AHS 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 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
an invasis that dissolves hyaluronic acid which holds connective tissue cells together
Collagenase
invasin that Breaks down collagen in muscle
Kinase
invasis that dissolves blood clots
Phospholipases
invasin that breaks down phospholipids in cell membranes
Lysozyme
in perspiration, tears, saliva, nasal secretions and urine destroys bacterial cell walls
IgA
prevents attachment of microbes preventing penetration of mucous membranes
he first line of defence in the resistance against infection
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
phagositosis
Basophils
important in inflammation and allergic responses
degranulation
release of histamine
cytokines and enzymes
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 which:
kills infected cells & releases microbes for destruction by phagocytes; active against tumour cells
Leukocytosis
Increased total no. of WBC in most infections; especially bacterial infection
• During active stage of infection number smight increase 2 – 4-fold
• Meningitis, infectious mononucleosis, pneumococcal pneumonia & gonorrhea
• Also occurs in autoimmune disease (RA) ,leukemia& in drug toxicity
Leukopenia
Decreased WBC count from impaired WBC production or increased sensitivity of cell membranes to complement
• Salmonellosis, some viral and rickettsial infections
• Septicemia – extremely severe bacterial infection
• Also occurs in autoimmune disease (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
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
A dark field microscope is ideal for viewing objects that are unstained, transparent and absorb little or no light. These specimens often have similar refractive indices as their surroundings, making them hard to distinguish with other illumination techniques.
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 – 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
Ageing – vision loss
- Cataracts
- Iris atrophy
- Retinal degeneration
Ageing – hearing loss
• Degeneration of nerve cells
Ageing – vocal change
Muffled/weak bark:
• Degeneration of nerve
cells in the larynx
Ageing – 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)
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
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
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.
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 microorganism.
- Phagocyte attaches to microbial cell surface – facilitated by interaction of PAMPs with PRRs on phagocyte surface
• 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
multicelular metazoan parasites
• Requires antibody-dependent cellular cytotoxicity (ADCC)
Fc receptorson Mo, Eos and neutrophils interact with antibodies coating helminth
• Stimulates releaseof toxic 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
infalmation is a defence used by the body
it increses the permiabilty of caplaries
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
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.
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
Immunoassays
Presence of infection
• Looking for pathogen / antigen
Evidence of exposure
• For diseases where antibodies are created
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)
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
steps of PCR
denaturation: 94-95C
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
annealing: 50-56C
• 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
extension/elongation: 74°CC
• 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
- 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
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
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
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.
method:
Crystal violet added to a heat-fixed bacterial smear stains all cells purple (primary stain);
After washing, the smear is covered with iodine which forms a purple CV-I complex in the cytoplasm;
Washing the slide with alcohol-acetone ( a decolouriser) disrupts the outer LPS layer of G-ve cells and the CV-I complex is washed away through the thin peptidoglycan; these cells are then colourless until counterstained with safranin (pink);
Alcohol-acetone does not wash the CV-I complex out of G+ve cells (thicker peptidoglycan), so they remain purple.
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,
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.
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
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 for parasites
• Skin scrapes/brushings/hair plucks – range of ectoparasites
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.
• 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.
- 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).
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.
Blood diagnostic method considerations
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.
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.
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.
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
AST
antimicrobial suseptibility testing
a widely-used method of evaluating antibiotic resistance and determining patient treatment plans in clinical settings. There are a number of different methods of AST such as agar dilution, broth dilution and disc diffusion assays
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
parasite
an organism that lives in or on an organism of another species (its host) and benefits by deriving nutrients at the other’s expense:
Definitive host
an organism which supports the adult or sexually reproductive form of a parasite.
intermediate host
an organism that supports the immature or non-reproductive forms of a parasite.
Paratenic host
paratenic host a potential or substitute intermediate host that serves until the appropriate definitive host is reached, and in which no development of the parasite occurs; it may or may not be necessary to the completion of the parasite’s life cycle.
Obligate parasite
obligate parasite (obligatory parasite) one that is entirely dependent upon a host for its survival.
Facultative parasite
an organism that may resort to parasitic activity, but does not absolutely rely on any host for completion of its life cycle.
mechanical vector
here are certain vectors where the parasites (germs) are attached to the outside of their body, such as in legs and thus transmit the germs or parasites from one host to another without involving any developmental stages of the parasites in their body.
Biological vectors
biological vector an animal vector in whose body the pathogenic organism develops and multiplies before being transmitted to the next host. mechanical vector an animal vector not essential to the life cycle of the parasite.
Endemic
regularly found among particular people or in a certain area:
Hyperendemic
exhibiting a high and continued incidence —used chiefly of human diseases hyperendemic malaria
Epidemic
widespread occurrence of an infectious disease in a community at a particular time:
Anthropozoonosis
An infectious disease acquired by humans from vertebrate hosts of the causative agents. Examples are rabies and trichinosis.
