Term 1 Flashcards
Commensal microbes
help defend the first line of defence:
• Secrete antimicrobials (S. epidermidis)
• Alter surface chemistry (Cutibacterium acnes)
induce protective responses that prevent colonization and invasion by pathogens. On the other hand, these bacteria can directly inhibit the growth of respiratory pathogens by producing antimicrobial products/signals and competing for nutrients and adhesion sites.
invasins
Pathogens may overcome these defences by the production of invasins (proteins associated with the penetration of bacteria into mammalian cells),
Hyaluronidase: Dissolves hyaluronic acid which holds connective tissue cells together
Collagenase: Breaks down collagen in muscle
Kinase: Dissolves blood clots
Phospholipases: Break down phospholipids in cell membranes
Hyaluronidase
: Dissolves hyaluronic acid which holds connective tissue cells together
Collagenase
: Breaks down collagen in muscle
Kinase
: Dissolves blood clots
Phospholipases
: Break down phospholipids in cell membranes
mucous membranes
Made up of epithelial layer and underlying connective tissue layer
• Secretes fluid, viscous glycoprotein (mucus)
• Prevents tracts from drying out (barrier)
• Traps potential pathogens
Lysozyme
in perspiration, tears, saliva, nasal secretions and urine destroys bacterial cell walls
IgA
prevents attachment of microbes preventing penetration of mucous membranes
Sebum
Lowers PH of skin inhibiting growth of pathogenic bacteria and fungi
Bacterial IgA proteases
Immunoglobulin A protease degrades IgA, allowing the organism to adhere to mucous membranes
Neutrophils
(polymorphonuclear leukocytes) active in initial stages of infection – enter infected tissues
Basophils
important in inflammation and allergic responses
Eosinophils
mainly act against parasites – numbers increase upon parasitic worm infection/hypersensitivity reactions
Monocytes
only actively phagocytic once they have entered tissues and matured into macrophages
Granules of NK cells release perforins and granzymes
– kills infected cells & releases microbes for destruction by phagocytes; active against tumour cells
Leukocytosis
Increasedtotalno.WBCinmostinfections;especially bacterial infection
• Duringactivestageofinfectionnumbersmight increase 2 – 4-fold
• Meningitis, infectious mononucleosis, pneumococcal pneumonia & gonorrhea
• Alsooccursinautoimmunedisease(RA),leukemia& in drug toxicity
Leukopenia
DecreasedWBCcountfromimpairedWBC production or increased sensitivity of cell membranes to complement
• Salmonellosis,someviralandrickettsialinfections
• Septicemia – extremely severe bacterial infection
• Alsooccursinautoimmunedisease(lupus), lymphoma, radiation therapy, anticancer drugs, antibiotics & diuretics
Leukocidins
cytotoxin that destroys both neutrophilic leukocytes and macrophages.
Humoral
Antibody-mediated response Extracellular fluids B cells Fast response upon detection Act on Extracellular pathogens Antibody-mediated destruction or neutralization MHC class II proteins
Cell Mediated
T cell-mediated response Location of antigen-presenting tissue T cells Slow response Acts on Intracellular pathogens, cancer cells Cell lysis and programmed death MHC class I proteins
Adaptive immune response - evasion
Concealment of antigens from the host:
• Staying inside host cells without displaying antigens (e.g. latent bovine herpesvirus)
• Infecting ‘privileged sites’ (e.g. microbes that colonise the skin, intestinal lumen, CNS, host cell DNA (retroviruses),
etc.)
Antigenic variation
• During the course of infection in a given individual (e.g. gene switching in brucellosis)
• During spread through the host population, e.g:
- ‘antigenic drift’ as influenza spreads through a community
- ‘genetic shift’ in influenza A virus as human and avian virus strains recombine
Immunosuppression:
Direct action on immune cells (e.g. paramyxovirus (cattle plague) on T cells) or release of immunosuppressive molecules
Cause a rapid ‘hit and run’ infection (e.g. rhinoviruses): invade, replicate and be passed on faster than immune system can respond
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What is another name for humoral immunity?
Antibody mediated immunity
After production in bone marrow, where do B cells mature?
Spleen
- Provide on example of a secondary lymphoid organ where mature B cells will be found?
Lymph nodes, spleen, lymph node nodules
- What feature of antibodies provide their uniqueness for binding with a specific pathogen?
Variable region
- What feature of antibodies distinguishes the major antibody classes?
Constant region
- What is the role of MHC-II proteins on the surface of B-cells?
Presentation of pathogen
- What is the role of follicular T-helper cells?
Bind to antigens on B cells and activate B cells
- What type of cells produce large quantities of antibodies?
Plasma cell
- What is antibody class switching?
When activated B cells switch from igM class to another class
- What is the purpose of the antigen-binding test that takes place in the B-cell germinal centre?
To select and preserve cells with the highest affinity for the antigen to then go on and become memory B cells
Compound light microscope
Compound – consists of two lens systems
Light – uses beam of light to view specimens
Light path of a microscope:
• The optimal set up for a light microscope is referred to as ‘Kohler
illumination’.
• In this case the iris diaphragm of the lamp, the specimen and the primary
image are simultaneously in focus.
• The objective forms a magnified primary image of the specimen in the
image plane, which is viewed and further magnified by the eyepiece.
Bacterial smears Blood smear Histology slides Swabs
Fine needle aspirates
Dark-field microscope
Special condenser set-up scatters light causing it to reflect off the specimen at an angle
Results in bright specimen on a dark background
Phase-contrast microscope
Light waves that are diffracted and shifted in phase by the specimen (termed a phase object) can be transformed by phase contrast into amplitude differences that are observable in the eyepieces
Good for observing live organisms as allows visualisation of transparent cells and structures without the use of stains
Ageing
An accumulation of physical changes over time that render organisms more susceptible to disease and death
A progressive loss of physiological integrity, leading to impaired function
Ageing – hair loss
Hair loss/shedding:
• Atrophied hair follicles
Ageing - sarcopenia
Weight loss/muscle loss • Sarcopenia
• Reduction in muscle fibres
• Affects ‘normal’ activity
Ageing – skin conditions
Dry, flaky skin: • Sebaceous glands less productive • Skin dries out and flakes (dandruff)
Ageing - odour
Odour:
• Reduced immune function
• Recurrent secondary skin
infections
Ageing – immune system
Immune function: • Reduces levels of immune cells • Impaired ability to fight infection & target cancer cells 10
Ageing
Ageing – vision loss
Vision loss: • Cataracts
• Iris atrophy
• Retinal degeneration
Ageing – hearing loss
Hearing loss:
• Degeneration of nerve cells
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Ageing – vocal change
Muffled/weak bark:
• Degeneration of nerve
cells in the larynx
Ageing – incontinence
Incontinence: • Weaker anal and urinary sphincters • Changes in hormone levels can also affect urinary sphincter
Ageing - arthritis
Osteoarthritis: • Progressive degeneration of the joint • Inflammatory disorder • Pain and stiffness in joints
Ageing – cognitive disfunction
Cognitive disfunction: • Changes in behaviour • Secondary to age-related degeneration of the brain • Canine cognitive dysfunction rating (CCDR)
Cognitive disfunction: • Changes in behaviour • Secondary to age-related degeneration of the brain • Canine cognitive dysfunction rating (CCDR)
Ageing – cardiac failure
Cardiac failure:
• Dilated cardiomyopathy
• Valvular disease
• Arterial hypertension
Ageing - diabetes
Diabetes mellitus: • Insufficient insulin production • More common in overweight animals
Hallmarks of ageing – genomic instability
DNA is continually being damaged, mutated and altered
The longer an organism is alive, the greater the chances that a DNA change could lead to disease
Hallmarks of ageing - proteostasis
Impaired protein homeostasis. Proteostasis involves mechanisms for the stabilization of correctly folded proteins and the degradation of incorrect or unneeded proteins by the proteasome or the lysosome
Hallmarks of ageing – cellular senescence
“stable arrest of the cell cycle coupled to stereotyped phenotypic changes” The accumulation of senescent cells in aged tissues can affect function and cause inflammation
Stem cell exhaustion
• • •
Decline in the regenerative potential of tissues
One of the ultimate culprits of tissue and organismal aging
Recent promising studies suggest that stem cell rejuvenation may reverse the aging phenotype at the organismal level
Altered intracellular communication
Alterations in communications between cells and tissues can have widespread effects
Pro-inflammatory status (inflammaging) impacts many organ systems
Parallel dysfunction in the immune system can aggravate the ageing status
Leukocidins
: cytotoxin that destroys both neutrophilic leukocytes and macrophages.
fixed macrophages
Some fixed macrophages are resident in tissues and organs
(e.g. Liver (Kupffer’s cells), lungs (alveolarmacrophages), CNS (microglia), spleen (splenic macrophages), bone (osteoclasts), placenta (Hofbauer cells) etc)
pattern recognition molecules (PRMs)
The initiation of innate immune response relies on the recognition of pathogen-associated molecular patterns by pattern recognition molecules (PRMs), including the cellular pattern recognition receptors and extracellular soluble PRMs.
pathogen-associated molecular patterns (PAMPs)
PAMPs activate innate immune responses, protecting the host from infection, by identifying some conserved nonself molecules. Bacterial lipopolysaccharides (LPSs), endotoxins found on the cell membranes of gram-negative bacteria, are considered to be the prototypical class of PAMPs.
damage-associated molecular patterns (DAMPs)
Damage-associated molecular patterns (DAMPs)[1] are molecules within cells that are a component of the innate immune response released from damaged or dying cells due to trauma or an infection by a pathogen.[2] They are also known as danger-associated molecular patterns, danger signals, and alarmin because they serve as a warning sign for the organism to alert it of any damage or infection to its cells.
toll-like receptors in phagocytosis
detect invaders and activate other cells and processes in innate and adaptive immune system
4 steps of phagocytosis
4 main steps
• Chemotaxis & Adherence
• Chemical signals attract phagocytes to microorganisms
• Phagocyte attaches to microbial cell surface – facilitated by interaction of PAMPs with PRRs on phagocyte surface
• Ingestion
• Opsonization: microorganism is coated with serum proteins to facilitate ingestion
• Phagocyte forms pseudopods to engulf the microbe – formation of phagosome
• Digestion
• Phagosome fuses with a lysosome → phagolysosome where microbe is digested
• Discharge
• Residual body discharges indigestible material from the cell
Various anti-phagocytic mechanisms have evolved to avoid phagocytic killing mechanisms including:
Eluding contact (capsule) Inhibiting or killing the phagocyte (e.g. organism releases toxin) Protection against intracellular death, e.g. - resistance to killing (e.g. staphylococci produce antioxidants) - inhibition of phagolysosome fusion (e.g. Mycobacterium tuberculosis); - escape into the host cell cytoplasm (e.g. leishmaniasis)
Helminths
Multicellularmetazoanparasites
• Requires antibody-dependent cellular cytotoxicity (ADCC)
• FcreceptorsonMo,Eosandneutrophils interact with antibodies coating helminth
• Stimulatesreleaseoftoxic chemicals/proteins
inflammation
Four main signs and symptoms:
• Redness
• Swelling (oedema)
• Pain
• Heat
Three main functions:
• To destroy injurious agent and to remove it/its by-products from body
• To limit its effects on the body by confining/walling off the agent
• To repair and replace tissue damaged by the injurious agent
- Vasodilation & increased permeability of blood vessels
- Phagocyte migration & phagocytosis
- Tissue repair
What are the Chemical signals released by damaged cells, pathogens and activated macrophages cause nearby capillaries to widen and become more permeable
Chemokines
• Cytokines
• Histamines
• Prostaglandins • Leukotrienes
Increasing the permeability of capillaries helps as it:
Increased permeability allows defensive substances in blood to enter injured area:
• Fluid (oedema)
• Antimicrobial proteins
• Clotting elements
Vasodilation also results in redness and heat
Glucocorticoids
Anti-inflammatorymedication
• Suppresscertaincomponentsof the immune system
Fever
systemic response of body to injury
Most frequent cause of fever is infection by bacteria & viruses
Inflammation
local response of body to injury
fever
Complications
- Tachycardia – particularly if any underlying cardiopulmonary disease
- ↑ metabolic rate → acidosis
- Dehydration
- Electrolyte imbalance
- Seizures
- Delirium & coma
- Can be fatal
The compliment system
The complement system, also known as complement cascade, is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen’s cell membrane. It is part of the innate immune system,[1] which is not adaptable and does not change during an individual’s lifetime. The complement system can, however, be recruited and brought into action by antibodies generated by the adaptive immune system.
