cell division. e. Principle sites for respiratory enzyme & analogous to mitochondria. B\) Cytoplasmic components: DNA, Ribosomes, storage granules & viscous water solution. 1\. DNA: I. Lack nuclear membrane II. Concentrated in the cytoplasm as a nucleoid. The nucleoid consist of one long, double stranded,circular, coiled DNA molecules. DNA is haploid III. Feulgen staining use to seen under light microscope. IV. Plasmid/episomes. - Some bacteria may contain extranuclear genetic material in the cytoplasm consisting of circular DNA called plasmid. - Plasmid is circular but much smaller than bacterial chromosome. - Plasmid carries antibiotic resistant(R factor) genes &toxogenicity. 1. Ribosomes: - Ribosomes: 70s - Two subunit: 50 svedberg units or 50s & 30s - Protein synthesis function - External structure: Capsule, flagella, &pilli are found outside cell envelope. 1\. Capsule/glycocalyx: - It is an outer covering of thick jelly like material that surround the bacterial cell wall. - Firmly attached to cell surface called capsule or loosely attach called slime layer. a. Most bacterial capsules are composed of complex polysaccharide. b. Some bacteria have polypeptide capsules composed of glutamic acid amino acid e.g.: *Bacillus anthracis* c. Negative stain : 1) India ink, 2) Negrosine dye d. Loefflers alkaline methylene blue stain and Hiss copper sulphate stain are used for capsule stain. Quelling reaction: When suspension of a capsulated bacterium mixed with specific anti-capsulated serum the capsule become 'swollen' due to increase reactivity called capsule swelling or quelling reaction. **Functions:** - Protective layer against antibacterial substances such as bacteriophage, phagocytes & enzymes(antiphagocytic activity) - Protection against temporary dryness - Blocks attachment of bacteriophage - Enhance bacterial virulence. - Antigen capsule called 'K' antigen. 2. Flagella: - Hair like filamentous helical cytoplasmic appendages protruding through cell wall but originate from cytoplasmic membrane. - It is unbranched long thread like structures composed of protein. - The protein called flagellin. - Stain:-Leifson's method of staining is used. - Each flagellum consists of three distinct parts- **Filament, Hook and Basal Body.** **Function:** It is an organ of locomotion & responsible for motility of bacteria. Types of flagell: i. Monotrichous: single polar flagellum(*Pseudomonas aeruginosa*) ii. Amphitrichous: singleor clusters of flagella attached to each end. (*Pseudomonas fluorescens*) iii. Lophotrichous/Periplasmic/Axial fibrils: tuft of flagella at one or both end(*Pseudomonas* spp; *Vibrio*spp) iv. Endoflagellum: amphitrious flagellum is tightly wrapped around spirochetes. v. Peritrichous: numerous flagella all over their surface(*Salmonella,E. coli, Proteus*) vi. Atrichous: no flagella.  3. **Fimbriae/pilli/pilus:** - These are thin short filamentous appendages extruding from the cytoplasmic membrane & extended through cell wall. - Found only in some Gram negative bacteria. - Protein called pilin **Types:** a. Common pilli b. Sex or 'F'(fertility) pili(bacterial conjugation) c. Col 1 colicin pilli. **Function:** i. Bacterial adhesion/attachment to the surface of cells. ii. Transfer of genetic material(by using sex pilli/F pilus). \*\*\*\*\* **BACTERIAL SPORES** - Under unfavorable conditions like starvation some bacterial cells transforms themselves into spores. - *Bacillus* and *Clostridium* are the two most common endospore-forming genera - Spore are metabolically dormant form of organism that are resistant to high temperatures, radiation, drying and chemicals because of very low water content & high concentration of **calcium dipicolinic acid**. - Resistance of Bacterial spore may be mediated by dipicolinic acid, a calcium ion chelator found only in spores. - Each bacterium form one spore, which on germination forms a single vegetative cell. - Sporulation in bacteria is **not a method of reproduction** - Spores remain viable for centuries. - Fully developed spore has core the nuclear body, surrounded by the spore wall, a delicate membrane from which the cell wall of the future vegetative bacterium develops. - Outside the spore wall surrounded by thick spore cortex which enclosed by a multilayer though spore cells. - Some spores have additional outer covering called exosporium. - Stain:- Schaeffer-fulton method used for staining of spore  **Types of spore** - Spores are endospore and exospore - Certain species of bacteria produce spores either within the cell (endospores) or external to the cell (exospores) i.e. spores external to vegetative cell. - **Central or equatorial, giving the bacillus a spindle shape (eg.*Clostridium bifermentans*)** - **Sub-terminal, the bacillus appearing Club shaped (eg. *Clostridium perfringens)*** - **Oval and terminal, resembling a tennis racket (eg. *Clostridium tertium*)** - **Spherical and terminal, giving a drumstick appearance (*Clostridium tetani*)** **Sporulation in bacteria** **The process of endospore formation is called sporulation** - **Steps in sporulation** 1. **Vegetative cell** 2. **Chromosome is duplicated and separated** 3. **Cell is septated into a sporangium and forespore** 4. **Sporangium engulfs forespore for further development** 5. **Sporangium begins to actively synthesize spore layers around forespore** 6. **Cortex and outer coat layers are deposited** 7. **Mature endospore** 8. **Free spore is released with the loss of the sporangium** 9. **Germination spore swells and releases vegetative cell** **Germination of spore:** The formation of vegetative state of bacterial cell from an endospore is called germination - Under favorable condition spore loses its refractivity & swells. - Spore wall is shed by rupturing the spore coat & elongates to form vegetative bacterium. \*\*\*\*\* **BACTERIAL NUTRITION** **Nutritional requirements** - All organisms require a source of energy. Those dependent on chemical compounds for energy are called *chemotrophs*. Those which use radiant energy are called phototrophs. Both types are present in bacteria. - All organisms require a source of electrons. Based on the source of electrons bacteria can be grouped as - Lithotrophs use reduced inorganic compounds as electron donors. Chemolithotrophs and photolithotrophs. - Organotrophs use organic compounds as electron donors. Chemoorganotrophs and photoorganotrophs. - All organisms require carbon in some form for synthesis of cell components. Based on the source of carbon bacteria can be grouped as - Autotrophs use CO~2~ as major or even sole carbon source. - Heterotrophs require organic compounds as their carbon source. **Energy source** **H/ electrons** **Carbon source** -------------------------------------------------------- ------------------------------------ ------------------ ------------------- **Photo lithotrophic autotrophs/** Photoautotrophs Light Inorganic CO~2~ **Chemolithotrophicautotrophs/**Chemoautotrophs Chemical energy Source (inorganic) Inorganic CO~2~ **Photo organotrophic heterotrophs/**Photoheterotrophs Light Organic Organic compound **Chemo organotropic heterotrophs/**Chemohetrotrophs Chemical energy source (organic) Organic Organic compounds **\ ** - Bacterial growth involves both an increase in the size of the organisms as well as an increase in the number of individual. The net effect is an increase in the total mass of the culture - The minimum nutritional requirements comprises of the carbon, nitrogen, hydrogen, oxygen and inorganic salts. - Bacteria are autotrophs or heterotrophs on the bases of synthesizing capability i. Nutritional requirements ii. Environmental conditions regulating growth i. Nutritional requirements - The most important elements necessary for synthesis of bacterial structural components like carbohydrate, lipid, protein, and nucleic acid are hydrogen, oxygen, carbon and nitrogen. - Phosphorus and sulphur are also required for bacterial growth - Trace elements like K, Ca, Mg, iron, Cu, Co are requires for enzymatic function - Growth factors or bacterial vitamins are also required in some bacteria. ii. Environmental conditions regulating growth / affecting growth a. Water:- 80% of the bacterial cell consist of water b. Oxygen:- According to oxygen reqirement it divides into (A) aerobes and (B) anaerobes A. Aerobes:- require oxygen for growth - **Obligate aerobes**:- Grow only in presence of oxygen Eg. Pseudomonas and Bacillus - **Facultative anaerobes**:- they are aerobes but can also grow without oxygen but less abundantly Eg.