Infection

Infection

1. Viruses

   • Virus Structure
     • Naked vs Enveloped
       • Naked
         • made of viral nucleic acid, viral enzymes surrounded by capsid
         • stable to temp change, pH change, proteases, detergents and drying
         • need to lyse cell to escape so cause more acute infections. can be dry and still spread and can survive gut
       • Enveloped
         • Same stuff as naked but also has an envelope derived from host cell embedded with viral glycoproteins
         • envelope makes vulnerable- destroyed by acid, detergents, drying and heat
         • must stay wet to spread, released from the cell by budding so cause more persistent infections
     • Capsid Structure
       • Icosahedral
         • 12 vertacies, 20 sides
         • 5:3:2 symmetry
         • Extremely stable made of capsomeres
           • e.g. parvo
       • Helical Capsids
         • 1 capsid protein structural unit
         • Always enveloped e.g. paramyxoviruses
       • Complex Capsid
         • neither icosahedral nor helical- complex symmetry
         • only animal one of any significance is poxviruses
     • Viral Genome
       • DNA viruses can be single stranded or double stranded
       • RNA viruses (all need RNA dependant RNA polymerase)
         • Positive single stranded- can be converted directly into protein
         • negative single stranded needs to synthesise positive before it can produce protein
         • double stranded
     • Viral Proteins
       • Structural
         • capsid proteins- form capsid to protect nucleic acid. in naked virus it must deliver nucleic acid to host cell so has recptors
         • envelope proteins- often glycosylated embedded in membrane from host. contains receptors which allow virus to bind to target cell
         • virion associated enzymes
       • non-structural
         • often enzymes for viral repliacation
         • made in infected ell often proteins involved in transcription and translation
   • Virus Replication
     • success= attach, enter, uncoat, synthesise mRNA, replicate nucleic acid, assemble and release
     • 1- attachment. viral proteins bind to receptor on cell surface. virus- receptor determines specificity of virus
     • 2- Entry= receptor mediated endocytosis. virus then released from endosome sometimes triggered by pH change. some envelopes fuse directly with cell membrane and release capsid into cell. some use direct genetic injection
     • 3- Uncoating, release of viral nucleic acid from viral capsid. variable process but must escape endosome.
       • sometimes virus capsid only partially disintegrates 
     • 4-- Synthesis of mRNA
       • Double stranded DNA just transcribed to mRNA using host RNA polymerase
       • Single stranded DNA viruses use host DNA polymerase to make double stranded DNA which can then be transcribed
       • Double stranded RNA undergo transcription via a viral transcriptase (RNA dependant RNA polymerase)
       • -ve sense single strand uses RNA dependant RNA polymerase to convert to +sense
       • +sense RNA viruses can be transcribed directly
     • 5-Nucleic Acid Relication
       • make copies of the genome
       • DNA viruses in the nucleus
       • RNA viruses in cytoplasm using virus encoded enzymes
     • 6- Assembly
       • package nucleic acid and viral proteins into new viruses
       • non enveloped- proteins form procapsid which nucleic acid is added to
       • enveloped must get an envelope from the host membrane often done in the budding phase
     • 7- release
       • Budding from cell- enveloped viruses get envelope
       • exocytosis - take advantage of cell pathway
       • lysis of cell due to toxic effect (Non enveloped cells)
   • Virus Cultures
     • could also use live animals or eggs
     • need living cells to replicate
     • 1-prep sample, take tissue sample and cut into bits, treat with enzymes to break tissue up into cells and set in culture flask with growth medium
     • 2- cell culture maintenance- treat with trypsin to detach from plastic
     • primary cell culture- fresh from sample, after a few divisions differentiation will prevent further growth
     • continuous cell line- immortal cells often from tumours- may lack the receptors viruses need to infect them