Interferons
Interferons are proteins that are part of your natural defenses. They tell your immune system that germs or cancer cells are in your body. And they trigger killer immune cells to fight those invaders. Interferons got their name because they “interfere” with viruses and keep them from multiplying
Antimicrobial peptides
Antimicrobial peptides (AMPs) are a class of small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. AMPs have a wide range of inhibitory effects against bacteria, fungi, parasites and viruses.
Dominant allele
an allele that produces the same phenotype whether its paired allele is identical or different.
Recessive allele
only expressed if the individual has two copies and does not have the dominant allele of that gene.
Heterozygote
an individual having two different alleles of a particular gene or genes, and so giving rise to varying offspring.
Homozygote
an individual having two identical alleles of a particular gene or genes and so breeding true for the corresponding characteristic.
Autosomes
An autosome is any of the numbered chromosomes, as opposed to the sex chromosomes.
Sex chromosomes
A sex chromosome is a type of chromosome that participates in sex determination. Humans and most other mammals have two sex chromosomes, the X and the Y
Mutation
A mutation is a change in a DNA sequence. Mutations can result from DNA copying mistakes made during cell division, exposure to ionizing radiation, exposure to chemicals called mutagens, or infection by viruses.
Differential regions
Parts of a chromosome that havenocounterpart on the other sex chromosome
• Genesfoundindifferentialregionshow ‘sex-linked’ inheritance patterns
Genetic linkage
Gene loci that are closer together are less likely to be separated onto different chromatids during crossing over
Liability (in the context of genetic disease)
the combined effect of all factors (environmental and genetic), that render an animal more or less likely to develop that disorder
Immunodiagnostics
, Diagnostic tests that use antibodies
Tests either:
• Detect antibodies in a sample OR
• Detect antigens in a sample using antibodies
What is an Antigen (Ag) ?
- Specific portion of pathogen
* Protein found on surface of pathogen • Non-self
What is an Antibody (Ab) ?
- Self
- Protein
- Immunoglobulins
- Designed to “fit” onto specific Ags and so neutralise them
- Specific binding sites
Ig molecules bind specifically to an antigen and eliminate it
For infectious diseases, can test for one of two factors:
Presence of infection
• Looking for pathogen / antigen
Evidence of exposure
• For diseases where antibodies are created
Immunoassays
Immunoassays test for or measure: • Presence of antigen • Presence of specific antibodies • Levels / quantities of antibody to determine level of protection, stage of disease (getting worse or better)
What is required of a test?
Accuracy Repeatability Reliability Quality control
Practicality?
Examples of Immunoassays
Precipitation
Agglutination with latex beads Radio-immunoassays
Agar Gel Immunodiffusion (AGID) Complement Fixation (CF)
Precipitation
Precipitation reactions are based on the interaction of antibodies and antigens. They are based on two soluble reactants that come together to make one insoluble product, the precipitate. These reactions depend on the formation of lattices (cross-links) when antigen and antibody exist in optimal proportions.
Binding of antibodies to the antigen forms precipitate
Agar Gel Immunodiffusion
An antigen and an antibody are placed in separate wells of an agar gel
Antigen and antibodies diffuse towards each other
A thin white line is formed due to the precipitation of antigen/antibody complex
Agglutination
addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination . Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells.
Complement Fixation
The antibody from the patient serum and the antigen are mixed with fresh complement. Sensitized sheep cells are then added. When the patient antibody is absent, the complement will be able to bind to the antibody-coated sheep cells and cause hemolysis. But when the antibody is present, the antigen-antibody complex binds to the complement, and therefore, no hemolysis will occur. When there is no hemolysis, it indicates a positive reaction.
Enzyme Linked Immunosorbent Assays
2 types of ELISA:
• Direct test - Antibodies used to test for antigen
• Indirect test – Antigens used to test for antibody Can test for:
• Bacteria or bacterial toxins
• Viruses
• Protozoa
• Ab to any of these or Ab to parasites, yeasts,
Direct Elisa
Antibodies used to test for antigen
Indirect Elisa
Antigens used to test for antibody
Immunohistochemistry (IHC)
To detect antigens in cells of a tissue section
Antibodies introduced that bind specifically to the antigens in questions in situ in the tissue sample
Antigen-antibody complex visualized in different way
Immunofluorescense
Radio-immunoassays
The basic principle of radioimmunoassay is competitive binding, where a radioactive antigen (“tracer”) competes with a non-radioactive antigen for a fixed number of antibody or receptor binding sites.
A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.
Nucleic acids can be detected by
staining and visualisation through gel electrophoresis (and other methods)
The only way to know if a particular sequence of DNA (a gene) is present is to selectively amplify it
Polymerase chain reaction (PCR)
- Uses oligonucleotide primers to amplify region of interest (gene)
- Cycles of heating and cooling drives each step
- Millions of copies can be produced in minutes
- Number of copies provides information on presence and/or amount of starting material
PCR - denaturation
High temperature breaks hydrogen bonds holding base pairs together
‘Melts’ double-stranded DNA revealing bases in specific order
Fun fact! The polymerase enzymes needed for this procedure were identified in thermophilic bacteria so they could cope with the high temperatures
PCR - annealing
• At cooler temperatures, complementary bases can bind
• Oligonucleotide primers ‘match’ small regions of the target
area (gene of interest)
• They bind to the matching areas (anneal)
Primers must be designed so that one matches the sense strand and the other matches the antisense strand
PCR – extension/elongation
• Temperature raised to approximately 74°C
• Synthesis of new complementary DNA strand from 3’ end
of primer
Only regions where primers bound will be amplified/copied. So it’s really important that they only match the region we’re interested in
PCR – cycling
- In one cycle (denaturation; annealing; extension) we have gone from one copy to two
- Cycle is repeated multiple times and product number increases exponentially
- Average PCR run is 40 cycles
The amount and size of the PCR product can be visualised using
staining and gel electrophoresis
This visually confirms if our pathogen / gene of interest / strain is present
Known as semi-quantitative PCR
qPCR
method by which the amount of the PCR product can be determined, in real-time, and is very useful for investigating gene expression.
does not rely on any downstream analysis such as electrophoresis or densitometry and is extremely versatile, enabling multiple PCR targets to be assessed simultaneously
fluorescence is measured after each cycle and the intensity of the fluorescent signal reflects the momentary amount of DNA amplicons in the sample at that specific time. In initial cycles the fluorescence is too low to be distinguishable from the background. However, the point at which the fluorescence intensity increases above the detectable level corresponds proportionally to the initial number of template DNA molecules in the sample. This point is called the quantification cycle
What are the four distinct phases within the qPCR curve?
Lag
Exponential
Linear and plateau
Reverse transcriptase PCR (RT-PCR)
Uses reverse-transcriptase enzyme to produce double stranded DNA from RNA
• This provides template for normal PCR
• Can also be incorporated into qPCR = RT-qPCR
The Lawrence Livermore Microbial Detection Array (LLMDA)
Livermore scientists analyzed the genetic code of every microbe that has been sequenced (about 6,000 species and strains in all) and then selected the roughly 360,000 most important genetic markers.
In one microarray configuration, 360,000 probes—short stretches of DNA or RNA that complement the isolated genetic markers—are arrayed in a microscopic square grid on a 2.5- by 7.5-centimeter glass slide.
When a fluorescently labeled fluid sample containing the genetic material of microbes contacts the microarray’s probes, only the squares with DNA or RNA unique to a particular organism are activated.
The activated squares produce a fluorescent pattern, from which species present in the sample are identified.
In this way, multiple pathogens are detected simultaneously, with typical processing times of less than 24 hours.
The current-generation LLMDA can identify 3,111 viruses, 1,967 bacteria, 94 protozoa, 136 fungi, and 126 archaea (primitive bacteria).
A good quality clinical specimen should:
- Be collected before the start of antibiotics (where possible)
- Be representative of the infection site
- Be collected in a sterile manner
- Transported properly and quickly
Microscopy
Stained preparations alow you to see
Morphology
- Size
- Shape
- Arrangement
- Staining affinity - Spores
- Capsule
Microscopy Unstained preproductions allow you to see
motility
Simple staining
involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail, in contrast to negative staining where the bacteria remain unstained against a dark background
Differential staining
Differential Staining is a staining process which uses more than one chemical stain. Using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organism. … One commonly recognizable use of differential staining is the Gram stain.
Special staining
“Special stains” are processes that generally employ a dye or chemical that has an affinity for the particular tissue component that is to be demonstrated. They allow the presence/or absence of certain cell types, structures and/or microorganisms to be viewed microscopically.
gram stains
Gram stain or Gram staining, also called Gram’s method, is a method of staining used to distinguish and classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria
Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out on addition of ethanol. They are stained pink or red by the counterstain,[2] commonly safranin or fuchsine. Lugol’s iodine solution is always added after addition of crystal violet to strengthen the bonds of the stain with the cell membrane.
Ziehl-Neelsen stain
Differential stain to distinguish between acid fast and non acid fast cells
Initially, carbol fuchsin stains every cell. When they are de-stained with acid-alcohol, only non-acid-fast bacteria get de-stained since they do not have a thick, waxy lipid layer like acid-fast bacteria. When counter stain is applied, non-acid-fast bacteria pick it up and become blue (methylene blue) or green (malachite green) when viewed under the microscope. Acid-fast bacteria retain carbol fuchsin so they appear red.
Differential culture media
Differential media contain compounds that allow groups of microorganisms to be visually distinguished by the appearance of the colony or the surrounding media, usually on the basis of some biochemical difference between the two groups.
Biochemical tests – oxidative-fermantative
The oxidative-fermentative test determines if certain gram-negative rods metabolize glucose by fermentation or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the bromthymol blue indicator in OF media from green to yellow in the presence or absence of oxygen .
Biochemical tests – catalase test
The catalase test is primarily used for gram positive bacteria and can be utilized to distinguish Staphylococcus spp.
and Micrococcus spp., which are catalase positive from Streptococcus spp.
and Enterococcus spp., respectively, which are catalase negative
The presence of the catalase enzyme can be demonstrated by adding hydrogen peroxide to the bacterial inoculum, which results in the rapid liberation of oxygen bubbles. The lack of enzyme is demonstrated by the absence of such bubbles.
Citrate test
Some bacteria can utilize citrate as the only carbon source and the citrate test shows if the actual bacterium has this capability.
Positive test result: growth in citrate medium or growth with colour change to blue in Simmon’s citrate tube.
Negative test result: no growth in citrate medium eller growth but no colour change (still green colour) in Simmon’s citrate tube.
Use
The citrate test is used to distinguish between, among others Citrobacter freundii and Escherichia coli.
Coagulase test
Some bacteria produce coagulase, which is an enzyme that converts fibrinogen to fibrin, which means that it can coagulate plasma. The ability to produce coagulase is assumed to be associated to the virulence of staphylococci. The test is used to distinguish between coagulase positive and coagulase negative staphylococci.
Positive reaction if the plasma coagulates and the coagulate is stable. It must not be dissolved upon stirring.
Negative reaction if the plasma does not coagulate or if the coagulate is dissolved again upon stirring.
The coagulase test is used to distinguish between Staphylococcus aureus from coagulase negative Staphylococcus spp
DNase test
Many bacteria have enzymes that break down nucleic acids. The bacteria can then use the resulting nucleotides to build up their own nucleic acids. DNase is such an enzyme, which thus hydrolyzes DNA. Existence of DNase is characteristic for certain species or strains of bacteria and can be used for typing.
Presence of DNase can be determined by cultivation on an agar plate, which contains DNA. If the bacterium has DNase and if the bacteria are allowed to grow over night, the DNA will be hydrolyzed into the constituting nucleotides. Diluted hydrochloric acid (HCl) is then poured onto the plate and there will be a clear zone close to the colonies or the streak, because individual nucleotides are soluble in diluted HCl, but not DNA, which precipitates in the rest of the plate.
Use
The test is useful to distinguish between:
Serratia spp. and Enterobacter spp.
Staphylococcus aureus (most strains are coagulase positive) and coagulase negative Staphylococcus spp.
Moraxella catarrhalis and Neisseria spp.
Hippurate test
Some bacteria can hydrolyze hippurate to the amino acid glycine and benzoate by means of the enzyme hippuricase. Glycine can be detected with ninhydrin (2,2-Dihydroxyindane-1,3-dione), which reacts with free amino groups (-NH2) and a blue product is formed.
Positive test resultat: Deep blue colour.
Negative test result: Pale blue colour.
Use
The hippurate test is primarely used to distinguish between Campylobacter jejuni (hip+) and Campylobacter coli (hip-) and to distinguish between different streptococci (see figure).The test is also used, in combination with other methods, to type Brachyspira spp.