*E. coli* - Most of pathogenic bacteria are fucultative anaerobes - **Microaerophilic/Microaerophiles:-** Grow in presence of low oxygen tension. Grow with trace of both oxygen and Co~2~Eg. Streptococci B. Anaerobes:- Grow only in absence of oxygen. Oxygen is toxic for them and die on exposure to oxygen. Eg. Clostridium and Bacteroides - **Non stringent or tolerate anaerobes:-**Some bacteria can tolerate low levels of oxygen called **non stringent or tolerate anaerobes** - **Stringent or strict anaerobes:-** Some bacteria cannot tolerate even low levels and may die upon brief exposure for air **stringent or strict anaerobes** c. Carbon dioxide:- Small amount of Co~2~ is required **Capnophilic organism/Capnophiles:-** Those organism which requires 5-10% Co~2~ for their growth. Eg.Haemophilus and *Brucella abortus* d. Temperature:- Temperature at which growth occurs best is called optimum temperatue - Optional temperature range varies for growth of bacteria - **Psychrophiles**:- able to grow below zero degree, although better at 15^o^C-20^o^C. Optium temperature is 15^o^C. Eg. Certain Pseudomona species - **Mesophiles:-**Grow best at 25^o^C-40^o^C. Most of pathogenic bacteria grow best at **37^o^C**, close to body temperatre. - Thermophiles:- Grow best between 45^o^C-60^o^C or above 45^o^C. Optimum temperature is 60^o^C. Eg*Bacillus stearothermophilus* e. Hydrogen ion concentration (pH):- Majority of pathogenic bacteria grow best at optimum **pH 7.2-.7.4.** f. Light:- Bacteria (except phototrophic species) grow well in the **darkness** g. Osmotic pressure:- Bacteria are able to withstand a wide range of external osmoticpressue due to mechanical strength of cell wall. - **Halophilic bacteria:-**Requires 10-15% salt (NaCl) in growth medium.*Staphylococcu aureus* require 7.5% sodium chloride in the medium **\*\*\*\*\*** **\ ** **REPRODUCTION AND GROWTH RATE OF BACTERIA** REPRODUCTION - Bacteria divide by transverse binary fission in which a single cell divides after transverse septum - When a bacterial cell reaches a certain size, it divides to form two daughter cells - Transverse binary fissionis an asexual reproduction process Figure:-Binary fission in bacteria  GENERATION TIME / POPULATION DOUBLING TIME - Interval of time between two cell division or time required for bacterium to give rise to two daughter cells under optimum condition - *E. coli*-- 20 min - *Staphylococcus aureus --* 30min - *Mycobacterium ­*-- 20hrs - *Lepra bacilli --* 20 days BACTERIAL COUNT / MEASUREMENT OF BACTERIAL COUNT - **Total count or Total growth:-**Total number of bacteria in the sample, including living as well as dead bacteria - Methods for total count are i) Direct microscopy ii) Direct counting in stained smear - **Viable count:-**Measure the number of living organisms in the sample which are capable of multiplication - Methods are i) Dilution method ii) Plating method **BACTERIAL GROWTH CURVE** - When a bacterium is seeded into a suitable medium and incubated, it growth follows a definite pattern. If bacterial counts are made at interval after inoculation and plotted in relation to time, a growth curve is obtained. - The growth curve has four phasesi) Lag phase ii) Log phase iii) Stationary phase and iv) Decline phase - There is an initial period of what appears to be no growth (the lag phase), followed by rapid growth (the exponential phase or logarithm phase), then a leveling off (stationary phase) and finally a decline in the viable population (decline or death phase). Between each of these phase there is a transition period (curved period). **i) Lag phase** - Addition of inoculum does not follow doubling of population which remains unchanged - During this stage individual cells increase in size, synthesize new protoplasm, enzymes and coenzymes and adjust in the surrounding environment **ii) Log phase/ Logarithm phase/Exponential phase** - Maximum rate of cell division and increase in cell mass - Population is nearly uniform in chemical composition of cell and metabolic activity **iii) Stationary phase** - Log phase decline after some time in gradual manner due to depletion of nutrient and accumulation of toxic substances - In this phase there is balance between cell growth, division and cell death. - Population remain constant **(iv) Decline phase / Death phase** - Bacterial lyses and cell destruction faster leads to reduction in cell number due to constant accumulation of toxic materials and stoppage cell division - Beside nutritional depletion and toxic accumulation, cell death may also be caused by autolytic enzymes - Hence for storage of culture, periodical sub culturing of the organism is done into fresh medium to restore the nutrient and other requirements of the medium \*\*\*\*\* **Definition** - - - - - - **TYPES OF MEDIA** **(i) Classification according to composition of media** **a) Chemically or synthetic defined medium:-** the exact chemical composition of the medium is known - A medium **composed of known chemical compounds**. - It is needed for cultivation of autotrophs. - It is also useful for defining nutritional requirements of heterotrophs. **b) Complex medium or non-syntheticmedium:-**the chemical composition of the medium is not known - For routine cultivation of heterotrophs raw complex materials like peptones, meat extract and yeast extract are used in the media. Such media support the growth of many heterotypic bacteria. - *Beef extract* - It is an aqueous extract of lean beef tissues concentrated to a paste. - It contains water soluble substances from animal tissues. This includes carbohydrates, organic nitrogen compounds, water soluble vitamins and salts. - *Peptone* - It is prepared by acid or enzymatic digestion of proteinaceous substances like meat, casein and gelatin. - Peptone is the principle source of organic nitrogen. - Vitamins and carbohydrates may also be present. - *Agar* - It is the complex carbohydrate obtained from seaweed which are not degraded by most bacteria. - Unique properties that is it melt at 98^o^C and solidify at 42^o^C - It is used as a **solidifying agent for media**. - It is **not provide source of nutrient for bacteria**. - *Yeast extract* - It is an aqueous extract of yeast cells. - It is a very rich source of B vitamins. - It also contains organic nitrogen and carbon compounds. - *Other supplements* -- **blood (5-10%),** serum, extracts