Zooanthroponosis
The transmission of disease from humans to animals. Specifically it refers to diseases that are primary infections of humans but which can be naturally transmitted to animals. Examples include tuberculosis and human metapneumovirus.
Amphixenosis
A zoonosis that can be passed from humans to other species as well as being passed from another species to a human
Anthroponoses
refers to pathogens sourced from humans and can include human to non-human animal transmission but also human to human transmission.
Cyclozoonosis
A zoonosis that requires more than one vertebrate host (but no invertebrate) for completion of the life cycle
Metazoonosis
A zoonosis that requires both a vertebrate and an invertebrate host for completion of its life cycle; for example, the arbovirus infections of humans and other vertebrates
Saprozoonosis
A zoonosis, the agent of which requires both a vertebrate host and a nonanimal (food, soil, plant) reservoir or developmental site for completion of its life cycle
How do parasites affect the host?
Compete for nutrients Depress appetite Damage skin or internal organs Diarrhoea Liver failure Respiratory problems Increase chances of secondary infections Stimulate immune system so that the animal is more susceptible to disease
nematode lifecycle
egg l1- free living/ in intermediate host l2- free living/ in intermediate host l3- free livivng in intermediate host- infective stage! l4- within host l5- within host adult reproduction- repeat
any unsegmented worm of the phylum Nematoda, having an elongated, cylindrical body; a roundworm.
hypoboisis
Hypobiosis
A stage of parasite larval dormancy where nematode parasite larvae escape harsh environmental conditions by remaining in the wall of the abomasums.
Inhibited development stage with mass emergence = longer PPP
No inhibited stage = shorter PPP
Environmental or external stimulus at free living stage?
Mass emergence
Examples:
Toxocara canis
Cyathostomins- small red-worms
Teladorsagia
basic cestode lifecycle
Adult
(within definitive host, e.g dog)- gravid proglottids shed
Embryophore
(in environment)- Ingested by intermediate host
Oncosphere
(within intermediate host, e.g sheep)- Breaks through gut wall of intermediate host and travels to site to form a …
Metacestode
(cyst within intermediate host)- Remains within intermediate host until it is ingested by definitive host
ndirect lifecycle
Hermaphrodites
Reliant on host – no free living stage
No mouth / anus – absorb pre-digested nutrients through tegument a parasitic flatworm of the class Cestoda, which comprises the tapeworms
Taenia saginata, T.solium, T. Ovis, T.multiceps
Echinococcus multilocularis, E. granulosus
Trematode Lifecycle
Eggs-Passed in faeces onto pasture
Miracidium -Miracidium
hatches
Within intermediate host-Develop to sporocyst, rediae and cercariae
Leave intermediate host
Cercariae
Metacercariae- Ingested by grazing animals
Lifecycle – 5 months
PPP – 3 months
Liver and stomach flukes
Indirect lifecycle - snails
Hermaphrodites
Paedogenesis - production of many new individuals from a single larval form
Any of numerous parasitic flatworms of the class Trematoda, having a thick outer cuticle and one or more suckers or hooks for attaching to host tissue.
Protozoa Eimeria Lifecycle (example)
single celled organisms
Unsporolated oocyst-Nucleus divides - sporocysts
Sporolated oocyst
(infective)- Ingested – liberation of sporocysts and sporozoites within them…
Sporozoites-Penetrate gut wall cells and reproduce asexually…
1st generation merozoites-Gut cells burst when full of 1st gen merozoites…
2nd generation merozoites-Invade more gut cells…Gut cells burst when full of 2nd gen merozoites…
Male / female -Fuse = oocysts!
Cryptosporidium
Sarcocystidae (Toxoplasma, Neospora)
Babesiidae (Babesia)
Transmission of parasites
Faeco-oral Grazing, bedding, coat, Fungi Intermediate host Paratenic hosts
Pre-Patent Period (ppp)
Time taken from ingestion of eggs/ larvae/ cysts to eggs being present in faeces
Ectoparasite groups
Arachnids: Mites Ticks 2. Insects: Flies Lice Fleas
Arachnid Lifecycle
egg
larvae
nymph
adult
insect lifecycle
Gravid female lays eggs
Eggs hatch - larvae- 12 hours
Larvae feed, grow and moult - maggots-Moult 3 times in 3-10 days
Maggots drop to ground and pupate– Pupate for 3 -7 days
(may overwinter)
Effects of ectoparasite infestation
Irritation / annoyance
Damage to skin / hide / fleece
Bites / wounds (painful!) and possibly anaemia if blood sucking
Disease transmission – vectors and 2ndry bacterial infections
Allergic reactions to saliva / faeces of ectoparasites
Myiasis
Creating a control plan for parasites
Need to know:
Which parasites are present, any resistance?