     • 3- isolate virus from clinical case
     • 4- innoculate cultures and wait
     • Infectivity Assays
       • quantify the amount of infectious virus produced in cell culture, egg or sample from infected animal
       • 10 fold dilution steps
       • inoculate cell cultures
       • TCID tissue culture infective dose. TCID50 = 50% tissue culture infective dose
       • or plaque forming units
   • Classification
   • pathogenesis
     • stages of a viral infection
       • Entry
         • must overcome host immune defences- tend to attack mucosal surfaces
         • Skin good barrier unless cut
         • Resp tract- ciliated epithelium, mucous, commensal bacteria, immunological defences
         • Alimentary tract- pH, Bile, Enzymes, commensal bacteria
         • Blood- haemophagus bacteria or iatrogenic
         • genital
         • trans-placental
       • Viral replication and spread
         • local- in epithelium of initial infection, site of infection = site of shedding. acute infection
         • Systemic- local followed by spread to another site via lymph/ blood/ nerves. allows different entry and exit routes. longer incubation and more severe pathology
       • Shedding
         • highest viral titre
         • critical to find a new host
     • mechanisms
       • cytopathic effects- (visible changes to cell following infection)
         • Host cell shut off
         • Lysosomal damage
         • Cell membrane abnormalities
         • syncitia formation
         • formation of inclusion bodies (made of viral proteins)
       • Cytocidal Effects- cell death
         • Cell lysis
         • apoptosis
         • deadly CPE
       • Immune mediated pathology
         • immune cytolysis of infected host cells required for recovery
     • factors
       • Host: age/ gender/ nutrition/ productivity/ species/ immunity/ origin/ stress
       • Virus: Virulence/ endemic/ exotic/ transmission route immuno-suppression/ co infections/ infectiousness
       • Environment: contamination, climate, insects, quality of pasture, stocking density, vaccination programs
     • Acute vs Persistent infections

2. Bacteria

   • Structure and Function
     • Round (COCCI)
       • long chains of round blobs in same plane= streptococci
       • Like a bunch of grapes- dividing in 3 planes (staphylcocci)
       • Groups of 4/8 = Micrococcus
       • Neisseria are in pairs- diplococci
     • Rod shaped (bacilli)
       • Coliforms- length 2/3 times width simple chain
       • Actinomyces -filamentous fibres- long look like fungi
       • Bacillus- big thick chains
       • Flexibacter- spiral shape v mobile
       • coccobacilli- small almost cocci
       • spirochaetes- cork screw shape
     • GRAM STAINING
       • used to classify bacteria- gram positive stain purple gram negative stain
       • put bacteria on slide, kill and stain with gram stain
       • wash then apply iodine to fix the gram stain
       • wash off with solvent- gram stain will be removed from gram negative
       • add pink dye so we can still see gram negative
       • Gram +ve
       • Gram -ve have Cytoplasmic membrane and outer membrane
     • Bacterial Cell Structure
       • Cell wall- protect from osmotic pressure etc.
       • Mucoid polysaccharide capsule- protect from phagocytosis
       • DNA- free floating
       • Mesosome- invagination of cell membrane helps in parting daughter chromosomes
       • fimbrae- projections help stick to things
       • Flagellae- motility
         • flagellae are made up of flagellin and increase in length at the top 
       • Bacterial Envelope
         • Peptidoglycan- main component of cell wall. linear glycan chains with peptide side chains joined by covalent cross links
         • Lipoprotein only found in gram negative since only found in the outer membrane
         • Lipopolysaccharide on outer membrane
           • can be an antigen lipid is the toxin in lots of lipopolysachharides
       • bacterial spores
         • non- reproductive
         • Resting inert/ dormant form of bacteria
         • only clostridium + bacillus genus
         • DNA dehydrated in middle
         • Specialised cortex of peptidoglycan and protein coat
         • Resistant to drying out, heat, cold, freezing, boiling, radiation/ uv light