of plant or animal tissues. They may be needed for cultivation of some fastidious heterotrophs. **(ii) Classification according to consistency of media** - Liquid media - A liquid medium is called a broth. Eg: Nutrient broth - Semisolid media - The media contain agar at a concentration of 0.5 percent or less. It has soft gel like/curd like consistency. - It is useful for cultivation of microaerophilic bacteria and determination of motility. - Solid media - They are useful for isolation of bacteria and determination of the characteristics of colonies. - **Agar** is used at a concentration of **1.5 -- 2 percent** level. - Eg: Nutrient agar. **(iii) Classification according to the requirements** - **General purpose media/ basal media:-** used for routine cultivation - Nutrient broth and Nutrient agar **Composition of nutrient broth and nutrient agar** **Component** **Nutrient broth** **Nutrient agar** ----------------- -------------------- ------------------- Beef extract 5 g 5 g Peptone 10 g 10g Sodium chloride 5 g  5 g Agar     - 15 g  Water  1000 ml  1000 ml  - **Enriched media** - These media contain extra nutrients in the form of blood, serum, egg yolk etc, to basal medium. - Enriched media are used to grow nutritionally exacting (fastidious) bacteria. Eg: Blood agar (5-10% blood), chocolate agar, serum agar  - **Selective media** - The medium is designed to suppress the growth of some microorganisms while allowing the growth of others (i.e., they select for certain microbes). - Addition of antibiotics, dyes, chemicals, alteration of pH or a combination of these added.  - Eg.: Mannitol salts agar (allow to grow Staphylococci), MacConkey agar (allow to grow gram-negative) - **Enrichment media** - Enrichment culture is used to increase the relative concentration of certain microorganisms in the culture prior to plating on solid selective medium. Unlike selective media, enrichment media are liquid media. Eg.: Selenite F broth, tetrathionate broth for *Salmonella* isolation - **Differential media** - Certain reagents or supplements when incorporated in to the culture media allow differentiation of different kinds of bacteria. Eg: blood agar. Some bacteria may hemolyse the red blood cells and others do not. So the hemolytic and non hemolytic bacteria can be differentiated. MacConkey agar-"lactose fermenters" bacteria utilize the media's lactose and create an acidic by-product that causes the pH indicator, neutral red, to turn pink, colonies of lactose fermenting bacteria that appear pinkEg.*E. coli*. Non-lactose fermenting bacteria will be colorlessEg.*Salmonella*. - **Special media** - Media used for the cultivation of specific organisms. Eg: Dorset egg medium for *Mycobacterium tuberculosis*, PPLO agar for *Mycoplasma* and EMJH medium for *Leptospira*. - **Anaerobic media:** Media used to grow anaerobic organisms. Eg.Robertson's cooked meat medium used for growth of anaerobic bacteria like Clostridia - **Assay medium:-**used for detection of assessment or sensitivity of antibiotics. Eg Mueller Hinton agar for antibiotic sensitivity test. - **Enumeration medium:-**Used for counting of microorganism. Eg Plate count agar \*\*\*\*\* **SOURCE AND TRANSMISSION OF INFECTION** **MICROORGANISM AND HOST** - Infection and immunity involves interaction between animal body (host) and the infecting microorganism - Based on host relationshipto microorganism,microorganism can be classified as *saprophytes* and *parasites* - *Saprophytes*:-are free living microbes that subsist on dead or decaying organic matter. They are found in soil and water and play important role in degradation of organic material in nature - *Parasites*:- are microbes that can establish themselves and multiply in hosts. - Parasitic microbes may be pathogens or commensalism - *Pathogen*:- are microorganisms that are capable of producing disease in the host - *Pathogenicity*:- the ability of a class of bacteria to produce disease - *Virulence*:- It is the measure of degree of pathogenicity. It refers to the capacity of a microbial strain to produce disease - In parasites there are different host parasite relationships. - *Commensal*:- are microbes live in complete harmony with the host without causing any damage to host. Commensals constitute normal bacterial flora of the body such as *Staphylococcus epidermidis* of skin and *E. coli* of gastrointestinal tract - *Opportunistic infection*:- some commensals or saprophytes can produce disease when the body resistant lowered - *Symbiosis* -- Mutually beneficial relationship - *Obligate* *pathogen* -- The organism always causes disease - *Infection*:- Lodgment and multiplication of an infectious agent in or on the body by microorganism **SOURCES OF INFECTION** Sources of infection are animal, Insect and inanimate in nature **Animal sources** - Normal flora - Animals in incubation period -Animals in incubation period may excrete the pathogen. - *Incubation period:-* the time interval between entry of infective agent and the onset of clinical manifestation of disease. - Animals with clinical disease. - *Convalescent carrier animals* -- In these animals shedding of the pathogen occurs for varying periods after clinical recovery. The period may vary from weeks to months - *Contact carrier or subclinical infections* -- They acquire pathogenic organisms from other animals with infectious disease without contracting the disease themselves.Such animals are called as contact or subclinical carriers. The carrier state may be temporary for a few days or lasting for months. **Inanimate sources** (fomites) - Through contaminated utensils, feed and water **Insects:-**Blood sucking insects may transmit pathogen to animal. The diseases called *arthropod borne diseases*. Insect such as mosquitos, flies, tick, mites, flea and lice transmit the disease and called *vectors*. - Contact - Inhalation - Ingestion - Inoculation or infection through the skin or mucous membrane - Through coitus or contaminated instruments, catheters, semen in Artificial insemination. - Congenital:-Trans-placental infection from uterus to fetus. - *Nosocomial infections:-*Hospital acquired infections are called nosocomial infections. - *Iatrogenic and laboratory infection*:-Physician induced infections are known as iatrogenic infections. Laboratory personnel handling infectious agent can get infection. During surgery or treatment if proper precaution not taken then animal gets infection. - Nosocomial (Hospital-Acquired) Infections:- Hospital-Acquired Infections during.Acquired as a result of a hospital stay.5-15% of all hospital patients acquire nosocomial infections. 2 million infections, 90,000 deaths **BACTEREMIA** - **Bacteremia is the presence of bacteria in blood.** - The pathogenic organisms may gain entry in to a blood capillary or venule by active or passive means from the initial site of entry. - Once in blood stream the organism can spread to various parts of the body and cause localized infections.Eg: *Leptospira* can reach the kidneys by means of bacteremia. - Organisms can directly gain access to the blood by first infecting the lymphoid system. **SEPTICEMIA** - **In septicemia the bacteria in the blood stream actively multiply and produce toxic products.** - One of most severe septicemia is anthrax in which the number of bacteria in blood may often exceed the erythrocytes in blood. **BACTERIOCIN**:- Are proteins liberated by bacteria that kills the same or other closely related species of bacteria Eg.