Perform faecal egg counts
Times of year those parasites cause problems
Best time to treat against them or use management to avoid them
e.g. Nematodirus, fluke, midges,
Which management strategies apply
Which chemicals are effective against the parasites that are present
Fleas
Fleas are wingless insects with laterally compressed bodies.
They have six legs that are well adapted for jumping.
Two of the most common companion animal fleas are -
Ctenocephalides felis (cat flea) - the cat flea is the most common in the UK and infests the cat, dog, rabbits, ferrets, small rodents and man.
Ctenocephalides canis (dog flea) found on some dogs in the UK and most common flea on dogs in Ireland. Diagnosis of fleas.
treatment targets: Kill the adult flea on the host.
Kill the developmental stages in the environment.
scientific name of human and pig flea
Pulex irritans
scientific name of rabbit flea
Spilopsyllus cuniculi
scientific name of bird related flea
Ceratophyllus gallinae
scientififc name of rodent flea
Xenopyslla cheopis
scientific name of cat flea
Ctenocephalides felis
scientific name of dog flea
ctenosephalides canis
Significance of fleas as vectors of disease.
Besides the direct effects resulting from blood feeding, Ctenocephalides species are important as vectors for a wide range of pathogens, many of which are zoonotic, for example -
Yersinia pestis (plague),
Rickettsia typhi (flea borne typhus in humans),
Rickettsia felis (flea borne spotted fever in humans),
Rickettsia conorii (boutonneuse fever in humans),
Bartonella henselae (cat-scratch disease in humans)
Fleas also act as intermediate hosts for cysticercoid larvae of Dipylidium caninum tapeworms.
Lice.
Lice are small wingless insects which can occur in large numbers on many companion animal species including dogs, cats, guinea-pigs and rabbits.
Lice are generally host-specific
There are two main types of louse - sucking and chewing.
The “sucking lice” have a pointed head with a piercing proboscis, and feed regularly on blood.
Linognathus setosus (dog).
The “chewing lice” have a broad head bearing strong chewing mouth parts and feed on epidermal scales, scurf and wool.
Trichodectes canis (dog). Felicola subrostratus (cat). Significance of lice infestation -
Re-infestation can best be prevented by ensuring that treatment of the host is effective.
As lice do not live well off the host, transmission is usually by direct contact.
Several products available, including spot-ons.
scientific names of suckcing lice
Linognathus setosus (dog).
scientific names of chewing lice
Trichodectes canis (dog). Felicola subrostratus (cat)
rabbit lice
Haemodipsus ventricosus.
Ticks.
Ticks are arachnids.
They have, as adults, eight legs and are flattened dorsoventrally with a hard shield on the back.
The ticks most commonly found on dogs and cats in the UK belong to the genus Ixodes (I. ricinus and I. hexagonus are the most common, but I. canisuga and occasionally I. frontalis and I.trianguliceps have been seen).
They have no wings.
I. ricinus is a three-host tick and the life cycle requires three years.
The main importance of ticks is their role as vectors of pathogenic agents which cause a range of tick-borne diseases.
Babesia spp (babesiosisi)., Borrelia burgdorferi (lyme) sensu lato, Hepatozoon canis, Acanthocheilonema (Dipetalonema) spp., Bartonella spp., Ehrlichia spp., Anaplasma phagocytophilum, A. platys, Rickettsia spp., flaviviruses and others can all be transmitted by ticks.
Removing ticks inappropriately can lead to tick granulomas.
Physical removal – tick hook.
Several products available including collars and spot-on preparations
dont lay eggs on host
Mites
The entire life cycle of the mite takes place on the animal or within its skin, but many of the stages can remain infective for several days off the animal.
Otodectes cynotis
(ear mites).
These are commonly found in the dog and cat
Cheyletiella.
Often referred to as fur mites.
walking dandrus
Cheyletiella yasguri most common in the dog and Cheyletiella blakei in cats.
The entire life cycle takes approximately three weeks and is spent on the host, although female mites can survive for up to ten days in the environment.
Transfer from host to host occurs readily and rapidly between animals in close contact and cheyletiellosis is common in kennels with young and weak animals being more susceptible.
Demodex
Demodex canis in dogs and Demodex cati in cats.
Demodex lives in hair follicles and sebaceous glands
Diagnosed via deep skin scrapings or hair plucks
Cat notoedric mange
Notoedres cati
Occurs mainly in cats.
Although infestation with N. cati has been reported from all European countries it is rare in some and tends to be local in distribution in others.
sarcoptic mange
Sarcoptes scabiei
Sarcoptic mange mites are small, round parasites (up to 0.4 mm in diameter) which spend their entire life cycle on the host, so transmission is mainly through close contact.
