APBI 419

Fins

• anatomical features on body of fish and provide stability in the water

  • supported by skeleton of fish

  • are either paired or unpaired

    • paired = pectoral + pelvis

    • unpaired = dorsal, caudal, adipose, anal

  • rays on either can be soft + flexible or modified into spines to strengthen fins

Streak plate isolation

• Most material obtained from fish contain mixed pop of microorgs

  • to make detailed study of characteristics of indiv species comprising mixture you have to isolate them in pure culture

• 4 diff kind of streaks

• plate now has mixed culture that you will now be able to remove individual and discrete colonies

  • each one represents one original bacterium

• you can re-streak them and obtain pure culture bacteria after another pass to run tests for full ID

Toxins

• produced by gram+ and gram- bacteria ; may produce rapid death of invaded host or can reduce effectiveness of host immune response in variety of ways

  • some gram +ve orgs produce exotoxins in cytoplasm that're released from dead / live bacteria

  • gram -ves may produce endotoxins that're cell membrane components and released only on death of cell

Systemic bacteria

• some bacteria only cause disease once inside organ / tissue

  • they might be fine external of the fish but if where they aren't supposed to be --> bad

  • once inside they can use bloodstream to travel to all organs and tissues

  • getting in: through gut / intestinal wall, skin, lesions or sores, natural openings like lateral line / sensory pores, and parasites

External bacteria

• cause skin + fin damage

  • not all are pathogenic; many bacteria existing normally on the skin will never cause disease

  • normal flora = protective and acts as competition for pathogenic bacteria

  • need to know what's normally present and which may be disease causing

Enrichment media

• adding certain special growth enhancing substances to a basic nutrient medium so it supports growth of certain fastidious microorgs

Selective media

• addition of specifical chemical substances that'll prevent growth of 1 group of bacteria without inhibiting others

  • ex. acids, antibiotics, dyes, etc.

Differential media

• adding certain chemicals / reagents that'll result in a kind of growth / change in medium; lets observer differntiate between types of bacteria

  • like adding substance which can be precipitated or used in the growth of the colony so there's a visible change in the medium

Gram’s stain

• cell wall staining characteristics that lets you classify bacteria as Gram positive or gram negative

Gram-positive

•  stain purple because they have a dye called Gram's violet in their exposed peptidoglycan layer

  • retains crystal violet-iodine complex during alcohol decolourization

Gram-negative

• stain red cuz peptidoglycan layer hidden below outside layer and so they don't retain violet dye

• thin peptidoglycan between inner/outer membranes, alcohol disrupts outer membrane, crystal violet washes out → safranin counterstain taken up → red/pink

Capsules

• thin external envelopes produced by some cells that help to protect them from host immune cells (like avoiding phagocytosis)

Caudal venous puncture

Blood smears

• Blood films used by some to provide permanent record of cell dynamics

 

1. Taking blood sample w/ heparinized syringe or whatever

2. Take portion of blood out into tube

3. Expel small drop onto pre-cleaned microscope slide frosted at one end for ID marking w/ pencil

4. place drop @ end of slide that's frosted; smear towards non-frosted end

5. W/ second slide, spread blood by backing second slide held at 30-40 deg angle into drop

6. smear blood

   • takes practice to get films nicely thin

   • goal is to get layer of blood cells that's one cell layer thick

   • if too thin counts won't be relevant as they aren't really representative

   • good clean film w/ feathered leading edge is the goal

7. Air dry slides

8. Stain slides using Diff-Quik method / Giemsa stain (more differential blue and purple shading within cells; better for photographs)

Cardiac puncture

Blood sampling site

• not the best way unless v large

• heart is small, not easy to hit accurately

Cumulative cases

total to date; propogated outbreak; sigmoidal S-curve

Point source curve

• sharply spiked; shows you that number of events rose rapidly to a peak and then fell to baseline levels

  • relative in length but time leans shorter than other curves

  • shape indicates all susceptible indivs exposed to common source in short timeframe

  • sharp peak = cases appearing after relatively uniform incubation period

    • contaminated batches

    • water treatment failures

    • acute toxic exposures

Broad source curve

•  indicate exposure to disease agent continuous / prolonged exposure to disease agent (chronic response), slow transmission rate, variable incubation periods, endemic presence of pathogen

  • curves relative in scale but these tend to have longer time scale

  • for chronic infections this curve could be years of data points (like BKD)

  • one w/ multiple spikes = frequent intermittent exposures to disease

Epidemiology

• scientific study of factors affecting health and illness of indivs + pops

  • foundation of interventions made in interest of public health and preventative medicine

  • highly regarded in evidence-based medicine for ID-ing risk factors for disease and determining optimal treatment approaches to clinical practice

Biosecurity

• describes preventative measures undertaken to minimize disease intro and spread

  • goal is to minimize both exposure to and loss from disease-related causes, physics-chemical insults, nutritional parameters, predation, etc.