         • detects if in right conditions and starts to grow active bacteria again
   • growth
     • Types of culture + growth mediums
       • Batch culture- fixed volume of growth medium- lag, exponential, stationary and decline phase
       • continuous culture- continually provide nutrients and remove waste more like a real infection
       • Defined Growth Medium- Glucose and buffered salts
         • e.g ammonium sulphate
       • complex medium- provides more biologically occuring ompounds- for metabolically lazy bacteria
       • medium can be liquid broth or solidified in agar
     • Temperature
       • 37 for most bacteria
       • thermophiles grow in really hot
       • psychotrophs- like it hot but can survive cooler temp too
     • Atmospheric requirements
       • experiment to find ideal atmosphere
       • innoculate and evenly disperse bacteria in semi solid medium open to environment
       • Strict/ Obligate aerobe- will grow at top of container
         • e.g. bordetella bronchispetica 
       • Facultative anaerobe- likes oxygen but has capacity to grow anaerobically, higher conc at top of container but spread through
         • E.coli
       • microaerophil- needs oxygen but at a reduced tension- grow just above middle of container
         • campylobacter
       • obligate anaerobe- just ato bottom of container
         • fusobacterium necrophorum 
       • Aerotolerant- doesn't need oxygen to grow but isn't poisoned by it either- grows through whole container
         • streptococcus dysgalactae 
     • pH
       • most like neutral
       • Acidophillic
       • Alkophillic
   • genetics
     • Gene transfer- Conjucation
       • sometime plasmid integrates in chromosomal DNA
       • Donor copies plasmid
       • Donor attaches sex pillus to recipient, recipient gets plasmid copy
     • Transformation- Uptake of naked DNA, can be natural or induced
       • induced example e.coli treat with calcium chloride and ice, then heat shock and put on ice again- will take up DNA
       • could also use electrophoration 
     • transmission- using phages
       • Mispackaged bacterial DNA transferred via a bacterial virus
       • 1-phage infects bacterium and slices up the bacterial DNA, it makes copies of it's own
       • 2- Accidentally packages DNA from bacterial cell into new virus
       • 3- new virus infects bacteria, injecting the DNA into the cell. DNA may be taken up into acceptor chromosome. in this case the phase is called a transducing particle
     • transposable elements
       • specialised insertion sequences with a marker. has transposase and inverted repeats but cannot self replicate
     • Restriction and modification
       • Restriction enzymes - cut at restriction sites way of protecting bacteria for foreign DNA
       • Modification- site specific methylation to protect from restriction enzymes- often part of the same enzyme system
   • sterilization and disinfection
     • vegitative bacteria= non spore forming normally killed at 60/70 heat for 5 mins
     • endospores and prions are thermostable very resistant hard to get rid of
     • Sterlization
       • COMPLETE DESTRUCTION OF ALL LIVING ORGANISMS
       • AUTOCLAVE- "WET HEAT"
         • steam must be able to penetrate packs and must be allowed to reach appropriate temperature 
         • Boil water under pressure of 15 pounds per square inch
         • 121 degrees for 15 MINUTES
         • Testing the autoclave
           • 1- autoclave tape goes black when been through cycle- detects heat and steam not quality of cycle
           • 2- Browne' s tubes detect appropriate heating and time. GO FROM RED TO GREEN
           • 3- Spore strips- try and culture after cycle- if cycle has been effective the spores will not germinate
       • DRY HEAT- hot air oven 160 for 45 MINUTES
         • can't use on plastics
       • Tyndalisation- only on liquids and not used that often
         • steam on 3 consecutive days at 100 degrees
         • Day 1- vegitatve spores killed and spores activated
         • Day 2- germinated spores are killed
         • Day 3- just in case
       • Radiation- UV used small scale not very penetrating. Gamma uses cobalt 60 source more industrial
       • ****Filtration****
         • Filter at positive pressure through pores of 0.22 micrometers.