*E. coli* -- colicin &*Pseudomonas* -- pyocin **TOXIN:-**Poisonous substances produce by bacteria that directly damage to host tissues. - Toxins are of two types, exotoxin and endotoxin. **Toxemia:-Toxemia is the presence of toxins in blood** **Exotoxins** - Exotoxins:- are proteins produced and releases extracellularly from the bacterial cell to cause toxicity. - Eg. Neurotoxins -- tetanus toxins, Enterotoxin -- *S. aureus*. **Endotoxins** - The lipopolysaccharides of the Gram negative bacterial cell released in to the medium cause the toxic effect. This is due to the lysis of cells which occurs during late growth stages of culture. - Biologicalactivation of endotoxins causes pyrexia, complement activation, leucocytosis and shock. **Fever production** **DIFFERENCE BETWEEN ENDOTOXINS AND EXOTOXINS** **Feature** **Exotoxins** **Endotoxins** -------------------------- --------------------------------------------- ---------------------------------------------------------------- Chemical nature Protein Lipopolysaccharide Source Secreted by bacterial cells Part of bacterial cell wall release by lysis of bacterial cell Action on heat Heat labile Heat stable Antigenicity Strong Weak Produced by Gram positive ; some Gram negative bacteria Gram negative bacteria Lethal dose Small Large Convertibility to Toxoid Yes No 15-04\_Toxins\_1.jpg 000163BEMacintosh HD ABA78158: \*\*\*\*\* **\ ** **BACTERIAL GENETICS** **Basic principles of molecular biology** - The"central dogma" of molecular biology is that deoxyribonucleic acid (DNA) carries genetic information which transcribed onto ribonucleic acid and then translated as the particular polypeptide (DNA to RNA to Protein) **MUTATIONS** - A change in the nucleotide sequence of a gene is called mutation. - Cell or organism which shows the effect of a mutation is called a **mutant**. Unmutated cells are called **wild type**. - Mutation rate of individual genes in bacteria range from 10^4^to 10^10^ per bacterium per division. **Types** - Point mutations  - Frameshift mutations Point mutation - Substitution of one nucleotide for another is called point mutation. - Transition - replacement of a purine by purine or replacement of a pyrimidine by a pyrimidine - Transversion - replacement of a purine by pyrimidine or vice versa. - Point mutation can result in - Missense mutation -- An altered protein is produced. - Nonsense mutation -- Stop codon formed leading to nonproduction of protein - Neutral mutation - There is no change in the protein sequence. Frame shift mutation - Addition or deletion of one or more nucleotides in a gene is termed insertion or deletion. This results in a shift of the reading frame. - Generally this causes production of nonfunctional proteins because an entirely new sequence of amino acids is synthesized from a frame shift reading of nucleotide sequence of m RNA. **Mutagens** - Mutations commonly occur during DNA replication. Any agent which increases the mutation rate is called a mutagen. Mutation obtained by the use of mutagens is called induced mutation.Chemicals and Physical UV, X-ray, Gamma-rays act as mutagens **Methods for Mutant selection / detection** - Replica plating - Penicillin enrichment - Ames test **PHENOTYPES OF BACTERIAL MUTANTS** - Antibiotic or drug resistance - Nutritionally deficient or auxotrophic mutants - Changes in colonial form or ability to produce pigments - Antigenic mutants - Resistance to bacteriophages - Changes in ability to produce spore, capsule or flagella - Loss of function but retaining the intracellular enzymatic activities to catalyse the reactions. - Altered fermentation ability **TRANSFER OF GENETIC MATERIAL / BACTERIAL RECOMBINATION** - Recombination:-The process or act of exchanges of genes between chromosomes, resulting in a different genetic combination and ultimately to the formation of unique gametes with chromosomes that are different from those in parents. - Recombination results from three types of gene transfer. They are - Transformation:- uptake of naked DNA. - Transduction:- infection by bacteriophage virus carrying bacterial genes. - Conjugation:- plasmid mediated mating between cells in contact. **TRANSFORMATION** - Griffith in 1928 inoculated mice with a mixture of a few live rough (noncapsulated, nonpathogenic) pneumococci and a large number of heat killed smooth (capsulated, pathogenic) pneumococci. The mice died of pneumonia and live smooth bacteria were isolated from their blood. This showed that some factor responsible for pathogenicity of the smooth bacteria (even though they were dead) was transferred to the living rough bacteria and transformed them in to pathogenic smooth bacteria. The transforming factor could be passed from the transformed cells to their progeny and thus had the characteristics of a gene. Later this transforming principle was identified as DNA by Avery Macleod and Mc Carty in 1944. - Transformation is the process in which cell-free or naked DNA containing limited amount of genetic information is transferred from one bacterial cell to another. DNA is obtained from donor cell by natural cell lysis or by chemical extraction. In the recipient cell recombination occurs. DNA is taken in through cell wall and cell membrane. After entry into the cell one strand of the DNA is degraded by deoxyribonucleases while the other strand undergoes base pairing with a homologous portion of the recipient cell chromosome. Since complementary base pairing takes place between one strand of donor DNA and a specific region of the recipient cell chromosome. Only closely related strains of bacteria can be transformed. **The Griffith's experiment**  **TRANSDUCTION** - This DNA transfer mechanism was found out by Zinder and Lederberg in 1952 when studying sexual recombination in *Salmonella*. - It is the transfer of a portion of DNA from one bacterium to another by bacteriophage. - *Bacteriophage*is a virus that infects and multiplies in bacterial cell. - Phages exhibit two types of replication, lytic and lysogenic. - In lytic mode after entering into the bacteria they direct the synthesis of phage genomes and proteins which assemble to produce new phages. The newly formed phages are then released by lysing the bacterial cell. - In lysogenic mode carried out by temperate phages they have their DNA like an episome in bacteria. This viral DNA can be integrated in the bacterial chromosome. Such DNA is known as prophages. - The phages particles may have some bacterial DNA incorporated or in some phages there will be only bacterial DNA. When such phage infects another bacterium the bacterial DNA in then is transferred to the recipient bacterial cell.  **Two types of Transduction:-GeneralizedTransductionand Specialized Transduction** - All fragments of bacteria DNA have a chance to enter in to the phage in generalized transduction while in specialized transductionspecific DNA fragments. **CONJUGATION** - The process was discovered in 1946 by Lederberg and Tatum. - In conjugation, transfer of DNA occurs between a mating pair of cells followed by separation of the cells. - In this process large fragments of DNA can be transferred. In *E.coli* the donor (male) cells contain a small circular piece of DNA which is extrachromosomal. It is called the sex factor or fertility (F) factor. - These cells are referred as F^+.