  • development of a series of protocols to respond to disease exposure risks that’ve been assessed through careful onsite audit

• Not implementing good management plans: fluctuation in environmental conditions and encourage spread of opportunistic pathogens

  • includes reasonable assurance that disease is not occurring

  • consists of preventative medicines, standard operating procedures, and management plans to prevent spread of disease, and adequate diagnosis; + treatment and regulatory oversight if outbreak occurs 

• Protective layers:

  1. environmental controls + husbandry

  2. physical barriers and access controls

  3. health interventions like vaccination

  4. elite broomstick protection in isolated systems

  5. emergency response protocols (treatment + containment)

 

Disease surveillance

• more action-oriented; intended to detect changes in disease trends or distribution in order to initiate investigations or control programs

  • surveillance is continuing scrutinize of the occurrence and spread of disease that’s relevant to its control

    • involves collection + analysis of data and distribution of results so control can be implemented

  • emphasizes detection of changes in risk factors

    • monitoring programs more focused on reporting disease cases over time

  • goal: prompt actions to control the spread; focused on increased freq or impact of disease

  • uses methods that’re rapid and repeatable

  • precision of monitoring programs may be sacrificed in surveillance programs to get timely reports of potential changes in patterns of disease occurrence

Active surveillance

• members of surveillance agency generate disease data through special surveys

  • able to produce more systematic and verifiable info but usually more expensive (dollars, time, personnel)

  • wild fish disease info comes from active surveillance

    • tend to be infrequent and limited in size;  methodological limitations to prevent disease-monitoring program for wild fish which creates limits in ability to document changing disease risk

Passive

•  relies on voluntary provision of info from indivs / organizations outside of surveillance agency

  • ex. reportable disease programs relying on fish health personnel, submissions to diagnostic laboratories, voluntary reporting by culturists to private or public orgs                                                                                    

  • are inexpensive but can suffer from accuracy and delays in reporting

Biofilms

accumulation of inorganic and organic materials that can attach to most surfaces

• can build up, fall apart, carry pathogens within them to other areas

  • they contain living + dead cells within protective matrix

• Resistance increases as it matures; and whole point of cleaning is to remove biofilms

  • responsible for most surface contamination

• 4 factors to achieve removal

  1. formulation + conc of agent being used

  2. exposure time

  3. temp

  4. mechanical activity

Chlorine

PRO: inexpensive and residue is readily eliminated

CON:

• caustic and v toxic to fish

• causes brittleness and deterioration of nets

Effective against:

• bacteria

• fungi

• viruses

• some protozoa

Sodium hydroxide

PRO: inexpensive

CON: caustic

Effective against:

• bacteria

• fungi

• viruses

• protozoa

Quarternary ammonium compounds

PRO: effective

CON: residue elimination difficult

Effective against: bacteria

Apply to:

• nets

• clothing

• hands

Iodophors

PRO:

• might remain active while solution remains coloured

• resides detectable by colour - colour changes brown to light yellow

CON:

• tendency to stain

• corrosive: may require rinsing

Effective against:

• bacteria

• viruses

• fungi

Citric acid

PRO:

• non-corrosive; safe to use on paint, metal, wood, skin whatever

• suitable for personal and protecting clothing disinfecting

CON: don’t use with soaps,alkalis, washing soda, ionic detergents

Virkon

potassium monopersulfate 21.4%

PRO: active against all virus families and penetrates organic matter

CON: oxidizing agent

Effective against:

• bacteria

• viruses

• fungi

Apply to:

• footbaths

• pre-cleaned equipment

• vehicles

Outbreak

• a change in the normal pattern of disease in a population

  • typically involves:

    • Increased number or rate of cases over time

    • Clustering of cases in time and/or space

    • May include:

      • Unusual or unexpected disease presentation

      • A single alarming case (“outbreak of one”)

      • Any situation that raises concern

Ovadine

• usage: buffered 1% iodine solution for disinfecting fish eggs; reduce transmission of diseases between generations of fish

• also good at disinfecting for inanimate surfaces (gears, nets, etc.)