         • Not true sterilization since some small bacteria and viruses may pass through
       • Chemical
         • 10% formaldehyde solution
         • Hypochlorite. hypochlorus acid formed powerful oxidising agent
         • ethylene oxide gas
     • Disinfection
       • removal of most infective organisms
         • BUT NOT ALL
       • heat treatment- boiling at 100 degrees- endospores not killed
       • Pasturization- slow 63/66 degrees for 30 mins. fast 73 degrees for 15 SECONDS
       • antiseptics are sufficiently non-toxic to apply to skin

3. Parasites

   • parasitism- intimate relationship between two organism where one lives at the expense of the other
     • interaction involves nutritional depndance, immunological defence and integration of life cycles
   • Micro parasites multiply inside the host, macro parasites do not multiply in host
   • Basic life cycle template
     • 1- locate susceptible host using sensors (e.g. heat/light/vibration /carbon dioxide
     • 2-be able to survive in environment if host not found- eggs can survive for years, larvae and pupae for months
     • 3- invade host when found e.g. ingestion/ innoculation/ skin penitration
     • 4- micro- multiply in host e.g. hydatid cyst
     • 5-macro- multiply outside of host usually involves intermediate
     • 6- survive inside the host if the outside environment is poor (arrested development)
   • ARTHROPODS
     • split into insets (fleas/ lice/ flies) and acarina (mites, ticks)
     • INSECTS
       • STRUCTURE AND FUNCTION
         • hard exoskeleton
         • split into 3 segments: head, thorax and abdomen
         • Head
           • made of fused plates
             • like a football
           • compound eyes and simple eyes (ocelli)
           • antennae- lots of variation some have bristles called aristae
           • mouth parts: Labellae, the lower lip is sponge lke, can't penitrate skin
           • Larvae heads have hooks to hold on to surfaces with
             • ephalopharyngeal skeleton for identification
         • Thorax
           • split into pro/ meso/ meta. each has a pair of legs= six legs attached by coxa
           • normally have 2 pairs of wings but the second may be vestigial/ balancing organs called "halteres"
             • wing veination important for ID
         • Abdomen
           • clearly segmented and soft
           • lots of apendages- houses most body systems
         • BODY SYSTEMS
           • Alimentary tracts. Still has fore mid and hind gut.
             • fore- oesophageus, crop and proventriculus
             • mid- food storage and digestion
             • Hind- water absorption
           • Circulatory system- a dorsal "heart" no capillary system instead their organs are bathe din blood, valves only allow blood to move anteriorly
           • Nervous system- small brain with a chain of fused ganglia and basic branching nerves
           • Fat body- cells containing fat vacuoles line body cavity and act as a food reserve
           • Reproductive systems- sexes are separate. female adapted has spermatheca to store sperm and use later
       • LIFE CYCLES
         • 1-Either lay eggs or live nymphs/ larvae
         • Simple metamorphoses- egg->nymph-> adult. (nymph looks like adult but is not mature)
         • Complex metamorphoses- egg->larvae->pupa (dormant)-> adult
           • insect develops to adult form in pupa before emerging
       • EXAMPLES OF VET IMPORTANCE
         • Blowfly (lucilia spp.) cause fly strike especially important in sheep
         • Warble fly - look like holes have been punched in hide. cause economic losses and drop in milk yield
         • Mosquitoes important disease vectors
         • fleas- flea allergy dermatitis
     • ACARINA
       • 2 body divisions not 3
       • no wings, antennae or compound eyes
       • generally smaller than insects
       • life cycle
         • 1- egg
         • 2- larvae 6 legs
         • 3- nymph 8 legs
         • 4- adult 8 legs
       • examples of veterinary importance
         • tick (ixodes) vector of disease
         • mite (otodectes) common cause of Otitis Externa in dogs
   • HELMINTHS
     • CESTODES (tape worms)
       • Structures
         • head= SCOLEX has suckers and hooks
         • body = STROBILA
         • body segments- PROGLOTTIDS bud from head
         • mature segments have both reproductive organs
           • mate by cross fertilization
         • gravid segments are full of eggs
           • the eggs have thick shells and hooks
       • 2 groups: pseduophyllidea/ Cyclophyllidea
       • life cycle
         • 1- Adult tapeworm in final host sheds segments
         • 2- eggs swallowed by intermediate host
         • 3- larvae develop in intermediate host (metacestodes)
         • 4- intermediate host tissues eaten- metacestode swallowed and develop into adult in final host
       • METACESTODE TYPES
         • Cysticercus- 1 inverted scolex in fluid filled bladder
         • Cysticercoid- 1 scolex and fluid filled bladder just not inverted
         • strobilocercus - 1 scolex joined to fluid cyst by chain of segments
         • coenurus- like cysticercus but multiple inverted scolices
         • hydatid cyst- coenurus but thousands of inverted scolices
         • Alveolar cyst- hydatid but can have endogenous and exogenous budding
     • TREMATODES (flukes)
       • split into monogeneans and digeneans - mono tend to be parasites on fish focus on di-
       • Digeneans- dorsoventrally flattened worms
         • 2 suckers- both attach to host but only one has a mouth
         • muscular pharynx pumps food into blind ending gut sack
         • most are hermaphrodites
           • exceptions- Shistosomes- not hermaphrodites- pathogenic when shed
         • epidermis is a syncitium and is invovled in nutrient uptake
           • also called tegument
       • life cycle
         • 1 egg
         • 2- micacidium first motile stage
         • 3- sporocyst----> cercariae free living stages
         • 4- metacercaria -> adult
     • NEMATODES (roundworm)
       • structures
         • tail important for identification- bursate vs non bursate
         • Cuticle modifications- Alae are wing like structures. Vesicle is a ring like swelling
           • think queen elizabeth collar
         • head structures
           • leaf crown
           • teeth
           • pharynx
           • either plug feeders or blood feeders
       • life cycle
         • egg- > 3/4 moults-> adult.