Hydrogen sulfide production test
Some bacteria can metabilize certain sulfur containing compounds under production of hydrogen sulfide (H2S). Hydrogen sulfide is a toxic, flamable and badly smelling gas (smells like rotten eggs). If soluble iron or lead salts (for instance ferric citrate) is used in a so-called H2S-medium, which should also contain sodium thiosulfate (Na2S2O3), they can react with H2S, if present, under formation of black insoluble iron and lead sulfide, respectively.
Positive test result: a black precipitate in the medium.
Negative test result: no precipitate in the medium.
Use
The test can be used for differentiation of, among other bacteria, certain Campylobacter spp.
Kovac´s reagent
Positive test result: The indole reagent change colour to cerise red.
Negative test result: The indole reagent remains pale yellow.
Use
Confirmation of suspected E. coli-strains. Typing (species determination) of Brachyspira spp. in combination with other tests. Kovac’s indole reagent is more sensitive than the indole spot reagent, but it is not recommended for use with anaerobic bacteria. The indole spot reagen is suitable for both aerobic and anaerobe use.
Lecithinase test
Many bacteria have enzymes which can break down lipids, so-called lipases. Lecithinase, which is also called phospholipase C, is such an enzyme that splits the phospholipid lecithin (= e.g. phosphatidylcholine). Phospholipids, which are charged are usually soluble in water, but one of the products which is formed by the splitting, namely a diglyceride, is not charged and it has two long hydrocarbon chains. It is, therefore, unsoluble in water and this is utilized in the lecithinase test, where bacteria are cultivated on egg yolk agar. Egg yolk contains a lot of lecithin.
Method
Apply the bacteria in the form of a streak onto the egg yolk agar.
Read the plate after 24 h.
Positive test result: Precipitation around the streak of bacteria.
Negative test result: No precipitation.
Can among other things be used to differentiate between certain species within the genus Bacillus.
Mixed acid fermentation test
Some bacteria can ferment glucose to a mixture of the following organic acids: formic acid, acetic acid and lactic acid. This is called mixed acid fermentation and it causes highly decreased pH in the medium. Mixed acid fermentation can, therefore, be detected by addition of the pH indicator methyl red (MR). The test method is sometimes called the MR test.
Positive test result: red colour change
Negative test result: no colour change.
Use
Some members of the family Enterobacteriaceae have mixed acid fermentation (see the respective bacterial page), which can be used to differentiate these bacteria.
Oxidase test
Bacteria, which have aerobic respiration, often have cytochrome c and a cytochrome c oxidase. The presence of these components can in combination with other methods be used for typing. A commersial test, which contains an artificial electron acceptor (N, N, N’, N’-tetramethyl-p-phenylenediamine, see Fig. 1), is often used. This artificial electron acceptor change colour depending upon redox state.
Positive test resultat: Dark blue-purple colour change within 10-30 sec.
Negative test resultat: No colour change or colour change after more than 30 sec.
The oxidase test is used for identification of gram negative bacteria. For instance to identify members of the family Enterobacteriaceae, which are oxidase negative, except members of the genus Plesiomonas (oxidase positive). Members of the family Pseudomonadaceae, and the genera Aeromonas and Campylobacter are oxidase positive.
Potassium hydroxide test
The purpose of the potassium hydroxide test (KOH test) is to identify gram negative bacteria. KOH dissolves the thin layer of peptidoglycan of the cell walls of gram negative bacteria, but does not affect gram positive cell walls. Disintergration of gram negative cell walls lyses the cell and release its contents, including the DNA. The DNA will make the solution very viscous and the solution will stick to the plastic loop when touched. Gram positive bacteria will not be affected by KOH, because they have thicker peptidoglycan layer in the cell wall. Thus, the cells will not be lysed, the DNA not released and no viscosity will be observed.
Positive results: The solution with the bacteria (gram negative) will be viscous
Negative results: The solution with the bacteria (gram positive) will not be viscous
Use
The purpose of the KOH test is to quickly distinguish between gram negative and gram positive bacteria as a complement to Gram staining. The test is not useful for anaerobic bacteria.
Urease test
Some bacteria have the enzyme urease, which in the presence of H2O converts urea (=carbamide) to NH3 (ammonia) and CO2 (carbondioxide), which forms ammonium carbonate in the presence of water.
Positive test result: colour change to pink.
Negative test result: no colour change.
Klebsiella spp. and Enterobacter spp. has the capacity to perform butanediole fermentation in contrast to Escherichia coli, Salmonella spp. and Shigella spp.
refractometer
instrument that measures the refractive index of a liquid. The more particles there are in a liquid the more a beam of light will be bent (refracted) as it passes from one medium to another e.g. from air to urine. The result is the formation of a shadow line between the illuminated and dark areas. The result is read from where this shadow line crosses the scale on the refractometer
Veterinary clinical refractometers typically have two or three scales (figure 3). The scale used to measure specific gravity is normally found on the righthand side and is typically labelled as U.G. (urine gravity) or S.G. (specific gravity) with a range of 1.000-1.030 or 1.000-1.040
The scale on the left is for serum protein (S.P.). It is used to measure the total protein levels present in a serum or plasma sample. Its units are typically g/dl (g/100ml). These units may also be printed on the lid of the refractometer case.
3
The central scale is the refractive index scale (nD or ND). It can be used with appropriate conversion charts to measure the concentration of many other solutions. It is not present on all clinical refractometers.
Specific gravity
The specific gravity of a substance refers to its density divided by (or relative to) the density of water. This is why specific gravity has no units, as it is based on the ratio of one density to
another, so the units cancel each other out. The specific gravity of pure (distilled) water is therefore expressed simply as 1.000
In veterinary medicine we frequently want to know the specific gravity (S.G.) of liquids such as urine or colostrum because this information can be clinically useful.
Urine typically has a S.G. in the range of 1.003 to 1.035. This means it is slightly denser than water. This makes sense as we know that urine consists of a mixture of excess body water and waste products of metabolism such as urea and creatinine, along with some crystals etc. Even very dilute urine contains some of these waste products so it will always have a S.G. >1.000. Very dilute urine is often seen in animals suffering from polyuria & polydipsia (PU/PD).
Tips for accurate urine S.G. results:
• If the sample was refrigerated allow it to come to room temperature before testing.
• Ensure that you are using the correct scale(!).
• Ensure that distilled water is reading 1.000 on the correct scale before testing.
• Ensure the urine sample is well mixed before testing it.
Increased urine S.G. is associated with
dehydration, shock, acute renal failure, reduced water intake and/or increased fluid loss e.g. vomiting, panting, sweating or diarrhoea, as well as increased excretion of urine solutes (such as glucose in cases of undiagnosed or poorly controlled diabetes mellitus).
Reduced urine S.G. is associated with
increased fluid intake (polydipsia), overzealous administration of fluid therapy. Diseases that reduce the ability of the kidney to reabsorb water (pyometra, diabetes insipidus, some liver conditions, kidney disease) will result in low S.G., as will the administration of diuretic drugs.
Isothenuria
occurs when the urine S.G. is the same as that of the glomerular filtrate (1.008-1.012). This indicates renal disease severe enough to result in the loss of the ability to concentrate or dilute the urine e.g. chronic renal failure. Even if these animals are deprived of water, their urine S.G. will not rise. Animals with less severe renal disease may have moderate but persistent dehydration and a urine S.G. that remains slightly higher than isothenuria (1.015-1.020).
California mastitis test
The California Mastitis Test (CMT) is a cow side test to estimate the somatic cell count of milk. It is a diagnostic tool to aid in the quick diagnosis of mastitis in dairy cows, and for an udder health management program.
The CMT is performed to;
• Detect the presence of subclinical infections at the beginning of or during lactation as part of an udder health management program.
• Additional diagnostics for cows with clinical signs of Mastitis.
The CMT will only trigger a visible reaction with a concentration of 400,000 cells/ml or more Observing results:
Mastitic milk tends to gel when tested by the CMT procedure. The degree of gelling indicates the presence and severity of mastitis. The change in colour indicates the pH variation of the milk and therefore, the level of inflammation
When infection occurs, white blood cells, or leukocytes, gather to engulf bacteria and stop the spread of infection. High leukocyte counts in milk strongly indicate mastitis-causing bacteria are present. The
CMT reagent, when added to milk, disrupts cell membranes allowing the DNA in the cells to react with the reagent and form a gel. The greater the mastitis infection, the more leukocytes present and the more gel-like substance that forms
Urinary dipsticks
The chemical examination of urine is usually performed by the use of reagent strips. These are multiparameter strips that contain a number of pads, each designed to test for a specific urine constituent:
Glucose
Bilirubin
Ketones
Blood
pH
Protein
Urobilinogen
Nitrite
Leukocytes
glucosuria
The term used to describe glucose in the urine is glucosuria. The reagent strip method for detecting glucose is specific for glucose, meaning no other sugar will cause a positive result. Possible causes of false positives with the reagent strips are contamination with hydrogen peroxide, as well as other strong oxidizing agents, such as chlorine. False negatives (or decreased values) can be seen in urine samples preserved with formalin.
bilirubinuria
When bilirubin is present in the urine, the term “bilirubinuria” is used. Bilirubin is unstable in sunlight or artificial light and should therefore be tested immediately. Delaying testing will result in false negative values.
Ketones: urinary dipsticks
There are three ketone bodies that are produced: acetone, acetoacetic acid, and β -hydroxybutyric acid. Most reagent strips are sensitive to acetoacetic acid, less sensitive to acetone, and do not detect β -hydroxybutyric acid. The presence of ketone bodies in the urine, or ketonuria, is often accompanied by a fruity odor
Blood urinary dipsticks
Reagent strips have the ability to detect intact RBC (hematuria), free hemoglobin (hemoglobinuria), and myoglobin (myoglobinuria) in a urine sample. The test is quite sensitive, and positive samples may appear normal in color during the physical exam.
pH urinary dipsticks
pH testing through reagent strips offer analysis of the urine’s acidity or alkalinity over a pH range of 5.0– 8.5. Results are read at 0.5 intervals on the pH scale. Care should be taken when using the reagent stick as not to contaminate the pH pad with the acidic buffer contained within the neighbouring protein pad.
Protein urinary dipsticks
When protein is present in the urine, the term proteinuria is used. Reagent strips are not specific for any particular protein. They primarily detect albumin and are less sensitive for globulins and mucoproteins. False negatives may occur due to the low concentration of protein contained within the sample therefore the concentration may fall below the sensitivity level of the reagent strip.
Urobilinogen urinary dipsticks
When testing for urobilinogen, the same light-sensitivity precautions used for bilirubin should be taken. Other false negatives can result from the use of formalin as a preservative.
Nitrite urinary dipsticks
Nitrites are products formed from nitrates by the actions of certain species of bacteria. Because of this, the presence of nitrites can suggest a bacterial infection, but the absence of nitrites should not rule it out. The reagent strips are specific for nitrites only.
Leukocytes urinary dipsticks
Leukocytes present in the urine are termed pyuria. The reagent strip method for detecting leukocytes relies on the detection of a leukocyte esterase enzyme. The presence or absence of leukocytes should always be confirmed with a sediment examination.
Microscope urinalysis
Sediment examination is the final step of a complete urinalysis. A sediment exam not only allows you to visualize structures such as cells, crystals, and casts, but it also serves to confirm suspected findings during the physical and chemical examinations previously conducted. As with all phases of testing, fresh urine is preferred, but appropriate preservation, typically refrigeration, is acceptable, especially when a delay in processing is anticipated.
Characteristics of a good smear
- Covers 3/4 of the slide
- Symmetrical, bullet-shape
- No tails or ridges
- Microscopically: should have an even distribution of cells
Blood smear stains
After a smear has completely air dried, it must be stained to identify cells and their characteristics. A Romanowsky-type stain, Wright’s stain, is used in hematology to stain blood smears. A commercial stain that is commonly used is Diff-Quick, which is a modified Wright’s stain (see Figure 2.9). The stain consists of three solutions that fix and stain various components of the cells. The first solution, a fixative, consists of 95% methanol. The second solution, eosin, has an acidic pH and stains cell cytoplasms and eosinophilic granules. The third solution, methylene blue, has an alkaline pH and stains the nuclei of the cells. The slide exposure time to each solution varies slightly depending upon the desired result or sample being stained. Typically, dipping a slide into each solution five times for one second each time dip will give adequate staining. This can be adjusted to alter the appearance of cells. It is important to rinse the slide after the last dip into stain #3. Distilled water is preferred. The now stained smear should be allowed to air dry. Rapid drying, such as a hair dryer or waving the slide, will cause crenation (distortion) of the RBCs.
slide agglutination
Immune-mediated haemolytic anaemia (IMHA) is one of the most common immune-mediated hematologic disorders in dogs and cats.
complement formation against RBCs causes accelerated cell destruction and subsequent anaemia. The anti-RBC antibodies can be either immunoglobulin G or M (IgG or IgM). In IMHA the body actually coats its red blood cells with antibodies and then the red blood cells end up sticking together.