^ The recipient (female) cells lack this factor and are referred as F^-^. Sex pili bind to the F^-^ cell and retract it into F^+^ cell. They also act as tubules through which DNA passes from F^+^ cell to F^-^ cell. Transfer of DNA can also occur at sites of contact between the cells. - The donor cell replicates its sex factor and one copy of it is transferred to the recipient. - Now the F^-^ cell is converted it into F^+^ cell and can act as a donor cell.  **Hfr:-Highy frequency recombination** - A strain of bacteria that has incorporated an F factor into its chromosome and can then transfer the chromosome during conjugation. - Plasmid integrated in to the bacterial chromosome and these cells called Hfr cell. - From the Hfr, genetic material transfer from fromHfr (F+) to F- cell. **PLASMIDS** - *Plasmids* are double stranded, closed circular DNA molecules capable of autonomous replication. One exception to circularity is the linear plasmid present in *Borreliaburgdorferi.*  - *Episomes *are plasmids that can integrate into bacterial chromosome. F factor is an example of episome. - *Conjugative plasmid:* A plasmid that can mediate its own transfer to a new strain. - *Non conjugative plasmid:* A plasmid which cannot mediate its own transfer to a new strain. - *Cryptic plasmids:* They have no identifiable function other than self replication. - *Resistance plasmids:* They have ability to impart resistance to drugs. R factor provides resistance to many drugs. A single R factor may carry traits for resistance to as many as seven or more chemotherapeutic agents. R factors in the normal flora of human and animals may be transferred to pathogenic organisms leading to sudden appearance of multiple resistant strains.R plamids play a major role in the development of resistance to antibiotics. **Genetic process** **Representative microorganism** **State of DNA as transfer agent** **Direction of transfer** --------------------- --------------------------------------------------- ------------------------------------ -------------------------------------------- Transformation *S. pneumonea, H. influenza*, *Bacillus* spp. Etc Naked DNA DNA donor to DNA recipient Transduction *S. aureus*, *E. coli*, *Shigella* Bacteriophage carrier Phage donor to phage recipient to bacteria Conjugation *E. coli*, *Salmonella, Proteus etc* DNA via pili / cytoplasm F+ cell to F- cell **MYCOLOGY** **Definition:-**Myco- fungus, Logy -- study.Study of fungi is known as mycology. The word mycology derived from *mykes*, the Greek word for mushroom **GENERAL PROPERTIES OF FUNGI** Fungi are eukaryotic, heterotrophic, and unicellular to filamentous, rigid cell wall, sporebearingorganisms that usually reproduce by both sexual and asexual means. Further they are insensitiveto antibacterial antibiotics. - Fingi are eukaryotic; cells contain membrane bound cell organelles including nuclei, mitochondria, golgi apparatus, endoplasmic reticulum, lysosomes etc. They also exhibit mitosis. - Cell membranes contains **ergosterols** whereas cholesterol in mammalian cell membrane. - Cell wall made of **chitin, glucans, mannans**and**complexpolysaccharides**which imparts rigidity to the cell wall and aretherefore non-motile, a feature that separates them from animals. - **Heterotrophs** (require organic compounds for both carbon and energy sources) and obtain nutrients by **absorption** - Tolerate high osmotic pressures and low pH - They obtain nutrients as saprophytes (live off of decaying matter) or as parasites (live off of living matter) - Reproduce both sexually and asexually with the production of spores - Branching **hyphae** and unicellular **yeasts** are the two major forms - Resistant to antimicrobial drugs which are effective against bacteria **Basic knowledge of fungi** **Feature** **Fungi** **Bacteria** --------------------- ----------------- --------------------- Size 4 µm (smallest) 1 µm Nucleus Yes eukaryotes No prokaryotes Organelles Yes No Membrane sterols Yes No (ex. Mycoplasma) Cell wall content Chitin Peptidoglycan Dimorphism Yes No Need Organic Carbon Yes Not always Obligate Anaerobe No Many **BENEFICIAL EFFECTS OF FUNGI** - Decomposition - nutrient and carbon recycling - Industrial fermentation based production of alcohols, fats, citric,oxalic and gluconic acids - Important sources of antibiotics, such as Penicillin - Used forrecombinant DNA technology,biochemical and genetic studies - Some fungi are edible (mushrooms) - Yeasts provide nutritional supplements such as vitamins and cofactors - Ergot produced by *Clavicepspurpurea* contains medically important alkaloids that help in inducing uterinecontractions, controlling bleeding and treating migraine - Fungi (Leptolegnia caudate and Aphanomyceslaevis) are used to trap mosquito larvae in paddy fields andthus help in malaria control **HARMFUL EFFECTS OF FUNGI** - Destruction and spoilage of food, vegetables,cereals lumber, paper, and cloth. - Animal, human and plant diseases, including allergies - Toxins produced some fungi like aflatoxin and poisonous mushrooms within food. - Laboratory contamination in bacteriological culture media and tissue culture media **MORPHOLOGY AND STRUCTURE OF FUNGI** - Fungi exist in two fundamental forms; Moulds (Molds): Filamentous (hyphal) fungi eg: *Aspergillus*sps, and single celled budding forms (yeast)eg.*Cryptococcus neoformans*. - Based on Morphology fungi are classified in four groups i. **Moulds (Molds):** Filamentous fungi Eg: *Aspergillussps, Trichophytonrubrum* ii. **Yeasts:** Single celled that buds Eg: *Cryptococcus neoformans, Saccharomyces cerviciae* iii. **Yeast like fungi**: Similar to yeasts but produce pseudohyphae. Eg: *Candida albicans* iv. **Dimorphic fungi:** Fungi existing in **two different morphological forms** at two different environmental conditions.They exist as **yeasts** in tissue and *in vitro* at 37^o^C and as **moulds** in their natural habitat and *in vitro* at roomtemperature 25^o^C. eg: *Histoplasmacapsulatum,* and,*Coccidioidesimmitis* **Moulds:** - The body of fungus called thallus. In moulds the thallus consists of the **mycelium and the spores** - Spore is basic structure and responsible for reproduction of fungi in suitable environment - Spore germinate enlarge in size and produce microscopic thread like structure called **hyphae** - Hyphea is thin transparent cylindrical tube like structure and contain protoplasm - Hyphae is the structural unit of mould - **The hyphae are two types** v. Nonseptate or aseptateorcoenocytichyphae which no cross partition eg.Mucor&Rhizopus - **Coenocytic (multinucleate)hyphae** fungal filaments that lack septa and so do not divide into uninucleate units vi. Septatehyphae in which protoplasm is divided at irregular interval with cross walls (septa) that divide them into numerous cells.Septate hyphae cells having single nuclei or multinucleate cells. Each cell has more than one nucleus in each compartment. - - Hyphae branch & produce a network or meshwork like structure called **mycelium** - Mycelium imparts color, texture and topography to the colony - **Mycelium are of three kinds:** i. **Vegetative mycelium**are those that penetrates the surface of the medium and absorbs nutrients. ii. **Aerial mycelium**are those that grow above the agar surface iii. **Fertile mycelium** are aerial hyphae that bear reproductive structures such as conidia or sporangia. **Yeasts:** - Yeasts are unicellular spherical to oval cells. - Yeasts reproduce by budding. In some cases, as the cells buds the buds fail to detach and elongate thus forming achain of elongated hyphae like filament called **pseudohyphae.