         • some larvae keep the skin from previous generations
       • taxonomy
         • non- bursate
           • big and fleshy
           • 3 lips
           • single bulb pharynx
           • e.g. ASCARIDS
         • Bursate
           • Strongloidea + hookworms
             • Stout
             • complex head structures
             • infect small and large intestine
             • larvae inside mucosal tissue- cause more damage when they emerge
           • Trichostrongloidea
             • thread like and simple head
             • infect stomach and small intestine
             • live in gastric glands/ crypts- cause less damage when they emerge
   • PROTOZOA
     • Structure and function
       • unicellular
       • locomotion
         • cilia beat in unison to move or waft food into mouth
         • flagellum- contractile fibres often attached to body by undulating membrane
         • psedopodia- extensions of cytoplasm. used to move but also to engulf things
       • nutrition- phagocytosis or pinocytosis
       • reproduction
         • homoxenous- one host heteroxenous more than one host facultativley heteroxenous- can have more than one host
         • asexual- can be simple binary fission or Shizogony/ Sporogony
         • some are sexual
     • examples of veterinary importance
       • eimeria spp- coccidosis in poultry
       • babesia spp. blood bourne and spread by ticks used to be very common in cattle some cases in dogs now
       • toxoplasma- abortion in sheep
       • cryptosporidium- infects animals and humans severe humans if immunosuppressed
   • PARASITIC GASTRO-ENTERITIS
     • associated with nematode species
     • clinical signs- diarrhoea and weight loss
     • sub-clinical- poor weight gain
     • Ostertagia ostertagi
       • causes Ostergaiasis
       • primary pathogen of cattle. 1 cm long and thread like
       • strongyle worm
       • infect abomasum gastric glands (1 larvae per gastric gland)
       • clinical disease over 40 thousand adult worms present
       • prepatent period 3 weeks (from infection to egg shedding) L3 are infective but sometimes keep the cuticle from L2
       • EPIDEMIOLOGY TYPE 1 DISEASE
         • L3 larvae are left on pasture from previous year
         • spring calves are turned out and infected by L3 larvae. 3 weeks later they start shedding eggs
         • Eggs shed early in the season take longer to develop into L3 than those shed later
         • summer auto-infection peak and clinical disease present
         • called auto infection because calves that shed the eggs are the same ones that are infected in the summer
       • Epidemiology Type 2 disease
         • infected calves housed over winter
         • L3 larvae feel temp dropping on pasture, infect calves then arrest development (EL4)
         • Disease in april/ may
         • larvae don't all wake up at one normally in 20% batches. characterised by intermitant diarrhoea
           • genetic clock determines when they wake up 
       • ostertagiasis in BEEF
         • Spring Calves- spring mortality occurs before most are turned out, turned out with adult cows who have immunity so v low disease risk
         • Autumn calves- graze before spring mortality but still have adult cows with them. higher disease risk than spring calves but still low compared to dairy
     • DISEASE CONTROL
       • Type 1
         • use clean pasture
         • turn out after spring mortality
         • dose and move to aftermath
         • strategic anthelmintic treatment
       • Type 2
         • give anthelmintics before in wintering if exposed to high challenge in late autumn

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