IMHA is a
type II immune reaction, where antibody and/or High levels of anti-RBC antibodies sometimes result in their attachment to more than one cell, causing spontaneous RBC agglutination. Agglutination may be appreciated as red speckles when blood is placed in an EDTA tube or onto a microscope slide.
haematology analyser
Resources on the haematology analyser can be found on the manufacturer’s website here
Haematology analysers are used widely in patient and research settings to count and characterize blood cells for disease detection and monitoring. Basic analysers return a complete blood count (CBC) with a three-part differential white blood cell (WBC) count.
The three main physical technologies used in hematology analyzers are: electrical impedance, flow cytometry, and fluorescent flow cytometry. These are used in combination with chemical reagents that lyse or alter blood cells to extend the measurable parameters. For example, electrical impedance can differentiate red blood cells (RBCs), WBCs, and platelets by volume. Adding a nucleating agent that shrinks lymphocytes more than other WBCs makes it possible to differentiate lymphocytes by volume.
Electrical impendence
The traditional method for counting cells is electrical impedance, also known as the Coulter Principle.
It is used in almost every haematology analyser. Whole blood is passed between two electrodes through an aperture so narrow that only one cell can pass through at a time. The impedance changes as a cell passes through. The change in impedance is proportional to cell volume, resulting in a cell count and measure of volume. Impedance analysis returns CBCs and three-part WBC differentials (granulocytes, lymphocytes, and monocytes) but cannot distinguish between the similarly sized granular leukocytes: eosinophils, basophils, and neutrophils. Counting rates of up to 10,000 cells per second can be achieved and a typical impedance analysis can be carried out in less than a minute.
Flow cytometry
Laser flow cytometry is more expensive than impedance analysis, due to the requirement for expensive reagents, but returns detailed information about the morphology of blood cells. It is an excellent method for determining five-part WBC differentials. A single-cell stream passes through a
laser beam. The absorbance is measured, and the scattered light is measured at multiple angles to
13
determine the cell’s granularity, diameter, and inner complexity. These are the same cell morphology characteristics that can be determined manually from a slide.
Fluorescent flow cytometry
Adding fluorescent reagents extends the use of flow cytometry to measure specific cell populations. Fluorescent dyes reveal the nucleus-plasma ratio of each stained cell. It is useful for the analysis of platelets, nucleated RBCs, and reticulocytes.
refractometer
instrument that measures the refractive index of a liquid. The more particles there are in a liquid the more a beam of light will be bent (refracted) as it passes from one medium to another e.g. from air to urine. The result is the formation of a shadow line between the illuminated and dark areas. The result is read from where this shadow line crosses the scale on the refractometer
Veterinary clinical refractometers typically have two or three scales (figure 3). The scale used to measure specific gravity is normally found on the righthand side and is typically labelled as U.G. (urine gravity) or S.G. (specific gravity) with a range of 1.000-1.030 or 1.000-1.040
The scale on the left is for serum protein (S.P.). It is used to measure the total protein levels present in a serum or plasma sample. Its units are typically g/dl (g/100ml). These units may also be printed on the lid of the refractometer case.
3
The central scale is the refractive index scale (nD or ND). It can be used with appropriate conversion charts to measure the concentration of many other solutions. It is not present on all clinical refractometers.
Specific gravity
The specific gravity of a substance refers to its density divided by (or relative to) the density of water. This is why specific gravity has no units, as it is based on the ratio of one density to
another, so the units cancel each other out. The specific gravity of pure (distilled) water is therefore expressed simply as 1.000
In veterinary medicine we frequently want to know the specific gravity (S.G.) of liquids such as urine or colostrum because this information can be clinically useful.
Urine typically has a S.G. in the range of 1.003 to 1.035. This means it is slightly denser than water. This makes sense as we know that urine consists of a mixture of excess body water and waste products of metabolism such as urea and creatinine, along with some crystals etc. Even very dilute urine contains some of these waste products so it will always have a S.G. >1.000. Very dilute urine is often seen in animals suffering from polyuria & polydipsia (PU/PD).
Tips for accurate urine S.G. results:
• If the sample was refrigerated allow it to come to room temperature before testing.
• Ensure that you are using the correct scale(!).
• Ensure that distilled water is reading 1.000 on the correct scale before testing.
• Ensure the urine sample is well mixed before testing it.
Increased urine S.G. is associated with
dehydration, shock, acute renal failure, reduced water intake and/or increased fluid loss e.g. vomiting, panting, sweating or diarrhoea, as well as increased excretion of urine solutes (such as glucose in cases of undiagnosed or poorly controlled diabetes mellitus).
Reduced urine S.G. is associated with
increased fluid intake (polydipsia), overzealous administration of fluid therapy. Diseases that reduce the ability of the kidney to reabsorb water (pyometra, diabetes insipidus, some liver conditions, kidney disease) will result in low S.G., as will the administration of diuretic drugs.
Isothenuria
occurs when the urine S.G. is the same as that of the glomerular filtrate (1.008-1.012). This indicates renal disease severe enough to result in the loss of the ability to concentrate or dilute the urine e.g. chronic renal failure. Even if these animals are deprived of water, their urine S.G. will not rise. Animals with less severe renal disease may have moderate but persistent dehydration and a urine S.G. that remains slightly higher than isothenuria (1.015-1.020).
California mastitis test
The California Mastitis Test (CMT) is a cow side test to estimate the somatic cell count of milk. It is a diagnostic tool to aid in the quick diagnosis of mastitis in dairy cows, and for an udder health management program.
The CMT is performed to;
• Detect the presence of subclinical infections at the beginning of or during lactation as part of an udder health management program.
• Additional diagnostics for cows with clinical signs of Mastitis.
The CMT will only trigger a visible reaction with a concentration of 400,000 cells/ml or more Observing results:
Mastitic milk tends to gel when tested by the CMT procedure. The degree of gelling indicates the presence and severity of mastitis. The change in colour indicates the pH variation of the milk and therefore, the level of inflammation
When infection occurs, white blood cells, or leukocytes, gather to engulf bacteria and stop the spread of infection. High leukocyte counts in milk strongly indicate mastitis-causing bacteria are present. The
CMT reagent, when added to milk, disrupts cell membranes allowing the DNA in the cells to react with the reagent and form a gel. The greater the mastitis infection, the more leukocytes present and the more gel-like substance that forms
Urinary dipsticks
The chemical examination of urine is usually performed by the use of reagent strips. These are multiparameter strips that contain a number of pads, each designed to test for a specific urine constituent:
Glucose
Bilirubin
Ketones
Blood
pH
Protein
Urobilinogen
Nitrite
Leukocytes
glucosuria
The term used to describe glucose in the urine is glucosuria. The reagent strip method for detecting glucose is specific for glucose, meaning no other sugar will cause a positive result. Possible causes of false positives with the reagent strips are contamination with hydrogen peroxide, as well as other strong oxidizing agents, such as chlorine. False negatives (or decreased values) can be seen in urine samples preserved with formalin.
bilirubinuria
When bilirubin is present in the urine, the term “bilirubinuria” is used. Bilirubin is unstable in sunlight or artificial light and should therefore be tested immediately. Delaying testing will result in false negative values.
Ketones: urinary dipsticks
There are three ketone bodies that are produced: acetone, acetoacetic acid, and β -hydroxybutyric acid. Most reagent strips are sensitive to acetoacetic acid, less sensitive to acetone, and do not detect β -hydroxybutyric acid. The presence of ketone bodies in the urine, or ketonuria, is often accompanied by a fruity odor
Blood urinary dipsticks
Reagent strips have the ability to detect intact RBC (hematuria), free hemoglobin (hemoglobinuria), and myoglobin (myoglobinuria) in a urine sample. The test is quite sensitive, and positive samples may appear normal in color during the physical exam.
pH urinary dipsticks
pH testing through reagent strips offer analysis of the urine’s acidity or alkalinity over a pH range of 5.0– 8.5. Results are read at 0.5 intervals on the pH scale. Care should be taken when using the reagent stick as not to contaminate the pH pad with the acidic buffer contained within the neighbouring protein pad.
Protein urinary dipsticks
When protein is present in the urine, the term proteinuria is used. Reagent strips are not specific for any particular protein. They primarily detect albumin and are less sensitive for globulins and mucoproteins. False negatives may occur due to the low concentration of protein contained within the sample therefore the concentration may fall below the sensitivity level of the reagent strip.
Urobilinogen urinary dipsticks
When testing for urobilinogen, the same light-sensitivity precautions used for bilirubin should be taken. Other false negatives can result from the use of formalin as a preservative.
Nitrite urinary dipsticks
Nitrites are products formed from nitrates by the actions of certain species of bacteria. Because of this, the presence of nitrites can suggest a bacterial infection, but the absence of nitrites should not rule it out. The reagent strips are specific for nitrites only.
Leukocytes urinary dipsticks
Leukocytes present in the urine are termed pyuria. The reagent strip method for detecting leukocytes relies on the detection of a leukocyte esterase enzyme. The presence or absence of leukocytes should always be confirmed with a sediment examination.
Microscope urinalysis
Sediment examination is the final step of a complete urinalysis. A sediment exam not only allows you to visualize structures such as cells, crystals, and casts, but it also serves to confirm suspected findings during the physical and chemical examinations previously conducted. As with all phases of testing, fresh urine is preferred, but appropriate preservation, typically refrigeration, is acceptable, especially when a delay in processing is anticipated.
Characteristics of a good smear
- Covers 3/4 of the slide
- Symmetrical, bullet-shape
- No tails or ridges
- Microscopically: should have an even distribution of cells
Blood smear stains
After a smear has completely air dried, it must be stained to identify cells and their characteristics. A Romanowsky-type stain, Wright’s stain, is used in hematology to stain blood smears. A commercial stain that is commonly used is Diff-Quick, which is a modified Wright’s stain (see Figure 2.9). The stain consists of three solutions that fix and stain various components of the cells. The first solution, a fixative, consists of 95% methanol. The second solution, eosin, has an acidic pH and stains cell cytoplasms and eosinophilic granules. The third solution, methylene blue, has an alkaline pH and stains the nuclei of the cells. The slide exposure time to each solution varies slightly depending upon the desired result or sample being stained. Typically, dipping a slide into each solution five times for one second each time dip will give adequate staining. This can be adjusted to alter the appearance of cells. It is important to rinse the slide after the last dip into stain #3. Distilled water is preferred. The now stained smear should be allowed to air dry. Rapid drying, such as a hair dryer or waving the slide, will cause crenation (distortion) of the RBCs.
slide agglutination
Immune-mediated haemolytic anaemia (IMHA) is one of the most common immune-mediated hematologic disorders in dogs and cats.
complement formation against RBCs causes accelerated cell destruction and subsequent anaemia. The anti-RBC antibodies can be either immunoglobulin G or M (IgG or IgM). In IMHA the body actually coats its red blood cells with antibodies and then the red blood cells end up sticking together.
IMHA is a
type II immune reaction, where antibody and/or High levels of anti-RBC antibodies sometimes result in their attachment to more than one cell, causing spontaneous RBC agglutination. Agglutination may be appreciated as red speckles when blood is placed in an EDTA tube or onto a microscope slide.
haematology analyser
Resources on the haematology analyser can be found on the manufacturer’s website here
Haematology analysers are used widely in patient and research settings to count and characterize blood cells for disease detection and monitoring. Basic analysers return a complete blood count (CBC) with a three-part differential white blood cell (WBC) count.
The three main physical technologies used in hematology analyzers are: electrical impedance, flow cytometry, and fluorescent flow cytometry. These are used in combination with chemical reagents that lyse or alter blood cells to extend the measurable parameters. For example, electrical impedance can differentiate red blood cells (RBCs), WBCs, and platelets by volume. Adding a nucleating agent that shrinks lymphocytes more than other WBCs makes it possible to differentiate lymphocytes by volume.
Electrical impendence
The traditional method for counting cells is electrical impedance, also known as the Coulter Principle.
It is used in almost every haematology analyser. Whole blood is passed between two electrodes through an aperture so narrow that only one cell can pass through at a time. The impedance changes as a cell passes through. The change in impedance is proportional to cell volume, resulting in a cell count and measure of volume. Impedance analysis returns CBCs and three-part WBC differentials (granulocytes, lymphocytes, and monocytes) but cannot distinguish between the similarly sized granular leukocytes: eosinophils, basophils, and neutrophils. Counting rates of up to 10,000 cells per second can be achieved and a typical impedance analysis can be carried out in less than a minute.