• non-staining, non-corrosive buffered 10% PVP Iodine complex

  • provides 1% available iodine

Parasite-S / Formalin-R solution

• formalin based solution containing methanol to prevent formation of paraformaldehyde which is toxic to fish

• usage: as a bath to control external parasites on gills, skin, fins of fish, surface disinfect eggs

Perox-Aid

usage: antifungal agent for use on fish eggs

Iodine solution

250 ppm iodine solution = diluting 25ml Ovadine into 1L of clean water

Pyceze

• antimicrobial agent

  • usage: preservative in health care, food-contact materials and cosmetic + antifungal treatment for salmonid eggs

  • available as prescription through vet

Humoral factors

adaptive immunity made up of this and cellular factors
soluble factors; antibodies and classical complement cascade

• cellular factors = B and T cells + macrophages

Cellular immunity

adaptive immunity made up of humoral factors and this; aka cell mediated immune response

• involves white blood cell types that act against specific antigens including macrophages and T-lymphocytes

 

Phagocytosis

•  some cells capable of engulfing foreign particles into cyto and digesting them

  • ex. macrophages, thrombocytes (for blood clotting) and capillary-lining cells in assorted tissues

  • cells activated to engulf anything and directed against specific invaders

  • also can be important processors; present digested invaders to other immune system cells for production of antibodies against invader

  • the removal of foreign particles from blood stream

Reticulo-endothelial system

• formed from phagocytic cell network

  • aka monocyte-macrophage system

  • system responsible for phagocytosis

  • if the foreign body is large then several phagocytic cells can encircle it and fuse trying to engulf it, making giant cells

  • foreign body reaction = histological observation of this response in tissues

  • action of phagocytes called cell-mediated immune response

    • can have specific and non-specific components

T-cells

part of adaptive immunity - the 3rd line of defence

• several kinds: helper T cells (T_h or CD4+), cytotoxic T cells (T_c, CD8+)

• CD4⁺ cells - recognize MHC-II associated antigens; set off chain of events that results in production of antibodies from B cells (plasma cells) and stimulates CD8⁺ cytotoxic T cells

• CD8⁺ cytotoxic T cells - recognize foreign peptide bound to MHC class-I , bind to them, and kills them through close contact lysis

B cells

part of adaptive immunity - the 3rd line of defence

• respond to T helper cells through cytokine signals (interleukins) and direct cell contact (CD40-CD40L)

  • have B cell reporters that recognize specific antigens

  • activate →  differentiate into plasma cells → secrete antibodies → mark pathogens for destruction

 

Constitutive

• when defence doesn't depend on an invading orgs identity

  • it's innate and heritable (transmitted in genetic code)

first branch of the immune system; non-specific, innate, natural immune system

Immunocompetence

• ability of fish to do specific immune response

  • in juv salmon doesn't occur till 4g

    • before this, it relies on non-specific defence mechanisms

Antigen

• any substance that can trigger immune response

  • can be bacterium / virus / portion or product of one of those orgs

Agglutination

occurs when immunoglobulins bind to several invaders → clumps them tgt → makes easier for other immune system components to remove clump

Precipitation

occurs when immunoglobulin activity directed against soluble molec → binding to molec precipitates it from solution

Neutralization

occurs when immunoglobulin activity directed against soluble molec → binding to molec precipitates it from solution

Opsonization

• refers to ability of immunoglobulins to make invaders more susceptible to engulfment by phagocytic cells

Cytophilic binding

immunoglobulins assist macrophage by binding to macrophage surface and assisting it to attack invading cells

Humoral immune response

1. antigen (bacteria / virus) activates macrophage

2. macrophage presents antigen to T-helper lymphocytes and B cells recognize receptors

3. macrophage produces interleukin-1 too that activates T-lymphocytes

4. those produce interleukins that will cause B-lymphocytes to produce antibodies like IgM

5. those bind to antigen → inactivate or make it more susceptible to phagocytosis by macrophages