** This property is seen in ***Candia albicans.*** - ***Candia albicans***have the ability to produce true hyphae, which is seen as **germ tube**. The difference between the pseudohyphae and true hyphaethere is a constriction in psueudohyphae at the point of budding, while the germ tube has no constriction. - The cellsof ***Candida albicans***are incubated in serum at 37^o^C form **Germ tubes**within 2 hours - Some yeast such as *Cryptococcus* and the yeast form of *Blastomycesdermatatidis* produce polysaccharide **capsule.** Capsules can be demonstrated by negative staining methods using **India ink or Nigrosin** **Yeast like fungi**: Some yeast grow partly as yeast and partly as chains of elongated budding cells joined end to end forming a psudomycelium/pseudohyphae. Eg: *Candida albicans* **REPRODUCTION ASEXUAL** **Asexual reproduction** - In asexual reproduction or somatic or vegetative reproduction there are no sex cells or organs and it does not involve union of nuclei. - It can occur byfission of somatic cells orbudding of somatic cellsfragmentation or disjointing of hyphal cellsspore formation. The different types of asexual spores are - Sporangiospores -- single cell spores formed within structures called sporangia at the end of special hypha called sporangiophores. Eg.Mucor&Rhizopus - Conidiospores or conidia -- small single celled conidia are called microconidia. Large multinucleated conidia are called macroconidia. Conidia are formed at the tip or side of a hypha.eg*Aspergillus*&*Penicillum* - Chlamydospores -- thick walled single celled spores formed from cells of vegetative hypha. They are resistant to adverse conditions - Blastospores -- they are formed by budding eg.*Candida albicans* **Sexual reproduction** - Sexual reproduction occurs by fusion of the compatible nuclei of two parent cells. The process begins with the joining of two cells and fusion of protoplasts (plasmogamy). - The two haploid nuclei fuse together to form a diploid nucleus. Then meiosis occurs to reduce the number of chromosomes to the haploid number - The sex organs are called gametangia. They may form differentiated sex cells, gametes or may contain instead one or more gamete nuclei. - If the male and female gametangia are morphologically different, the male gametangium is called antheridium and the female gametangium is called the oogonium. The different sexual spores are - Ascospores -- single celled spores produced in a sac called ascus. Usually eight ascospores in each ascus. - Basidiospores -- single celled spores are borne on a club-shaped structure called a basidium. - Zygospores -- large thick walled spores formed when the tips of two sexually compatible hyphae - Oospores -- formed in a special female structure called oogonium. Fertilization of eggs or oospheres by male gametes formed in an antheridium gives rise to oospores. One or more oospores are present in each oogonium.  **AscosporeBasidiosporeZygospore Oospore** **GROWTH, NUTRITION AND CULTURAL CHARACTER** - **Sabouraud's Dextrose Agar (SDA)** is one of the most common media used to culture fungi in laboratory. It consists of peptone, dextrose and agar. High concentration of sugar and a low pH (4.5-5.5) prevents growth of most bacteria and makes it selective for fungi. - Saprophytic fungi grow much quickly than pathogenic fungi (e.g, dermatophytes). In such situations the saprophytic fungi can be inhibited by the addition of cycloheximide (actidione) to the SDA. Addition of antibiotics such as Chloramphenicol, Gentamicin or Streptomycin to SDA serves to inhibit bacterial multiplication. - Fungi can grow both under aerobic and anaerobic conditions. - Optimum temperature for saprophytic fungi is 22-30 ^o^C. For pathogenic fungi it is 30-37^o^C. Some fungi can even grow at 0 ^o^C and can cause deterioration of meat and vegetables in cold storage. - Other specialized media used for different fungi include - Caffeic Acid Agar and Birdseed Agar for isolation of *Cryptococcus neoformans.* - Corn Meal Agar: Enhances production of chlamydospores in *Candida albicans*and formation of conidia in fungi. - Dermatophyte Test Medium: isolation of dermatophytes from clinical specimens. - 'CHROM agar Candida' is useful in identification of Candida species. - Cultural character of fungi - Colour of colony:- Top surface of the culture appear different colours and this is due to different spores produced different colour like black, green, yellow - Rate of growth:- Aspergillus, Mucor grow within 3 to 6 days and Microsporum, Trichophyton cause ringworm grow 1-2week - Pigment production:- Pigment production seen on the **reverse side**ie under the surface of the colony. Some fungi produced pigment - Surface structure:-Mycellium appear waxy, smooth, cottonwool or velvet **CLASSIFICATION OF FUNGI** i. ii. iii. iv. - - - - v. +-------------+-------------+-------------+-------------+-------------+ | **Character | **Deutromyc | **Ascomycet | **Basidiomy | **Zygomycet | | ** | etes | es** | cetes** | es** | | | Fungi | | | | | | imperfecti* | | | | | | * | | | | +=============+=============+=============+=============+=============+ | **Division* | Deutromycot | Ascomycota | Basidiomyco | Zygomycota | | * | a | | ta | | +-------------+-------------+-------------+-------------+-------------+ | **Septate | \+ | \+ | \+ | \- | | Hyphae** | | | | | | | | | | Coenocytich | | | | | | ypae | +-------------+-------------+-------------+-------------+-------------+ | **Sexual | \- | \+ | \+ | \+ | | State/Struc | | | | | | ture** | | Ascus | Basidium | Zygosporang | | | | | | ium | +-------------+-------------+-------------+-------------+-------------+ | **Sexual | \- | Ascospore | Basidiospor | Zygospore | | spores-** | | | e | | +-------------+-------------+-------------+-------------+-------------+ | **Asexual | Conidia | Conidia | Conidia | Sporangiosp | | Spores** | | | | ores | | | Arthrospore | Arthrospore | Arthrospore | | | | | | | | | | Chlamydospo | Chlamydospo | | | | | res | res | | | | | | | | | | | Blastospore | | | | | | s | | | | +-------------+-------------+-------------+-------------+-------------+ | | Majority of | Also known | Known as | *Zygomycete | | | animal and | as sac | club fungi | snigricans* | | | human | fungi. | e.g. Smut, | e.g. | | | pathogens | | Jelly | Rhizopus | | | | Important | fungi, | which grows | | | e.g. | in food | Mushroom, | on bread | | | Candida | spoilage. | puffballs | | | | | | etc. Some | | | | | Some member | are used as | | | | | are edible | food other | | | | | fungi | help to | | | | | | decompose | | | | | e.g. | plant | | | | | Saccharomyc | debris. | | | | | es, | | | | | | Aspergillus | e.g. | | | | | , | Agaricus,Am | | | | | Penicillium | antia | | +-------------+-------------+-------------+-------------+-------------+ \*\*\*\*\* **\ ** **VETERINARY VIROLOGY** Definition: Viruses are obligatory intracellular infectious with only one type of nucleic acid either DNA or RNA, unable to grow by binary fission, lack metabolic enzymes with an absolute dependence on living cells for their replication. **HISTRY** - In 1892, Dmitri Ivanovsky, a Russian scientist, reported that tobacco mosaic diseasecould be transmitted from a diseased plant to a healthy one using filtered leaf extractfrom the diseased plant as inoculums. The first virus discovered tobacco mosaic virus - In 1898, MartinusBeijerinck (a Dutch) demonstrated thefilterability of the agent of tobacco mosaic disease. The filterable diseasecausingagent was termed **'*virus'* meaning 'poisonous fluid'.