Flow cytometry
Laser flow cytometry is more expensive than impedance analysis, due to the requirement for expensive reagents, but returns detailed information about the morphology of blood cells. It is an excellent method for determining five-part WBC differentials. A single-cell stream passes through a
laser beam. The absorbance is measured, and the scattered light is measured at multiple angles to
13
determine the cell’s granularity, diameter, and inner complexity. These are the same cell morphology characteristics that can be determined manually from a slide.
Fluorescent flow cytometry
Adding fluorescent reagents extends the use of flow cytometry to measure specific cell populations. Fluorescent dyes reveal the nucleus-plasma ratio of each stained cell. It is useful for the analysis of platelets, nucleated RBCs, and reticulocytes.
polymorphisms
When there are multiple potential versions of a gene that are common in a population they are referred to as polymorphisms
Polymorphisms can be:
• Single nucleotide polymorphisms - differences in a single nucleotide
• Deletions (of large or small amounts of DNA)
• Copy number variation - chromosomal regions that differ in copy number of certain
regions from one to the next
• Microsatellites – tandemly repeated short DNA sequences (also called simple sequence repeats or short tandem repeats) which vary by how many repeats are present
(there are many other types as well!)
Single nucleotide polymorphisms
Single nucleotide polymorphisms (SNPs or ‘snips’) are a prevalent type of polymorphism
Single base-pair differences between individuals in a population
Every genome contains millions of SNPs
and they can be used to identify unique features in individuals (e.g. in a paternity test)
Some SNPs are the cause of inherited genetic disorders as the polymorphism can result in a faulty, inactive or overactive gene product.
Analysis of our patient’s DNA can be done in three main ways:
- Polymerase chain reaction (PCR) – to look at individual SNPs
- DNA microarrays – for analysing collections of known genetic variants
- Whole genome sequencing (WGS) – identifying the exact sequence of every nucleotide in a patients genome to pick out any polymorphisms linked to disease phenotypes (currently cost-prohibitive in veterinary medicine)
Linkage tests (DNA based)
Most DNA tests look for a particular gene that is known to cause a particular condition. Sometimes scientists are unable to find the exact gene, but are able to know approximately where in a dog’s genome it is located. Genes and other genetic markers are often inherited together because they are near one another on the same chromosome. While it may be difficult to identify the exact gene causing a condition, scientists are sometimes able to find sections of DNA that are usually linked to, and inherited alongside, the unknown gene. By identifying these linked genetic markers, breeders are able to know, with considerable confidence, the genetic status of their dogs.
Risk-based DNA tests (incomplete penetrance)
Most DNA tests look for a particular gene that is known to cause a particular condition. For some conditions, certain environmental factors, or other genetic influences can also contribute to whether a dog becomes affected. Having copies of the disease-causing genes will therefore not be a guarantee that the condition will occur. Similarly an absence of these genes will not be a guarantee that the condition will not occur.
These risk-based tests are sometimes not quite as accurate as other DNA tests, but can still be highly accurate and laboratories will often estimate how accurate their test is.
There are several methods available for assessing gastrointestinal samples for the
presence of parasites, which include -
Stained faecal smear – protozoan oocysts and trophozoites
• Passive faecal flotation – helminth eggs
• Centrifugal flotation – protozoan cysts
• Faecal sedimentation – helminth ova (especially trematode ova) • Baermann technique – lungworm larvae in faeces
• Vomit flotation – nematode ova
Blood analysis for endoparasites.
Assessment of blood samples can aid in the detection of certain parasite
species.
• Direct blood examination – heartworm microfilaria • Modified Knott’s test - heartworm microfilaria
• ELISA testing – heartworm antigens and antibodies • Stained blood smear – blood protozoa
Urine Analysis for endoparasites.
• Urine sedimentation – helminth ova
Skin Analysis.
• Skin scrapes/brushings/hair plucks – range of ectoparasites
Gastrointestinal diagnostic methods: Faecal Sampling for parasites
Many parasites pass ova, cysts or larvae in the faeces, which can then be detected by faecal analysis.
• Shedding of larvae and ova is often intermittent, so ideally, samples should be collected over three consecutive days for increased diagnostic sensitivity.
11
• Collection of faecal sample.
• Faecal samples can be collected from the ground
or directly from the rectum.
• From the ground - If the sample is collected from the ground, it must be picked up as soon as it is passed to avoid contamination with free-living nematodes and mites which may confuse diagnosis and obscure the field of view during microscopic examination.
• Directly from the rectum – Using a gloved hand/finger (species dependent) this method achieves a fresh, uncontaminated sample.
• Care must be taken to avoid damaging rectal mucosa.
12
• Storage of sample.
• Once collected, samples should either be examined immediately, or stored at 4°C to
prevent hatching of ova or larval development.
• Hookworm eggs will rapidly develop and hatch if faeces are left at room temperature.
• Ensure that faecal sample fills the container – too much air space encourages parasite eggs to hatch prior to examination.
• Store for no more than 7 days in sterile, airtight and clearly labelled container – if examination is delayed, dilution with 10% formalin stops endoparasite development.
Causes of sample deterioration or inaccurate results
General operator error
Incorrect sample collection technique
Delay between defaecation and examination
Contamination of sample (on collection, in storage or in the lab)
Incorrect handling, storage or sample preservation
Inappropriate package / storage for transport to external lab
Direct faecal smear
Direct smear is a very simple technique and can easily be
performed in practice.
• A small faecal sample (the size of the head of a match) is mixed with a drop of water on a microscope slide and examined with a cover slip under the microscope.
• The addition of a drop of lugol’s iodine will aid in the detection of Giardia cysts, which will be stained yellow.
• Direct smears only analyse very small volumes of faeces, and, as a result, are considered too insensitive for the detection of helminth ova, which are passed in relatively low numbers.
• Lung worm larvae such as Crenosoma vulpis and Angiostrongylus vasorum may be detected, and is useful as an initial screen for these parasites, however the low sensitivity (54% to 61%) for the detection of lungworm by this method means it should not be relied on as a sole test if negative.
Faecal flotation.
- Faecal flotation remains the most common method to detect helminth eggs and protozoan cysts and are commonly used in large and small animal faecal analysis.
- Flotation techniques allow much larger volumes of faeces to be examined by concentrating ova into small volumes of liquid whilst eliminating debris and allowing direct assessment of parasitic ova.
• Principle.
• The principle of faecal flotation is based on the specific gravity (SG) differences of the various parts of a faecal sample, i.e. faeces, ova, cysts and debris.
• The parasite eggs are lighter (i.e. a lower SG) than the flotation solution and so will float to the surface, whereas the heavier faecal matter (i.e. higher SG) sinks rapidly.
• Therefore,theflotationsolutionutilisedmusthaveahigherSGthan the parasite eggs or cysts.
16
- There are several faecal flotation solutions that are commonly used in diagnostic assessment.
- Many of these can be made quickly and easily in practice.
- Solution utilised should be chosen based on health history of animal and the expected findings.
Many different faecal flotation methods are described in the literature, however the Modified McMaster Technique (MMT) is commonly used (see practical sessions for details).
• The McMaster technique uses a counting chamber that has two compartments, each with a grid etched onto the upper surface.
• When filled with a suspension of faeces in flotation fluid, much of the debris will sink while eggs float to the surface where they can easily be seen and counted.
• If a known weight of faeces and a known volume of flotation fluid are used to prepare the suspension, then the number of eggs per gram of faeces can be calculated.
• (The MMT may have diagnostic sensitivities as low as 60% for some roundworm ova such as Toxocara species and has poor sensitivity for tapeworm egg detection, however pooling samples over a three day period will increase sensitivity)
Sodium chloride for fecal flotation
Common helminths, protozoan ova and cysts
Sg: 1.2
Sheather’s solution for fecal flotation
Common helminth, protozoan ova and cysts (particularly Cryptosporidium oocysts)
Sg: 1.2-1.25
Sodium nitrate solution for fecal flotation
Common helminth, protozoan ova and cysts
Sg:1.2-1.33
Zinc sulphate for fecal flotation
Common helminth (particularly Giardia), protozoan ova and cysts (particularly lungworm larvae) Sg:1.18
Magnesium Sulphate solution for fecal flotation
Protozoan ova and cysts
Sg:1.32
Centrifugal flotation technique.(fecal flotation)
- Centrifugal flotation increases effectiveness by spinning down faecal debris, allowing the eggs/cysts to float to the surface.
- Many research papers have demonstrated that correctly carried out centrifugal flotation results in significantly higher faecal egg counts than using flotation techniques alone.
Vomit flotation.
• While not common, it is possible to identify some nematode ova by evaluating
vomit using the same methodology as for faecal flotation.
• Likewise, vomit may also be scrutinized under a microscope to locate parasites common to the stomach.
• Vomit flotation is useful when parasites, such as Physaloptera species or Ollulanus tricuspis, are suspected in dogs and cats.
Faecal Sedimentation
- The majority of trematode (fluke) eggs are too large and heavy to float reliably in the flotation fluids normally used for nematode eggs, i.e. they have a higher SG, however they do sink rapidly to the bottom of a faecal/water suspension and this is the basis of the faecal sedimentation technique.
- Also, some parasites pass free larvae instead of eggs which cannot be detected by routine faecal flotation.
- The faecal sedimentation method allows detection of large/heavy eggs and certain free larvae.
- This method may also be used for ova that will be distorted or destroyed in the presence of the super saturated salt solutions used in flotation techniques.
The Baermann technique.
• The Baermann technique uses inexpensive equipment, much of
which can be reused, for the detection of larvae in faeces.
• A rubber hose is attached to a funnel and warm water is placed into the funnel into which the faecal sample, wrapped in gauze is placed.
• The warmth of the water activates the larvae in the sample, but they are unable to swim upwards against gravity and as a result will drop through the gauze into the tubing.
• This allows collection of the larvae which can then be centrifuged to concentrate the sample.
• Addition of Lugol’s iodine before examination kills the larvae, making identification easier.
• As well as Angiostrongylus vasorum,the larvae of other lungworms such as Oslerus osleri and Crenosoma vulpis may also be detected using this method.
Coproantigen testing.
• Coproantigen ELISA tests are available for the detection of excretory/secretory
products from intestinal nematodes.
• These tests allow infections to be detected when ova shedding is not occurring and so flotation methods will be ineffective.
• ELISA tests also avoid false positive results due to coprophagia.
• Testing for Giardia faecal antigens is a highly sensitive and specific test, as are recently commercially launched test kits for intestinal roundworms, whipworms and hookworms.
• However, this type of testing indicates the presence of nematodes but gives no indication as to what extent ova shedding is occurring.
• Coproantigen testing is being developed for commercial Echinococcus species testing, and PCR testing of faeces is now commercially available.
Considerations when assessing faecal samples.
• All faecal examinations that rely on the visual detection of parasite eggs, cysts, oocysts or larvae in the faeces have some implicit constraints and may not be indicative of the number of worms present –
• Inaccuracies in counting can occur.
• Microscopic examination of faecal samples cannot detect infestations involving immature
worms or those involving only males.
• Ideal flotation methods may differ for diagnostic stages of different parasites, but due to time constraints and the desire for standardized protocols, a single method is often used for all faecal testing.
• Even though centrifugal flotation has been shown to be superior for parasite recovery from faecal samples, many veterinary practices continue to use a standing, passive flotation.
Other, patient specific considerations include -
• The daily output of eggs by fertile females is influenced by host-physiological factors
such as stress or lactation (increased) or immunity (decreased).
• Chemotherapy can affect egg-production, e.g. corticosteroids (increased) or sub-lethal anthelmintic doses (decreased).
• Some food-stuffs may affect egg production e.g. tannin-rich forages (decreased).
• The concentration of eggs is influenced by the daily volume of faeces being produced by the host, the rate of passage by the ingesta through the intestine, and the distribution of eggs throughout the faecal mass.
• Some eggs from different species are indistinguishable (particularly trichostrongylids and strongylids) which complicates clinical interpretation.
25
- Coprophagic behaviour needs to be identified in dogs prior to testing,
- False positives can occur if dogs are coprophagic prior to testing.
- Strongyle eggs in ruminant and horse faeces will pass through the digestive tract of cats and dogs unchanged, giving the impression that the pet is infected with hookworm.
- Similarly, Toxocara cati eggs may be found in dogs that have eaten cat faeces.
Blood diagnostic methods.
• Dirofilaria immitis (heartworm) infects cats and dogs and is
found in the pulmonary artery of the heart.
• (Although D. immitis is not endemic in the UK, increasing numbers of infected rescue dogs are being imported from endemic countries.)
• Three methods are used to diagnose heartworm infection in dogs –
• Direct Smear – examination of blood on slide.
• Modified Knott’s Test (MKT) – detects and allows identification of microfilariae (larval form of D. immitus) of D. immitis via examination of buffy coat layer.