6. helper T-lymphocytes produce interleukin-2 too → induces proliferation of lymphocytes, and  lymphokines cause macrophages to migrate towards invading bacteria

7. B and T differentiate into memory cells; persist long-term; allow rapid response when re-encountering antigen

   • stay in bloodstream; identify same antigen next time

   • second time humoral response = quicker and more pronounced

8. memory abilities of specific immune system form basis of vaccines

MHC

•  these glycoproteins found on cell surfaces; serve as self-markers by presenting peptides

  • 2 classes:

    • MHC Class I - present on all nucleated cells; recognized by CD8+ T cells

    • MHC class II - found only on antigen-presenting cells

Anamnestic response

• ability of humoral response system to remember and act more efficiently second time it encounters invader

  • cells responsible = lymphocytes produced in renal and splenic haematopoietic tissue

  • cells belong to either of: B-lymphocytes or T-lymphocytes

    • both types thought to be present in fish

  • B and T means bursa and thymus locations in other vertebrates 
    

• For fish, B cells develop in kidney tissue

     T cells develop in thymus

 

• B-lymphocytes:

  • responsible for production of antibody and for memory of previous types of immunoglobulin produced 

• T-lymphocytes:

  • responsible for controlling cellular components of the immune response

B-lymphocytes

• responsible for production of antibody and for memory of previous types of immunoglobulin produced 

• encounters antigen → divides → produces cells that manufacture appropriate antibodies

  • some of those daughter cells remain in system ready to produce antibody if required in the future

T-lymphocytes

• responsible for controlling cellular components of the immune response

• encounter antigen → divide to produce cells sensitized against antigen

  • those are killer cells that can attack antigens on invading orgs

  • lymphocytes also produce soluble proteins that influence behaviour of other phagocytic cells → encourage them to migrate to and remain in area of invading microorganisms

Antibodies

•  3D structure of long chains of proteins

  • all immunoglobins = basic structure called constant region and variable region which gives them unique shape

  • classified into various types by pattern of these regions

  • fish: primarily use immunoglobin M (IgM) that form tetrameric structure

    • mammals lack IgM and IgA

Acquired

when defences are dependent on recognition of an invader

○ depends on host's previous encounter w/ infectious agent

second branch of the immune system; specific or adaptive immune system

Fish encounters pathogen → survives encounter (let's say) → responds to it w/ greater speed and intensity next time it encounters that particular pathogen

Constitutive immunity

innate and inherited in genetic code

• nonspecific immune responses can be considered to form first and second lines of defence against invading orgs

FIRST LINE OF DEFENCE within acquired immune system

External defences

involves physical, mechanical, chemical, and behavioural protective mechanisms that prevent entry of foreign agents

• mucus

• scales and skin

• behavioural protection

• chemical protection

• natural flora

Internal defences

• number of cells + proteins that have non-specific defence role

  • designed to prevent pathogens that've gained entry from spreading throughout body

  • has both humoral and cellular components

  • consists of cells and biologically active protein molecs present in the blood, serum, and tissue fluids

part of CONSTITUTIVE IMMUNITY - SECOND LINE OF DEFENCE

Lysozome

1. enzyme produced by various immune cells including macrophages, neutrophils, epithelial cells

   • breaks down cell wall of some bacteria by breaking down peptidoglycan layer of Gram-positive orgs

   • can't access peptidoglycan layer in gram-negative pathogens cuz needs to work with other molecs like cationic peptides / complement proteins

Cationic peptides

small 3D peptides w/ net positive charge; act as defensive molecs that bind with LPS (lipopolysaccharide) on surface of Gram-negative pathogen, disrupt bacterial surface, and create pores allowing antimicrobial compounds to access cell wall

Natural killer cells (NK)

• non-phagocytic; look like lymphocytes

  • have granular cytoplasm and involved in tumour destruction + viral immunity

  • work without prior exposure to virus and activated by interferon

  • recognize stressed / infected cells through changes in surface molecs

  • recognize and directly kill infected or abnormal cells by releasing cytotoxic granules containing perforin and granzymes

Granulocytes

• contain damaging molecs that're released at a site of infection

  • difficulty for animal: reactive molecs equally damaging to both invading cells and host cells

