animal health - diagnostics

what is diagnostic testing

• the process by which we can identify the presence of a disease or condition

what is diagnostic testing used for

• detecting diseases or pathogens

• identify a specific type or strain

• monitor the progression of a disease

• assess the severity of the disease

• guide treatment plans and biosecurity

• monitor success of treatment

types of diagnostic testing

• clinical exam

• blood tests - counts, smears, biochem

• urine + faecal tests

• cytology + histopathology

more types of diagnostic testing

• imaging techniques

• microbiological tests - bact + viral culture, fungal and parasite ID, PCR

• molecular diagnostics - PCR, sequencing

• immunological tests - serology, ELISA

clinical exam

• initial process - vet will evaluate animal health - sings, symptoms + physical findings

• history

• observation

• palpation

• auscultation

• percussion

how does diagnostic testing support clinical exam

• confirm clinical suspicions

• differentiate between sim conds

• identify sub clinical or hidden conds

• informs approp treatment plans

diagnostic imaging

• visualise the internal structs of animals to aid in diagnostics + treatment

• detect conds which not visible

• non invasive

X-ray - assess?

• bones

• chest cavity

• abdomen

• inc: fractures, infections, lung diseases + tumours

pro of xrays

• widely available

• cost effective

• quick results

con of x-rays

• 2D imaging - makes examining soft tissue difficult

ultrasound

• sound waves produce real time images

• abdominal + cardiac imaging

• ideal for soft tissues + liver, kidneys, heart + rep

pro of ultrasound

• no radiation exposure

• non invasive

con of ultrasound

• limited view of bone structs

CT imaging

• cross sectional 3D images

• detailed imaging of the brain, bones, soft tissues

• good for detecting tumours, fractures + neurological disorders

MRI imaging

• uses magnetic fields + radio waves

• create highly detailed images of soft tissues

• used for imaging the brain, spinal cord + joints

Light microscopes

• only objects larger than wavelength of light are visible - e.g. nucleus

• sides stained to enable colour to be viewed

• process of staining + fixing kills cells

transmission electron microscopes

• smaller objects visible - mitochondria, ribosomes

• usually monochrome

• not possible to view living specimens

• expensive

scanning electron microscope

• coats the sample w molecular layer of heavy metal e.g. gold

• monochrome

• not poss to view living specimens

• 3D

histology

• microscopic exam of tissues from the body

histology - biopsy sample

• collect from live animal - e.g. in surgical procedure

• samples are abnormal areas of growth

histology - necropsy sample

• samples from dead animal - from post mortem

• can inc wide array of tissue w suspected pathologies/lesions

tissue collection

• should be from fresh specimens

• should use sharp blade to cut specimen out

• tissue should be approp thickness w lesion inc

• should fix immed after collection

aims of tissue fixation

• preserves a sample as close to its nat state

• prevent postmortem degeneration

• stop bact growth

• harden the tissues - fix causes coagulation of proteins

• facilitate subsequent tissue staining

a good fixative

• preserve cell quick w/ shrinkage or swelling

• penetrate tissue rapidly

• inhibit bact decay + autolysis

• harden tissue + render it insensitive to subsequent treatment

• allow tissue to be stored for long time

• simple to prep + economical to use

tissue cassette

• cut small sections of fixed tissue w sharp knife

• put in a special tissue cassette

• wash 3h in water

tissue processing

• dehydration in acl series - 8h

• clearing in xylene - 3h

• wax infiltration - 1h

dehydration

• water must be removed before it is infiltrated w wax

• serues of acl solutions used

• sequence ensures water is replaced w alc progressively to avoid tissue distortion

embedding

• molten wax

• helps orientate tissue for sectioning

• avoid air bubbles in the section

sectioning

• wax block is fixed on to the microtome

• expose tissue by trimming wax out

• section 5-7nm thick sections

• lay on to a water bath at 40C

• lift onto glass slides

• dry on drying rack + then in oven at 60C

staining

• haematoxylin - basic dye - stains acidic structures a purplish blue - nucleus

• eosin - acidic dye - stains basic structs red or pink e.g. cytoplasm

special staining

• used to explore tissues/specimens in detail - e.g. presence of certain molecules/microorgs, distribution + intensity

microorgs special staining

• gram

• giemsa

• acid fast - mycobacteria

connective tissue special staining

• toluidine blue

• trichrome stain

carbohydrates special staining

• alician blue

• acid shiff

histopathology

• microscopic exam of diseased tissue allowing for accurate diagnosis of certain diseases

histopathology fixation + staining

• tissues must initially be fixed onto the slide so that the cells dont decompose after cell death

• trad use formalin

brain tissue, adrenal gland + eye tissue staining

• bouins

• karnovsky

diseases could be picked up thru histopathology

• arthritis/osetoarthritis

• leukaemia

immunohistochemistry

• labelled antibodies used to detect antigens win tissues

• can be used to check for signs of disease often following a biopsy

serology

• exam of blood - cells, antibodies, antigens

what is serology used for

• to detect whether an animal has been infected w a pthogen

• determine immunity

• test for prev exposure

• e.g. Feline Leukemia Virus to detect proteins from the virus

types of serology tests 1

• enzyme linked immunosorbent assay (ELISA)

• western blot

• agar gel immunodiffusion AGID

types of serology tests 2

• Indirect fluorescent antibody test IFAT

• complement fixation test CFT

• rapid diagnostic tests

serology - ELISA

• used to detect antigens or antibodies in the serum

• highly sensitive + widely used for disease diagnosis

examples of when ELISA used

• test for parvovirus

• leptospirosis

• heartworm

serology - western blot

• used to detect specific proteins from a pathogen

• gel electrophoresis will sep proteins for visual identification

microbiological testing

• bacterial + viral culture - selective media

• parasite identification

• PCR

PCR - polymerase chain reaction

• rapid technique of amplifying a specific DNA sequences

• DNA heated to high temp

• sep into single strands but reforms helix when cooled

• uses in vitro enzyme catalysed DNA synthesis to create mills of identical copies of DNA

PCR Method 1

• extracted DNA is added to a solution containing:

1. Primers

2. nucleotide bases

3. DNA polymerase

1. primers

• short DNA fragments complimentary to the target region

2. nucleotide bases

• A

• C

• G

• T

3. DNA polymerase

• e.g Taq polymerase isolated from Thermus Aquatics found in hot water springs

PCR Method step 1

• Denaturing

• heating to 94C causing DNA to separate into single strands

PCR method step 2

• Annealing

• cooling to 45C allowing primers to bind to the ends of ssDNA

PCR method step 3

• Extension

• heating to 72C causing DNA polymerase to synthesise new complementary strands to all ssDNA

• 

PCR cycles

• the 3 steps are cycled over + over to reach the required amplification

• the DNA is doubled at each cycle

• 32 cycles = 1 bill times amplification

• 1 cycle = 17 secs

identification

• gel electrophoresis

• sequencing/ DNA profiling

DNA profiling

• the use of molecular genetic methods to determine the exact genotype of a DNA sample to distinguish one org from another

how might DNA profiling be helpful

• species determination for diagnosis

• crime scene investigations

• genetic testing

steps in DNA profiling

• sample collection

• DNA extraction

• DNA amplification - PCR

• identification + analysis

considerations

• importance of contamination

• use of controls - pos and neg control

• also good to inc a DNA extraction control

why choose one over another

• type of disease or infection

• stage of disease

• reliability of tests - sensitivity + specificity

• cost

• availability

• speed

should we use more than one?

• when clinical signs are ambiguous we may use more than one tests

• make test results more reliable

• to confirm the prev diagnosis

• monitor disease progression

• surveillance purposes