• Antigen Test – detects adult female heartworm (ovarian) antigens in a serological assay (ELISA methods such as SNAP Heartworm test available).
Direct Smear
examination of blood on slide.
Modified Knott’s Test (MKT)
– detects and allows identification of microfilariae (larval form of D. immitus) of D. immitis via examination of buffy coat layer.
Antigen Test
– detects adult female heartworm (ovarian) antigens in a serological assay (ELISA methods such as SNAP Heartworm test available).
Blood diagnostic method considerations
- The antigen test is the most sensitive test however false negatives can occur –
- In animals with low burden of female heartworms (detects ovarian antigens) or when only male worms are present
- If certain types of wormers have been used that lead to the formation of immune complexes that can block detection of the antigen.
- Animals on heartworm preventive medication become amicrofilaraemic and so the MKT will be insufficient.
- Feline dirofilariasis cannot be reliably diagnosed by microfilaraemia or antigenemia tests, because heartworm numbers are typically too low.
Urine diagnostic methods.
- Urine testing for parasites less relevant for the UK.
- There are several parasites restricted to the urinary system, such as the giant kidney worm (Dioctophyma renale) and bladder worm (Pearsonema plica).
- Ova may be identified by examining urine sediment samples collected through cystocentesis.
Ectoparasite diagnosis.
- Animals displaying dermatoses should always be evaluated for the presence of ectoparasites or signs of their presence.
- Superficial close examination may reveal the presence of certain ectoparasites (lice, ticks, flies) but further methods are required for microscopic parasites or those that live beneath the surface of the skin.
- The techniques described can be used in a range of veterinary species but are most commonly utilised in companion animal species.
Skin Scraping.
- Skin scraping is an easy and effective method that can be used to make a definitive diagnosis of ectoparasitic infestation.
- The edge of a scalpel blade is gently scraped across the surface of the skin in order to collect material which can then be examined under a microscope, usually in a drop of mineral oil on a slide under low power-magnification.
- Addition of 10% potassium hydroxide solution may help to clear debris and allow better visualization.
- Some surface living ectoparasites such as Cheyletiella may be found with a superficial scrape, however those that burrow (Sarcoptes) or live in hair follicles (Demodex) will require a deeper scrape (capilliary ooze).
Interpretation of a skin scrape.
• Finding one mite, egg or deposit of faeces from
sarcoptic mites allows a definitive diagnosis.
• However, as demodex mites are commensals on several species, the significance of one or two mites is not clear.
• False negatives are common with skin scrapes, especially with the deep living forms, and so the absence of a parasite in a sample does not confirm absence on the animal.
Coat Brushings.
• Coat brushings are useful to indicate presence
of fleas and Cheyletiella mites.
• The simplest approach is to place a sheet of white paper below the animal and rub or comb the fur towards the paper.
• Debris removed by this method can indicate fleas through the presence of black, comma- shaped faeces which can be moistened to further confirm presence.
• Debris can also be examined with a microscope to identify presence of mites or eggs.
Tape Strips.
- Tape strips can be useful to indicate the presence of Cheyletiella mites, Trombicula autumnalis (harvest mite) larvae, Otodectes and lice.
- Demodex mites may be seen on these samples if infestation is severe.
- Clear adhesive tape is applied to several locations and then transferred to a microscope slide for examination.
Hair Plucks.
- Hair plucks can indicate the presence of Demodex mites, Cheyletiella eggs, lice and lice eggs.
- Small clumps of hair are plucked and examined under a microscope slide.
Sarcoptes serology.
• A serological (ELISA) test is available for the
diagnosis of sarcoptic mange in dogs.
• Serum IgG antibodies against Sarcoptes antigens are measured.
• It has been reported that the test has 95-98% reliability and a positive result indicates past or present exposure to Sarcoptes scabiei.
• Time to sero-convert to positive is approximately 4 weeks and so false negatives are possible.
• Time to sero-convert back to negative varies between individuals but can be several months thus affecting accurate assessment of treatment success.
Antimicrobial sensitivity testing should:
- Be standardised
* Include the most appropriate antimicrobials • Use the most appropriate methodology
Direct method of getting specimens for the testing of antibiotics
- pathological specimen (e.g. urine, swab of pus etc.)
- Sample is used to inoculate a culture plate
- Single colonies are selected to inoculate a liquid culture
InDirect method of getting specimens for the testing of antibiotics
- Pure culture already isolated
- Sensitivity plate created directly
from pure culture
Disc diffusion (Kirby-Bauer method)
• Solid agar plate (containing suitable nutrients)
• Discs, tablets or strips containing a known concentration of antimicrobial agent
• Pure culture of microbe to be tested
Measures zone of inhibition to find MIC
Minimum inhibitory concentration (MIC)
The lowest concentration of an antimicrobial that will inhibit visible growth of a microorganism after incubation
Dilution methods
- Dilution methods take a volume of antimicrobial to be tested and create a series of dilutions to produce individual tubes or agar plates with a range of strengths of antimicrobial
- A small amount of the microbe of interest is then added and incubated
- The lowest concentration at which there is no visible growth of microbes is identified as the MIC (minimum inhibitory concentration)
E.g agar and broth dilution method
Etest
Etest (previously known as the Epsilometer test) is a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate. A strain of bacterium or fungus will not grow near a concentration of antibiotic or antifungal if it is sensitive. For some microbial and antimicrobial combinations, the results can be used to determine a minimum inhibitory concentration (MIC) via where the elliptical meets the strip
Molecular methods of antibiotic sensitivity testing
- Many antimicrobial resistance genes have been identified in many different microorganisms
- Molecular methods allow us to identify if a pathogen in our patient sample possesses that gene and would therefore not be susceptible to that antimicrobial
- Offers very rapid, accurate and specific resistance testing
- Currently used alongside disc-diffusion AST for comparison
Clinical breakpoint
The concentration of antibiotic used to define whether an infection by a particular bacterial strain/isolate is likely to be treatable in a patient
efficacy ratio of different antimicrobials can be calculated by
comparing the recorded MIC from the AST with the clinical breakpoint MIC
Breakpoint MIC divided by measured MIC = efficacy
Describe the muscle composition of the Equine Oesophagus
Proximal two thirds skeletal muscle Distal third smooth muscle
Margo Plicatus
A region called the margo plicatus is present which separates the glandular and non-glandular parts of the equine stomach. The non-glandular area is lined with squamous mucosa (not columnar). The glandular portion consists of mucosa
Equine Stomach
- Simple monogastric stomach
- 10-20 Litres capacity
- Anatomy of oesophageal sphincter (the cardia) prevents eructation under normal conditions
- Squamousmucosainfundus–no digestive function
- 2-3 litres of gastric acid produced by glandular mucosa each day
- Veryacidicenvironment(pH4)
Glandular mucosa (equine)
- HCl produced by parietal cells in responseto;
- Parasympathetic action (acetylcholine from vagus nerve)
- Gastrin stimulation from G-cells in the gastric antrum
- Histamine stimulation
- Increase expression of H+/K+-ATPase proton pumps on apical surface of parietal cell
Equine Small Intestine
20-25 metres long – Duodenum(1metre) – Jejenum – Ileum(0.5metre) Function similar to other species • Digestion/absorption of Non-Structural CHO Proteins Fats
“Suspended” from roof of abdomen by the great mesentery
Normally positioned in
left dorsal abdomen
Relatively mobile with long mesentery can lead to;
• Volvulus
• Incarceration/herniation
• Intussusception
Equine Large Intestine – Ascending Colon
Large colon holds 70-80 litres of semi-liquid ingesta
Contains 10% of total body water content
Hind-gut Fermentation
• Starts in caecum
• Microbial digestion of structural CHO to release VFAs for absorption
Equine Taenial Bands
Concentrations of the external longitudinal musculature at various aspects around the circumference of the ascending colon
➢Creates segmentation of the colon = HAUSTRA Formation
➢Haustral ‘flow’ contractions of taeniae to mix
Ingesta
All sections have at least 1 taenial band at the mesenteric attachment
Identifying the number and position of additional bands is key knowledge for evaluating the equine GIT by trans-rectal palpation
How many taenial bands in the caecum
4
How many taenial bands in the right ventral colon
4
How many taenial bands in the left Ventral colon
4
How many taenial bands in the pelvic flexure
1
How many taenial bands in the left dorsal colon
1
How many taenial bands in the diaphragmatic flexure
2
How many taenial bands in the right dorsal colon
3
How many taenial bands in the transverse colon
2
How many taenial bands in the descending colon
2
Equine Caecum
First Section of the Ascending Colon up to 1m long, holding up to 30-35 litres of fluid ingesta
Base
• Fixed in position in right caudodorsal abdomen, occupying right PL Fossa
✓ Retroperitoneal attachment at level of right kidney
✓ Attachment to root of mesentery
✓ Attachments along lumber vertebrae
✓ Attachment to right dorsal colon
Body
• Cylindrical and curved, following right flank
Apex
• Tapered blind-ended apex which sits toward midline, extending to xyphisternum
Ileoceacal junction sits dorsal to the caecocolic orifice
Ingesta spills into the body of the caecum
Peristaltic contractions begin at caecal apex to move ingesta up to the caecal base and toward the caecocolic orifice
Enters into the Right Ventral Colon
Equine Ascending Colon – Sections and Flexures
Main body of the ascending colon is “divided” into 4 sections and 3 flexures, based on their topographical location within the abdomen Right Ventral Colon Sternal Flexure Left Ventral Colon Pelvic Flexure Left Dorsal Colon Diaphragmatic Flexure Right Dorsal Colon
Equine Ascending Colon
Ventral and Dorsal colons closely bound by mesocolon
Left Ventral colon narrows as it reaches pelvic flexure
Pelvic flexure is 180-degree bend in colon
Pelvic flexure and left dorsal colon only have 1 taenial band, and no haustra formation
Entire left colon sits ‘free’ within the abdomen, with no body wall attachments
Equine Descending Colon
Heavily segmented, narrow aspect of colon, leading to rectum
Final Reabsorption of water
Formation of faecal balls
Sits within left caudodorsal aspect of abdomen
Arteries in the equine GIT
Caudal Mesenteric Artery
Cranial Mesenteric Artery
Splenic Artery
Coeliac Artery
Nephrosplenic Ligament & Nephrosplenic Space
The nephrosplenic ligament connects the left kidney to the spleen in the horse.
Clinical significance as left colon can become entrapped in this space
Veterinary epidemiology
- thestudyofthedistributionanddeterminantsof
animal health, welfare and production
• The goals are to determine the frequency and distribution anb risk factors for the disease
• Epidemiology – mainly concerned with populations rather than individuals
• Key question often asked by epidemiologists – what is the denominator – the population from which cases came from
• Estimates the frequency of events by:
a. countingthenumberofhealth-relatedeventswhichoccur
within a specified time in different populations b. takingthedenominatorintoaccount
• Help compare occurrences between different populations.
Descriptive epidemiology:
- examine the distribution of disease in a population - observing the basic features of its distribution
Analytic epidemiology:
- investigate a hypothesis about the cause of disease by studying how exposures relate to disease
The epidemiologic triad/triangle
– consist of an external agent, a susceptible host, and an environment that brings the host and agent together.
• Disease is the result of forces within a dynamic system consisting of:
- Agentofinfection
- Host
- Environment
Host
- behaviours
- genetic predisposition
- immunologic factors
Agents
- biological - physical
- Chemical
• Influence the chance for disease or severity
Agents
- environment
- external conditions - physical/biological
• Contribute to the disease process
Heterodonty
heterodont (of an animal) possessing teeth of more than one kind, such as incisors and molars
four types of teeth
incisors
canines
pre molars
molars
decidous dental formulae of the cat
3130/3120
= 26 teeth
permanent dental formulae of the cat
3131/3121
= 30 teeth
decidous dental formulae of the dog
3130/3130
= 28 teeth
permantent dental formulae of the dog
3142/ 3143
= 42 teeth
Brachydont
-(‘short teeth’)-Carnivores, omnivores, incisors ruminants
Do not grow continuously.
Carnivores have specialised teeth for grasping and tearing: canines and carnassials.
Hypsodont
(‘long teeth’)- herbivores
Most teeth of herbivores except incisors of ruminants.
Erupt throughout life.
Have complex grinding surfaces: cement covers enamel of hypsodont teeth
horse dental formulae
3-1-4-3/ 3-1-4-3
the insisor is only sometimes present and mostly in males
Jaw size and shape of horse
Head of mandible is larger in species where lateral grinding of food matter is the predominant movement, and disk of TMJ thicker.