  • they'll damage any cells in vicinity

  • ex. neutrophils, eosinophils, mast cells, basophils

Macrophages

• body's first cellular line of defence; ingest and destroy foreign cells and molecs through phagocytosis by breaking them down in complex vacuoles containing highly reactive molecs

  • they can present some of these molecs to inform specific immune system that there's invader and response should occur

  • serve as antigen presenting cells (APC) by presenting antigenic peptides to B and T cells

    • play both non-specific and specific lines of defence

Thymus

• tissues that produces T cell lymphocytes that're involved in allograph rejection

  • enhances macrophage activities and assisting in production of antibodies through B cell stimulation

 

GALT

• (gut associated lymphoid tissue) - has diffuse lymphoid cells distributed throughout intestinal wall + organized lymphoid aggregates in some intestinal regions (varying by species) + plasma cells in gut mucosa

  • mammals have Peyer's + appendix; fish don't have

  • fish have functional mucosal immunity

Immune response

• cells + molecs responsible for immunity

  • provides defence mechs that prevent entry of disease causing orgs or help to destroy bacteria + viruses once they get in bod

Immunity

reaction of bod to foreign substance (microbe / molec)

Antigen

•  foreign substance capable of causing activation of immune response

  • protein

  • polysaccharide

Antibody

• protein produced by lymphocytes in response to presence of antigen

  • released into bloodstream; there're also mucosal antibodies (in gut and mucous membranes)

  • will bind to specific antigen which initiated its production

Immunocompetence

• ability of fish to mount specific immune response against foreign body/agent

  • differences for diff species of fish

  • first signs in salmonids appear around 1-2.5g body weight (temp-dependent) w/ fuller development by 4g

 

Immunological tolerance

• condition where host doesn't recognize antigen as foreign so doesn't respond against it; 'tolerates' its presence

  • vaccination shouldn't be done before immunocompetence develops or immunotolerance can happen

Immunology

integrated system of host defence; cellular and molecular events that occur after an org has encountered foreign substance

Brain

lies in cranial cavity located between eyes of fish

• protected by cartilage / bone surrounding organ + spinal fluid

• cranial nerves extend from brain through skull

Erythrocytes

• red blood cells = oval cells w/ central, oval nucleus; cells transport O2 from gills to tissue and transport CO2 away from tissues

• O2 carried by haemoglobin as in mammals

• cytoplasm = pinkish

• organelles lost in mature cell

• younger cells more basophilic aka blue and are rounder

Lymphocytes

• cells function in humeral (antibody mediated) and cellular immunity

• rounded nuclei, thin rim of blue cytoplasm

• larger ones w/ more abundant cytoplasm can be seen

• obvious nucleolus and plasma cells w/ eccentric nuclei + dark blue cytoplasm seen as well

• antigenic stimulation → induce lymphocyte proliferation

• most fish produce an IgM like antibody in response to antigenic stimulus

Granulocytes

• function like mammalian neutrophil and will migrate to sites of inflammation and have phagocytic capabilities in some species

• vary in morphology between species

• granules may be apparent w/ light microscopic examination, or ultrastructural examination

• nuclei maybe robed, roughly segmented, irregularly round

• phagocytic capability limited compared to monocytes/macrophages

  • more important antimicrobial mechanisms = respiratory burst and degranulation

Monocytes

• participate in inflammation, phagocytic, believed to be involved in antigen trapping + presentation to immune system

• large cells with abundant cytoplasm and often-irregular nuclei

Thrombocytes

• participate in blood coagulation

• small w/ round to oval nuclei

• colourless to pale-blue cytoplasm

• oval, tear drop shaped, or fusiform in shape

• tendency to clump

Kidney

• main filter of body; internally maintains salt/water balance of fish

  • for fish it only plays small part in excretion of waste products (ammonia etc.)