** - In 1898, Loeffler and Frosch showed that Foot-and-Mouth-Disease was caused by afilterable agent. The first animal virus discovered Foot-and-Mouth-Disease virus - In 1901, Walter Reed and his team identified Yellow Fever Virus as afilterable pathogen in humans. The first human virus discovered Yellow Fever Virus - In 1915, Frederick Twort& In 1917, D Herelle discovered virus infect bacteria & name "Bacteriophage" - Goodpastuer& Woodruff in 1931 -- First time use egg embryo for virus cultivation - Knoll & Ruska 1939 -- Invented electron microscopy - Enders 1949 -- developed cell culture technique for cultivation of virus - Cohen & Boyer 1973 -- Developed recombinant DNA technology - Kohler & Milstein 1975 -- Hybridoma technique for production of monoclonal antibodies - Kary Mullis 1983 - Discovery of Polymerase Chain Reaction (PCR) **GENERAL PROPERTIES** i. The genome contains either DNA or RNA. ii. Bacteria are measured in terms ofmicrometer whereas viruses are **measured in nanometer**. Viruses range in size from 17nm to 300nm. The circovirusare the smallest viruses (17nm) while the poxviruses are thelargest viruses (300nm). iii. Ultramicroscopic in size. Viruses cannot be seen by light microscope because of theirsmall size. They are seen only by the aid of electron microscope. However, poxvirusescan be seen by light microscope iv. Viruses do not possess cellular organization or ribisomes v. Viruses are completely dependent on living cells, either eukaryotes or prokaryotes forreplication and existence. They do not grow in inanimate/non-living media. vi. Do not divide by binary vii. Resistant to antibiotics Growth on artificial media Division by binary fission Whether they have both DNA and RNA Whether they have ribosomes Their sensitivity to antibiotics ------------------ ---------------------------- ---------------------------- ------------------------------------ ----------------------------- ---------------------------------- **Bacteria  ** Yes Yes Yes Yes Yes **Mycoplasma  ** Yes Yes Yes Yes Yes **Rickettsia  ** No Yes Yes Yes Yes **Chlamydia** No Yes Yes Yes Yes **Viruses** No No No No  No **STRUCTURE AND MORPHOLOGY OF VIRUSES** **Basic Components of Viruses** - A virus particle or virion is consists of **nucleic acid (DNA or RNA)** that is covered by aprotein coat called **capsid**. The combined nucleic acid and capsid is called **nucleocapsid**. - The nucleocapsid can either be naked or enclosed by a membrane termed **envelope**. Thecapsid itself is made up of subunits called **capsomere**. - Genome of virus is haploid except in retroviruses, in which it is diploid. - Virion: Complete virus particle with either DNA or RNA nucleic acid with protein coat - Viriod: Simple infectious agent which consist of only naked RNA molecule - In morphology most of animal virus is spherical while some virus are irregular & pleomorphic. - Rabies virus is bullet shape, pox virus is brick shape & Bacteriophage is sperm shape. **Viral Nucleic Acid** - The genome contain either RNA or DNA - It contains the information necessary for directing the infected cells to synthesis virusspecificproteins - It may be single stranded or double stranded - It may be linear or circular - It may be positive sense or negative sense ( a positive sense nucleic acid possesses thesame polarity as the mRNA and so can be translated directly into protein without firstbeing transcribed) - It may be a single piece or segmented - It is haploid except in retroviruses in which is diploid **Viral Capsid** - It is made up of proteins arranged in multiple almost identical units called capsomere - It offers protection for the nucleic acid against adverse conditions - It facilitates attachment and entry of the virus into host cell - It possesses antigens used for virus identification in serological tests **VIRUS SYMMETRY** - The orderly arrangement of similar protein - protein interfaces results in a symmetrical structure and it determines the symmetry of the virus. - Symmetricalpatterns which could be:1. Icosahedral (Cubic)2. Helical 3. Complex **1. Cubic/Icosahedral symmetry** - Highly structured capsid in which capsomers are arranged in form of an icosahedrons - In the icosahedrons, there is polygon with 20 triangular faces/sides, 12 vertices/corners and 30 edges - The capsomeres of each face form an equilateral triangle - When five capsomers unite/join at vertices it form pentons & at facets when six capsomere join to form hexons - All DNA viruses of animals except poxviruses as well as some RNA viruses possessicosahedral symmetry - Viruses with icosahedral symmetry could be naked (without envelope) or enveloped **Helical Symmetry** - The capsomeres& nucleic acid bound together to form helical or spiral tube, inside tube nucleic acid coiled itself - Single stranded RNA viruses such as paramyxoviruses, orthomyxoviruses andrhabdoviruses have helical symmetry **Complex Symmetry** - Some viruses like pox virus exhibit a complex symmetry with dumbbell shape core surrounded by complex membrane show icosahedral or helical symmetry **Envelope** - Some viruses have envelope or outer covering called envelope virus. Virus without envelope called non envelope or naked virus - Envelope is derived from derived from the plasma membrane of the host cell during the release of the virus from the cell by budding - Envelope of virus is lipoprotein in nature. The lipid is largely of host cell origin, while the protein is virus coded called glycoprotein - Protein subunit may be seen as projecting as spikes on the surface of the envelope and this structureis called **peplomer**. Peplomers often bind to cell surface receptor of host cell - Many virus have more than one type of peplomers like in Influenza virus carries haemagglutinin and Neuraminidase - The spikes are glycoprotein in nature - Enveloped viruses are usually susceptible to action of lipid solvents like ether, chlorophorm, bile salt &detergent which rendered noninfectious due to damage to the envelope **REPLICATION OF VIRUSES** Viruses rely completely on living host cells for their replication. The small genome sizeput them at disadvantage. Also, they lack organelles and other machineries required forprotein synthesis. Although some viruses enter the host cell with few virus-encodedenzymes, others do not possess any protein of their own and therefore depend completelyon those produced by the host cell. Virus replication is facilitated by the host cell whichprovides the required energy and synthetic machinery and sometimes essential enzymesfor replication and also by the viral nucleic acid which carries the genetic informationrequired for the synthesis of viral components. The replicative cycle of a virus can be divided into a number of stages: 1. Attachment to surface receptors on a susceptible host cell 2. Penetration into the host cell 3. Uncoating of the viral nucleic acid 4. Biosynthesis of viral nucleic acid and translation of mRNA for synthesis of virusencodedproteins 5. Assembly of newly formed virus particles 6. Release of the daughter virions from the host cell   **Steps in virus replication** **1. Attachment / Adsorption:** - The host cell surface should contain specific receptor sites to which the virus can gain attachment. For attachment virions must come into