Third joint at mandibular symphysis allows for crushing and cutting by the jaw of carnivores eg dog.
judging Ageing
of horses
By stage of eruption of permanent dentition- up to 5 years old
By shape owing to wear and eruption
Ageing by wear on incisors
angle of incisors
descriprion of horse teeth at 6 days
1st deciduous incisor erupts
descriprion of horse teeth at 6 weeks
2nd deciduous incisor erupts
descriprion of horse teeth at 6 month
3rd deciduous incisor erupts
descriprion of horse teeth at 1 year
3rd deciduous incisor in wear
descriprion of horse teeth at 2 1/2 year
1st permanent incisor erupts
descriprion of horse teeth at 3 1/2 year
2nd permanent incisor erupts
descriprion of horse teeth at 4 1/2 year
3rd permanent incisor erupts
descriprion of horse teeth at 5 years
First and second incisors level, all cups present
descriprion of horse teeth at 6 years
Cups begin to disappear
descriprion of horse teeth at 8 years
Cups gone, stars appear
descriprion of horse teeth at 10 years
Incisors become round
equine paranasal sinuses
frontal sinus
caudal maxillary sinus
rostral maxillary sinus
frontomaxilary sinus
Trephination
creating a hole in the skull- in this instance allows access to the sinuses to flush out debris and infection.
Nasolacrimal duct
he nasolacrimal duct (also called tear duct, latin: ductus nasolacrimalis) is a channel that is directly continuous with the lacrimal sac and opens into the nasal cavity, forming the final part of the tear drainage system of the lacrimal apparatus.
the horse has how many thoratic vertebrae
18
the equine heart sits between ….
the 3rd and 6th rib
the horse has how many lobes in the lung
none, it is unilobular
Tracheal wash
samples from trachea
Bronchoalveolar lavage
samples from lower airways
what do th extrenal inetercostal muscles do in respiration
elevating the ribs and expanding the chest cavity to trigger in inhilation
what do th internal inetercostal muscles do in respiration
assist with exhalation and moving the ribs and chest cavity back to their original position.
Visceral Piston Effect (equine)
Gastrointestinal tract swings cranially as forelimbs hit the ground, pushing against the diaphragm and compressing the thorax, forcing air out
Gastrointestinal tract swings caudally as hindlimbs are loaded, drawing the diaphragm caudally drawing air into the lungs
Links stride frequency to respiration
what does PAM stant for in regards to auscaultation the heat of a horse and whaere are these points located
Pulmonary valve- 3rd Intercostal space
Aortic valve- 4th Intercostal space
Mitral valve – 5th Intercostal space
(on the left side)
how and where do you auscultate the tricuspid valve of the horse
Tricuspid valve- 5th intercostal space on right hand side
describe the structures important to the pallate in the horse
Pharynx: oropharynx, nasopharynx and laryngopharynx
Nasopharynx something really unusual: entry to the auditory tubes (=guttural pouches)
Hard palate (palatine bone) and soft palate which separates oropharynx from nasopharynx
Soft palate free border stuck under the epiglottis: nasal breather and not able to vomit*
Palate muscles: levator veli palatini and tensor veli palatini, palatinus and palatopharyngeus
Lymphoid tissue in the palate
muscles controlling the soft pallet
tensor veli palatini
levator veli palatini
palatinus
palatopharyngeus
function and innervation of the tensor veli palatini
tenses rostral aspect of the soft pallet
innervaded by the mandibular branch of thr trigeminal nerve
function and inervaation of the levator veli palatini
elevates the pallet durning swallowing and closes the nasopharnyx
pharyngeal branch of the vagus
function and innervation of the palatinus
shortens and deperesses the palete
pharyngeal branch of vagus
function and inervation of the palatopharungeus
shortens and depresses the pallet
paharyngeal branch of the vagus
function and innervation of the Stylopharyngeal muscles (nasopharyngeal wall)
Elevates pharynx and larynx Glossopharyngeal nerve (CN IX)
equine Soft palate
regulates entry of air in upper airway If dysfunction of the SP goes above the epiglottis DDSP (“gurglers”?) Close relation with tongue and associated musculature (tongue tie) Lymphoid tissue (palate, pharyngeal wall and tongue tonsils)
name the bones of the hyoid appaeratus
stylohyiod epihyoid thyrohyoid cerahyoid basihyoid
name the hyoid muscles
geniohyodeus, sternohyoideus, omohyodeus, thyrohyoideus
Laryngeal cartilages:
Arytenoid
Epiglottis
Thyroid and cricoid (not visible on endoscopy but palpable during CE)
Rima glottis (or rima glottidis)
Intrinsic laryngeal muscles:
CAD CAL Crycothyroideus Vocalis Ventricularis Arytenoideus transversus
Extrinsic laryngeal muscles:
Hyoepiglotticus (controversy about epiglottic position?)
Thyroihyoideus pulls larynx forward
Sternothyrohyoideus pulls larynx caudally and ventrally
what nerves innervate the laryngeal muscles
Cranial and caudal laryngeal nerves (branches of the vagus, X)
what is th efunction and innervation of the crycothyroideaus
tenses vocal folds indirectly by pullung the cricoid caudally
crainail laryngeal branch of vagus nerve
functions and inervation of the CAD
opens rima glottis (abducts vocal process of ary tenoids and folds)
caudal recurrent laryngeal branch of vagus nerve
functions and innervation of the CAL
narrows the rima glottis
Caudal recurrent laryngeal nerve (branch of vagus
rima glottis
narrowest point of the cavity of the larynx
function and innervation of the Vocalis
rensing the vocal cords
Caudal recurrent laryngeal nerve (branch of vagus)
ventricularis
tensing the vocal folds
Caudal recurrent laryngeal nerve (branch of vagus)
arytenoideus transversus
adduction of rima glottis but opens vocal cords
Caudal recurrent laryngeal nerve (branch of vagus)
Dynamic endoscopy of the larynx can show
abnormalities during exercise (DDPS, RLN, EE, etc…)
Larynx: functions
Glottis closes forcing pressure from abdominal press leads to cough
Glottis closes when effort (defecation, micturition, parturition) maintaining intrathoracic pressure
Full opening during breathing
Phonation (vocal folds and ventricles)
(in cats purring = larynx muscles)
Protection from inhalation of food
Together with tongue and palate deglutition
Larynx: sections
Vestible
Glottic cleft (middle part)
Infraglottic
Guttural pouches
2 large, air-filled sacs ONLY IN THE HORSE connecting with the auditory tube and pharynx
Lined by mucous membrane
Close to vital structures: pharynx, larynx, arteries, nerves and hyoid apparatus
Strangles (bacterial infection), GP mycosis (fungal infection)
Thought to be to cool the brain during exercise (envelop the cranial arteries)
Cranial nerves affected in GP pathology
IX glossopharyngeal nerve, X vagus nerve, XI acessory nerve, XII hypoglossal nerve, Pharyngeal branch of glossopharyngeal nerve, pharyngeal branch of vagus nerve, cranial laryngeal nerve, cranial cervical ganglion
Guttural pouch mycosis and strangles
Effect on essential structures! Remember:
Internal carotid artery
Glossopharyngeal, vagus, accessory, hypoglossal nerves
Sympathetic trunk
External carotid artery
Internal carotid artery
clinically relevent Vessels and nerves
in the equine head
Jugular vein – iv injections
Transverse facial vein – blood collection
Common carotid artery – avoid during iv injection in neck!!!
External carotid artery – in guttural pouches
Internal carotid artery – in guttural pouches
Linguofacial artery – pulse
important veins in the equine head
external jugular vein dilation of transveres facia vein lingofacialus carotis communis lingofacial vein ext. jugular vein recurrent laryngeal nerve commin carotid artery
clinical relevence of the facial sinus in the horse
located just below the facial crest at the level of the middle of the eye
used for transvere facial blood sample collection
direct blood pressure monotoring in an the anethetized horse by
placing a catheter in the facial artery, transverse facila artery or dorsal metatarsal artery
equine Trachea
Rigidity from the rings but also susceptible to collapse due to air pressures
Smooth muscle contraction from the trachealis makes the trachea easier to collapse than in other species
Some breeds more prone to tracheal collapse (TC) (Shetlands for example)
clinical procedures of the trachea
Tracheal examination during CE
Endoscopic appearance, tracheal wash
Endotracheal tube for GA
Emergency tracheotomy for compromised airway
differences in the male horse compared to other species
Entire male = stallion. Castrated male = gelding
No splanchnic skeleton – no os penis
Additional accessory glands: 2 vesicular glands and 2 bulbo-urethral glands – produce seminal fluid (rest are the same)
name components of the equine penis
vesicular glands ampulla deferent duct epidydimus penis glans penis testis blader retractor penis muscle bulborethral glands prostate prepuce fossa glandis urethral process
what types of tissues would you see in a cross section of the equine penis
tunica slbugenea
corpus cavernosum
corpus spongeum
name componetnts of the testes
deferent mesoduct deferent duct deferent duct artery mesochium tail of epididimus ligament of tail of epeididimus proper ligament of testes liberal border of the testes testicular artery paniform plexus body of epididimus head of epeididimus
describe open vs closed castration in horses
During open castration, one incision is made over each testicle, but rather than being closed with sutures, they are left open so that they can drain and heal freely.
Closed Castration
Closed castrations must be performed under sterile conditions at your equine veterinarian’s surgery and under a general anesthetic. While the procedure is the same, in a closed castration the wounds are sealed using sutures. This significantly reduces the likelihood of hemorrhaging, but the wounds are unable to drain as well as those in open castrations and many horses will develop reasonable swelling at the castration site in the days or even weeks after the operation.
deferent duct
The ductus deferens is a muscular tube that is located within the spermatic cord and is a major component of the male reproductive system. It is a continuation of the epididymis and is involved in transporting spermatozoa from the epididymis to the ejaculatory ducts.
epidydimus
The epididymis is a long, coiled tube that stores sperm and transports it from the testes. It appears as a curved structure on the posterior (back) margin of each testis. It is comprised of three sections. These are the head, body,and tail.
name componetns of the Spermatic cord:
Deferent duct Testicular artery Testicular vein (p plexus) Lymphatic vessels Nerves Cremaster muscle - together with pampiniform plexus = cooling
descibe the technique used to castrate horses
During castration we separate the deferent duct and then the rest (cranial vascular part) to emasculate them separately (most common technique)
descirbe the vascular supply to the testes
Testicular artery (from abdominal aorta) well packed within the cord Testicular veins (forming the plexus pampiniformis) coil around the artery and reduced to a single vein which drains in the caudal vena cava Arteriovenous anastomoses within the cord
describe the lympahtics within the testes
Lymphatic vessels drain into lumbar lymph nodes and iliac lymph nodes (testicular hormones)
describe the innervation of the testes
Parasympathetic fibers (vagal nerve and pelvic plexus) Sympathetic fibers from cd mesenteric plexus and pelvic plexus
describe some complications that can occur involving the equine testes
Testicular haernia – wide inguinal canal. On CE testicular torsion and haernia difficult to tell apart unless ultrasound
Testicular torsion
Abscess/haematoma
describe equine sperm deposition
Erection>emission>ejaculation
Erection: relaxation of penile musculature and penis extrudes + increase of blood flow
Emission: semen passes from the epididymis and mixed with seminal plasma > ejaculation: passes through the penis and outside the male and into the mare repro tract (through intromission)
intra uterine (natural breeding) or the uterine body (artificial insemination) unless done transendoscopically (very unusual) when it is left in the papila
when does the stallion reach amximal reproductive capacity
5 years
when does the stallion reach puberty
2 years (sometimes earlier)
spermatogenisis in the stallion takes
55-57 days
libado and mating behaviour in the stallion is at its peak in….
april to september (long days)
name the stages of the equine reproductive cycle
pro-oestrus (inscrese in FSH) oestrus (increase in oestrogen and LH) metoestrus (increase in progesterone) (possibly birth) anoestrus (no hormones)
leanght of equine cycle
21 days
describe equine pro-oestrus
FSH is growing and growing promoting Graafian follicle growth. Oestrogens are starting to go up (produced by the follicle) which lead to the flirty behaviour (review the AMHP notes on breeding!). Lasts around 9 days
describe equine oestrus
kicks in with the surge of LH which leads to the egg being released. After the release of the egg into the oviduct, the tissue left behind starts scaring turning into CL which produces progresterone. It lasts 2-5 days
describe equine metoestrus
Metoestrus is a phase dominated by progesterone. Up to 56 days. This is when pseudocyesis occurs (false pregnancy) towards the end when P4 is decreasing and prolactin increasing. After CL the scar turns into a Calbicans
describe equine anoestrus
no activity. This could last months
decribe the componests of the equine female reproductive system
uterus crvix vagina vestebule ureathra vulva clitorus uterine horns ovaries broad ligamet
names the componets of the evuine ovary
ovarian artery ovarian veins medulla (vascular zone) cotex (parenchymatous zone) corpus luteam ovulation fossa graphian follicles
descibe the equine ovaries
Cranioventral to the iliac wings
Mesovarium allows mobility – laparoscopy through flank for Gran cell tumour
Up to 10 cm long with an ov fossa
CL and growing follicles more challenging to asses without U/S
descibe the three parts of the oviduct in the horse
: the infundibulum (funnel-shaped portion nearest the ovary), ampulla (expanded middle portion), and isthmus (narrowed portion connecting the ampulla (fertilisation point) to the uterine horn).
equine uterotubal junction (UTJ),
Sperm gain access to the oviduct through the uterotubal junction (UTJ), which is located in the center of the oviductal papilla that projects into the uterine lumen near the blunt end of the uterine horn. Deep, edematous longitudinal folds are present in the UTJ during estrus, and numerous sperm can be found “bound” to epithelial cells or “trapped” in these folds within 4 hours of breeding. The UTJ may play a role in the selection of morphologically normal sperm and may also act as a storage site for sperm awaiting transport into the oviduct.