  • freshwater fish: helps get rid of water (lots of v dilute urine)

  • saltwater fish: helps get rid of ions (low vol of v conc. urine)

• rich in lymphoid tissue and contains and produces antibody producing cells, phagocytes, macrophages

  • head kidney site for monocyte/macrophage cells

Head kidney

formation of O2 carrying red blood cells and white blood cells to fight off infection

Middle kidney

contains nephrons / renal corpuscles; these are blood-filtering units / traps that catch any foreign material and tries to destroy them

Heart

• 4 chambered organ situated @ base of throat; likes in pericardial cavity that's separate from body cavity

  • primary purpose: pump that pushes blood around system to carry imp molecules to and from the cells, tissues + orgs of the body

Spleen

•  small, dark, red-black organ that can be triangular / oval in shape; attached to wall of stomach or intestine

  • function similar to haematopoietic tissue of the kidney

  • composed of haematopoietic tissue similar to the kidney

• ellipsoids (small, thick walled capillaries) surrounded by clumps of immunologically active leucocytes and blood forming cells

  • blood trickels through ellipsoids → lining cells filter out old erythrocytes + foreign particles → macrophages pick up foreign material → present to immune cells to begin their response to foreign invaders

IMPORTANT:  is an organ frequently infected by bacterial diseases due to filtering activity

• rich in tissue containing and producing antibody producing cells

  • full of phagocytes and macrophages

Dorsal fin

• mainsail along back of fish between tail fin and head; provides stability so fish can swim in straight line

  • can't swim --> won't live long --> can't compete for food or evade predators

 

• some tropical fish have a doubled dorsal fin; part of it is a spine that resembles a straight razor and can inflict puncture wounds while second part is a stabilizer

  • used to intimidate + threaten

  • can also use an erect and locked fin as a wedge to jam into tight areas of coral so predator can't pull it out

Caudal fins

•  tail fin; sudden forward movements and for fast swimming patterns

  • also used to slow forward movement and to help make turns

  • can lengthen the fin through artificial selection → produced slower moving fish for aquariums → wouldn't survive long in wild

Anal fin

on the underside of fish between pelvic and caudal fins; provides stability, keeps fish from rolling over and going belly up

• in some species, male's anal fin acts as sexual organ and is known as gonopodium

some characins develop breeding tubercles / small hooks that're on the fins of males and not specifically for attaching to mates

Pectoral fins

•  stability as fish moves through water, hovers, and makes slow turns; near bottom of fish beneath gill openings

  • used for navigation and always in motion

  • many tropical species use them during incubation of eggs, fanning eggs w/ water during brooding period

Pelvic fins

• homologous to legs and are supported by pelvic girdle; stabilizers

  • might be highly modified like in freshwater angelfishes and gouramis so they're threadlike

  • some might use them as "feelers" to sense surroundings

Adipose fin

• few tropical fish species and most of aquaculture-reared fish (salmonids) have extra fin located on back between dorsal and tail

  • these contain mechanosensory and proprioceptive functions

  • helps detect water flow and might help with swimming performance

Lateral line

• on both sides of body and runs from back of eye to base of tail fin

  • made of small neuromasts that contain cilia in fluid-filled canals + superficial (surface-level) neuromasts

  • detects vibrations in water (low freq pressure waves)

  • fish feel → forms mental map for them; like hydrodynamic map / mechanosensory field

Skin

•  first barrier to environment; maintains osmotic integrity of fish

  • freshwater: osmotic challenge is water flooding IN (hypoosmotic environment) so fish has to expel excess water and retain ions

  • marine: vice versa, they lose water to the hyperosmotic environment

2 main layers:

1. outer epidermis overlaid by v delicate clear covering of secreted material that lays over the scales (the mucus layer)

2. inner dermis

Mucus

• protective and forms slimy outer covering of fish

  • can get small particles of material that can irritate fish (parasites, bacteria, heavy metal salts that're sloughed off)

  • increases when fish becomes stressed

Dermal skeleton

• various types of scales / plates that develop early in life (fry stage)

• salmonids: cycloid scales

• once fish has all scales no more develop as it grows

  • scales will grow and become larger w/ the fish

• not all fish have scales (catfish + eels)

Gills

• vascularized structures (blood moving systems) contained in chambers located on both sides of head region

  • water is continually passed over

    • imp site for excretion of nitrogenous wastes

Operculum

• bony flap; gill cover that protects gill structures

  • 4 cartilaginous branchial arches in each chamber that have forward facing cartilaginous projections called gill rakers

vary in morphology

• long and thin, short and knobby

species and diet dependent

Gill rakers

• help filter feeding fish trap food particles from the water and help piscivorous species prevent escape of prey through gill slits

  • tend to vary in #, length, spacing based on diet

  • gill arches support gill filaments that're directed away from mouth opening