collision with cells - Influenza virus the haeagglutinin on virus surface spikes get attached to glycoprotein receptor sites on the respiratory epithelium **2. Penetration** Following attachment, the virus gains access to the host cell internal environmentwhere replication takes place. Viruses employ three different mechanisms for thisinternalization: i. Receptor-mediated endocytosis (viropexis): The site of virus attachment to the plasmamembrane is coated internally with the protein clathrin and the virus-receptor complexis taken into the cell in a **manner similar to phagocytosis**. A cage-like lattice, in form ofendosome (vesicle) is then formed after internalization. (seen inrhabdoviruses, orthomyxoviruses and flaviviruses). ii. Fusion of viral envelope with the plasma membrane of the host cell (seen inretroviruses, herpesviruses and paramyxoviruses) iii. Direct introduction of viral genome into the cytoplasm (injection) through channels inthe plasma membrane. This is seen in some non-envelope viruses such asBacteriophage **3. Uncoating** - Release of viral genome from the nucleocapsid fortranscription to take place. - Uncoating may occur on the cell membrane,cytoplasm, or nucleus and is facilitated by celluar enzymes in some viruses. - Inpoxviruses, uncoating takes place in two stages. The initial stage is facilitated by host cellenzymes in which outer coat is removed by lysomal enzyme in the phagocytic vacuole while the later stage is mediated by virus-specified proteins in which virus containing internal protein & nucleic acid is released in to the cytoplasm. - In non-envelopeviruses, uncoating may be due to proteolytic activity of lysosomal enzymes. Uncoatingleads to loss of virus infectivity. **4. Biosynthesis of viral nucleic acid and viral proteins** This stage includes synthesis of not merely of viral nucleic acid and capsid protein but also of enzymes necessary in the various stages of viral synthesis, assembly and release. Biosynthesis consists essentially of the following steps: i. Transcription of mRNA from viral genome ii. Translation of mRNA into 'early protein' or 'nonstructural proteins' are enzymes which which initiate and maintain the synthesis of viruscomponents and shut down the host protein and nucleic acid synthesis iii. Replication of viral nucleic acid iv. Synthesis of 'late' or structural proteins which are the components of daughter virion capsid - In general most of DNA viruses synthesize their nucleic acid in host cell nucleus except pox virus which synthesize all component in host cell cytoplasm - Most of RNA viruses synthesize all components in cytoplasm except orthomyxo, paramyxo& retroviruses which synthesize partly in nucleus - In general most of animal viruses are RNA type in nature - Depending on the type of DNA or RNA, viruses use different strategies for transcription of mRNA - Viruses have been categorized into six classes by Baltimore (1970) based on their replication mechanisms - Adeno, herpes & papilloma viruses have double stranded DNA, replicating in host cell nucleus & depends on host cell DNA dependent RNA polymerase - Pox & African swine fever viruses have double stranded DNA but replicate in cytoplasm so they have to carry their own DNA dependent RNA polymerase - Parvo&circoviruses have single stranded DNA which becomes double stranded DNA by DNA polymerase enzyme and can enter nucleus& use host cell DNA dependent RNA polymerase - Hepadna virus is double stranded but part of its single stranded becomes double stranded before replication - - - - **5. Assembly/maturation** - Assembly of daughter virion may take place in the nucleus(DNA viruses) or in the cytoplasm (RNA viruses). The progeny nucleic acid isincorporated into the capsids. - Non-envelope viruses are afterwards present in the host cell asfully developed virion while envelope viruses acquire their envelope from plasmamembrane during their release. **6. Release** - Envelope viruses usually released by budding from the plasma membrane ofthe host cell. - In non-envelope viruses, release is by cytolysis of the hostcell. - Slow and prolonged release **Eclipse phase:** this is the initial stage of virus replication whereby the infecting virusloses its physical identity and most or all of its infectivity. At this time, no virus isdetectable in the infected host. The eclipse phase is followed by the productive stage asnew virus particles are formed and released from the cell. **CLASSIFICATION OF VIRUSES** - The International Committee on Taxonomy of Viruses (ICTV) established in 1973, for classification of virus. - The characteristics of virions are used to assign them to five hierarchical levels:- order, family, subfamily, genus and species. The hierarchical levels are denoted with the following suffixes: - Order: -*virale* - Family: -*viridae* - Subfamily: -*virinae* - Genus: -*virus* - Species: -*virus* - - - - - - - **VIRUS ISOLATION** Advantages: - It allows for further studies on the virus isolate - Required for preparation of vaccines - Required for preparation of antigens for rapid diagnostic kits Disadvantages: - It is slow,labor intensive and time consuming - It is expensive to acquire and maintain required facilities - Some viruses may not grow - Selection of appropriate media may require critical consideration **Methods of virus isolation** 1\. Virus isolation in cell culture 2\. Virus isolation in embryonated egg 3\. Virus isolation in laboratory animals 4\. Virus isolation in susceptible host 5\. Virus isolation in arthropods **Purification of viruses** i. ii. iii. iv. v. vi. vii. viii. **DIAGNOSIS OF VIRUSES** 1\. Electron microscopy 2\. Viral nucleic acid detection:-Dot-blot hybridization, In-situ hybridization, Southern blot hybridization, Northern blot hybridization, Western blot, Polymerase chain reaction 3\. Serology/detection of viral antigen and antibody:-Single radial diffusion, Double diffusion in agar (Ouchterlony test), Immunofluorescence, Enzyme Linked Immunosorbent Assay (ELISA) and Radioimmuno Assay (RIA), Virus neutralization test, Haemagglutination Inhibition TestandComplement fixation test **INTERFERENCE** When two viruses infect same host or same cell, most probably both the viruses multiply in the same host, but sometimes the replication of one virus is inhibited by another virus called interference. **Types of Interference** ***Heterologous:*** Interference: Interference between two completely different viruses. ***Homologous:*** Interference: Interference between two related viruses or two strains ofviruses. ***Autointerference***: Interference between a virus and its defective particles**.** **Mechanism of Interference** 1\. Change on receptor sites thereby preventing the attachment of the second virus 2\. Competition for cellular sites and enzymes 3\. Altering the metabolic pathway for the replication of the second virus 4\. Inducing the production of interferon **VIRAL GENETICS** Viral genetics contributed to a better understanding of virus host interaction & development of better viral vaccines Two main mechanisms for genetic modification in viruses are mutation & recombination **Mutation:-** - Heritable change in the nucleotide sequence of the genome of an organism or virus **Recombination:-** - Genetic recombination may occur when two different but related viruses infect a cell together. Two viruses exchange segments of nucleic acid between them so that a hybrid results, possessing genes from both paren