In the mare, the mesometrium attaches to……
the dorsal surface of the uterine horns, whereas in the cow the attachment is on the ventrolateral surface. Therefore in mares the free (unattached) surface of the uterus is ventral to the broad ligament, whereas in cattle the free surface is dorsal to the broad ligament.
what can be felt and done with a rectal exam on the mare
Uterine horns – easy to palpate Fluid/oedema on U/S ”Pinch” for terminating twin pregnancies Body short Cervix tone and oedema-lacerations Presence of all the structures Size ovaries Uterine tone and size Cervical tone Recognition of big uterine cysts Recognition of pain in ovary (behavioural consults) PD after 13-14 days with ultrasound – much later with RE and depends of experience of vet
caslick surgery
Caslicks is an operation to partially suture together the lips of the vulva. Caslicks are used to prevent fecal contamination problems in mares that have abnormal vulva conformation
Breeding soundness examination (‘MOT’)
Old mares (>12 y.o.) Thoroughbred mares: commonly done at the beginning of the breeding season to all mares Essential Programmes with artificial insemination and/or embryo transfer
Pregnancy diagnosis
in mares
Day 14 maternal recognition of pregnancy starts (otherwise PGF2a)
As early as 10 days (most VSs do 13-14 days) pregnancy vesicle on ultrasound PD
Unusual rounded embryo that moves about more than other species and it is covered in a “rounded” protective layer of sugars that disappears around day 23* (blastocyst capsule) when fixation occurs
A critical point is day 45 when CL can start to break down but it is replaced by accessory CLs (progesterone). After 5th month, placenta takes over the production of progesterone
Day 25 starts the formation of the chorionic girdle – cells on day 38 migrate inside the uterine wall to form the endometrial cups
Day 28 development of sinus terminalis (future umbilical cord)
Day 35 organogenesis completed (after day 35 we can call it foetus)
Day 45 early placenta leads to microplacentomes (microcotyledones with microcaruncles)
Endometrial cups = Circular horseshoe pale irregular outgrowths that reach peak size day 70 then regress (130 days disappear) produce eCG, thought to stimulate secondary CLs for pregnancy maintenance
Day 45 is critical: if pregnancy loss after this it takes up to 3 months to get back on heat because of the cups!
PD checks: 13-14 days (twins!! Mindful not to mistake embryo with cyst!!), then 21 (if AI or valuable as it will be inseminated for the next cycle), then 45 days.
describe the palcenta of the horse
Placenta is diffuse, microcotyledonary and epitheliochorial
descibe the mamory glands of the mare
Two mammary glands, much smaller than cows Small in non pregnant young mares Each gland has two separate duct systems Inside very similar to cattle External pudendal artery Pudendal vein + lateral thoracic vein Lateral thoracic vein very marked in pregnant mares
Useful kits to have for mares
: milk electrolytes test for predicting parturition (sodium drops below K, increase of Ca+2 are signs of imminent 24 to foaling)
IgG milk content for testing IgG to ensure foal has good passive immunity
List the main differences in the accessory glands between stallions, dogs and cats
dogs have: prostate gland
cats have: bulbourethral gland
and prostate gland
horses have: vesicular gland, ampulla of defertn duct and, prostate gland anf bulboourethral gland
Ovulation fossa
n the concave surface of the ovary is the ovulation fossa where the oocyte is expelled from the ovary
Endometrial cups
Circular horseshoe pale irregular outgrowths that reach peak size day 70 then regress (130 days disappear) produce eCG, thought to stimulate secondary CLs for pregnancy maintenance
vessels and nerves of the horse of clinical importance
Jugular vein – iv injections
Transverse facial vein – blood collection
Common carotid artery – avoid during iv injection in neck!!!
External carotid artery – in guttural pouches
Internal carotid artery – in guttural pouches
Linguofacial artery – pulse
Prevelance
Prevalence, sometimes referred to as prevalence rate, is the proportion of persons in a population who have a particular disease or attribute at a specified point in time or over a specified period of time
what bones make up the equine distal forelimb
3rd metacarpal bone (cannon bone) 4th and 2nd metacarpal bone (splint bones) proximal sessamoid bones proximal phalanx (p1, cannon bone) middle phalanx bone (p2, pastern) distal phalanx (p3, coffin bone)
extensor tendons of the equine distal limb
(not prone to injury)
Common Digital Extensor Tendon
Courses distally over dorsal aspect of cannon, fetlock and pastern
Inserts on extensor process of P3
Lateral Digital Extensor Tendon
Lies dorsolateral to cannon
Inserts on dorsal aspect of P1
extensor tendons of the equine distal limb
(not prone to injury)
Common Digital Extensor Tendon
Courses distally over dorsal aspect of cannon, fetlock and pastern
Inserts on extensor process of P3
Lateral Digital Extensor Tendon
Lies dorsolateral to cannon
Inserts on dorsal aspect of P1
flexor tendons of the distal equine limb
Superficial Digital Flexor Tendon (SDFT)
Inferior Check Ligament
Deep Digital Flexor Tendon (DDFT)
Suspensory Ligament
sessamoidean support ligaments of the fetlock joint
Sessamoidean Ligaments Straight Oblique Cruciate Short
annular support ligaments of the fetlock joint
Palmar Annular Ligament
Proximal Digital Annular Ligament
Distal Digital Annular Ligament
pints for nerve block on the equine distal forelimb
low 4 point
Abaxial Sessamoid
palmar digital
Low 6-Point
pints for nerve block on the equine distal forelimb
low 4 point
Abaxial Sessamoid
palmar digital
low 4 point nerve block
deep nerves course between the splint bones and the cannon bone on the axial surface of the splint bones-block just distal to the end of the splint bone
This block numbs the fetlock and distal cannon bone (palmar or plantar aspect).
Palmar Digital
(Heel Block) – The block targets the back of the foot. It is injected over the palmar digital nerve just under the skin. It blocks the heel bulbs, frog, navicular bone, navicular bursa, the coffin joint, and the phalanx.
low 6 point nerve block
blocks lateral and medial plantar nerves and lateral and medial metatarsal nerves the same as 4 point block in forlimb
indlimb hower has dorsal metatarsal nerve and hence doral surface is alo blocked
equine distal limb- digital flexor sheath
a synovoial flud space that surrounds the flexor tendons as they course around the bottom third of the cannon bone and the fetlock joint and disperse throughout the pastern region
case study
Observational, descriptive
Careful, detailed description of a single case or series of cases (typically by observant clinician(s))
Analysis: narrative description, simple descriptive statistics (case series)
• Select people based on outcome
• Select people with and without lung cancer and ask about habits
• Good for rare diseases
• Quick and inexpensive
• Lack temporality
• Recal bias- inaccurate data
• Can’t estimate prevalence or incidence
May be the first clues of new diseases, outbreaks, impact of a condition, unsuspected adverse effects, possible
exposures
no comparason group
crossectional study
- A snap shot in time
- Quick and cheap
- Data collected at one period of time
- Large Heath surveys
- Census data
- Do they smoke? Yes no
- Lacks temporality- what came first? Smoking or lung cancer
- Can estimate prevelance
Observational, analytical
Careful, detailed description of study population (time and place)
Exposures and outcome/disease status are assessed the same time
multiple exposures or outcomes
high suceptability to bias
cohort study
- Select people based on exposure
- Eg selecting people based on whether they smoke
- Has temporality
- Follows people over time
- Take a long time
- Expensive
- Loss to follow up issues
- Incidence can be measured
- Prospective or retrospective
Observational, analytical
Careful, detailed description of study population and exposures (risks)
Starts at the time of the exposure – follow-up until outcome occurs
randomised control trial
• Similar to cohorts
• People are randomly assigned to exposures
• Might be unethical to expose or not expose the group to certain factors
• Balance out confounders
• Long
• Expensive
Experimental, analytical
Prospective cohort design
Starts at the time of exposure (intervention) – follow-up until outcome occurs
Key features:
Control arm (no exposure) Random allocation of exposure to intervention groups: similar baseline characteristics; similar distribution of confounders Blinding of participants (e.g. owners) and clinicians (where possible)
randomised control trial
- Similar to cohorts
- People are randomly assigned to exposures
- Might be unethical to expose or not expose the group to certain factors
- Balance out confounders
- Long
- Expensive
Case control designs
Observational, analytical
Compares cases (diseased animals) and controls (non-diseased animals) with respect to their level of exposure to a suspected risk factor
Starts with the disease (or outcome of interest) and looks back at prior history of exposures
“all the effects are already produced before the investigation begins”
investigates multiple exposures
well suited to rare disease with long latency
quick and inexpensive
suseptable to bias: selectional bias, information bias
lacks temporality
Information bias
Exposures and outcomes are not measured well, or not in a similar way in all study participants (animals)
Information bias: assessment of exposure varies depending on risk of experiencing the outcome / disease status
Information bias
Exposures and outcomes are not measured well, or not in a similar way in all study participants (animals)
Information bias: assessment of exposure varies depending on risk of experiencing the outcome / disease status
describe the components of the equine forelimb
Scapula: scapular cartilage
Humerus: greater tubercle = point of the shoulder
Olecranon (ulna) = palpable point on elbow
Radio and ulna fused
Accessory carpal bone = palpable point on carpus/knee
Chestnut
Ergot
Equine joints of the forelimb
Proximal to distal: Shoulder Joint Elbow Joint Carpal joint – (Knee!) Metacarpophalangeal joint – (Fetlock joint) Proximal interphalangeal (PIP) joint – pastern Distal interphalangeal (DIP) joint – coffin joint
function and innervation of the trapezius
Protracts & abducts forelimb. The cervical part acting alone swings the scapula forward, which advances the limb, whereas the thoracic part acting alone swings it in the opposite direction. Accessory nerve.
function and inervation of the brachiocephalicus
Limb on floor: flexes the neck & bends it laterally. Not on floor: protracts foreleg. Accessory cervical and axillary nerves.
function and inervation of the brachiocephalicus
Limb on floor: flexes the neck & bends it laterally. Not on floor: protracts foreleg. Accessory cervical and axillary nerves.
function and innervation of the omotransversius
Draws the FL forward. Accessory nerve.
function and inervation of the lattisimus dorsi
Retracts limb (antagonist of brachiocephalicus) & flexes shoulder. Thoracodorsal nerve.
function and innervation of the pectoralis transversus
Adduction and protraction/retraction of limb. Pectoral branches brachial plexus
function of the rhomboideus
Extends and raises the shoulder; pulls the scapula cranially and dorsally. Raises the head
function of the serratus ventralis
Supports the trunk and rotates scapula. The cervical part rotates the bone so that the ventral angle is carried caudally, thus retracting the limb; contraction of the thoracic part advances this angle and thus the limb.
funtion and innervation of the pectoralis profundus
Adduction and protraction/retraction of limb. Pectoral nerves.
function and innervation of the subclavius
Adduction and protraction/retraction of limb. Pectoral nerves.
how to tap the shoulder joint
The cavity is relatively capacious. It may be tapped by inserting a needle at the cranial margin of the palpable infraspinatus tendon about 2 cm proximal to the caudal part of the greater tubercle. The needle is directed ventromedially and must be introduced about 4 or 5 cm before its tip penetrates the capsule.
The procedure requires some care because a cranial deflection may cause the needle to enter a quite separate synovial sac, the bursa that protects the biceps tendon within the intertubercular groove. This intertubercular bursa corresponds to the diverticulum of the joint capsule found in the dog and sheep. (Dyce)
describe the bones and muscles of the equine shoulder
Bones
Scapular spine and cartilage
Point of the shoulder (cranial part of the greater tubercle humerus)
Muscles:
Lateral: supraspinatus, infraspinatus, deltoideus (and teres minor)
Medial:
subscapularis, teres major, coracobrachialis (and capsularis)