They burrow in the superficial layers of the skin.
Transmission to new hosts from infested individuals is by direct or indirect contact, most likely by transfer of larvae from the skin surface - It is known that S. scabiei can survive for several weeks off the host.
Harvest Mites.
(Neotrombicula (Trombicula) autumnalis)
responsible for the condition known as trombiculosis.
Uncommon and characterised by their seasonal nature (July and August).
Control – spot-on preparations available although control is difficult due to the fact that reinfestations are frequent in animals exposed to these mites.
Only the larvae are parasitic and they do not transfer from animal to animal.
Harvest mites are resistant to adverse climatic conditions and female mites can live for more than 1 year.
Tapeworm – general features.
Flattened, tape-like segmented body.
Each segment is self-contained, containing one or two sets of male and female reproductive organs.
The end segment is released from the tip of the tail and can pass out in the faeces.
This contaminates the areas where the animal defaecates.
Tapeworms have an indirect lifecycle requiring an intermediate host where the larval stages develop.
Larval forms usually encyst within the tissues of the intermediate hosts and primary control measures therefore include preventing exposure to intermediate hosts, where possible.
Dipylidium caninum.
Primary host is the dog or cat.
Intermediate host is the flea.
The adult tapeworms live in the intestines, eggs develop in the segments (proglottids). segments are shed from the tail of the tapeworm and are then passed out with the faeces into the environment.
The segments (proglottids) can often be seen around the affected animal’s perineal/anus area - often described by owners as “grains of rice”.
Eggs ingested by flea larva (intermediate host) and cyst develops in the flea body cavity containing developing forms of the tapeworm.
Flea ingested by primary host.
Taenia tapeworms.
Primary host - most commonly cats but also dogs.
Intermediate host – birds, small mammals etc (prey of hunting cat).
The adult tapeworm is found in the small intestine of the final host, segments and eggs reach the exterior in the faeces, egg is ingested by the intermediate host.
Once in the intermediate host, egg hatches and the embryo moves into the blood, lymph or (in invertebrates) the body cavity.
The embryo then moves to its predilection site in the host, which can be lungs, brain or muscle.
Once in its predilection site the embryo develops into its larval stage (cyst).
Intermediate host ingested by primary host.
Taenia taeniaeformis, the species that occurs in cats, uses rodents as intermediate hosts and dogs or cats are infected when they eat tissues or viscera of infected hosts.
Usually well tolerated in dogs and cats, potentially some anal irritation due to motile segments.
hyatid disease
Echinococcus granulosus
Primary host – dog.
Intermediate host – the usual intermediate host is sheep however, any mammal in contact with dogs can become an intermediate host, including man (zoonosis).
The adult tapeworm is found in the gut of the dog, contamination of the environment occurs through the dog defaecating (eggs can survive in the environment for about a year).
Eggs are ingested by the intermediate host and encyst within the body (hydatid disease).
Cysts develops in lung, liver and body cavity containing developing forms of the tapeworm (hydatid disease).
Dog becomes infected by eating the intermediate host.
Echinococcus multilocularis.
Zoonotic tapeworm causing alveolar echinococcosis in humans that results in high fatality.
The definitive hosts are canids, mainly the red fox although domestic carnivores (dogs and to a lesser extent cats) can also be infected with the parasite.
Voles act as intermediate hosts.
The mandatory tapeworm treatment within the PETs scheme prior to entry into the UK is designed to prevent its entry.
Roundworms.
Roundworms have no segments and tend to be of a whitish or pinkish brown colour.
Prolific egg-layers and just a few worms can produce large numbers of eggs.
Toxocara spp. eggs can survive in the environment for months or even years.
Toxocara canis in zoonotic roundworm in dogs
Toxocara cati in zoonotic roundworm in cats
Toxascaris leonina - zoonotic roundworm in cats and dogs
Dogs and cats can become infected from a number of sources including -
Picking worm eggs up from grass or soil in public places.
By ingesting a paratenic host.
Pregnant bitches can pass infection to their puppies both in utero and via the milk.
Pregnant queens can pass infection to their kittens via the milk (but not in utero).
There are many different lungworm parasites whose adult stages occur in different anatomical areas within the respiratory tract of the host.
name an example
Angiostrongylus vasorum
A. vasorum is now endemic in much of the UK.
Slugs and snails act as intermediate hosts, although dogs may also acquire infection through ingestion of frogs and other amphibians acting as paratenic hosts.
nematode
l1 eaten by intermediate host
l1 deveops to l3 in intermediate host
Hookworms
Hookworms are small nematodes characterised by large mouthparts that are at an angle to the rest of the worm, hence the common name.
There are three species of significance in Europe –
Ancylostoma caninum,
Bunostomum spp
Ancylostoma tubaeforme
Uncinaria stenocephala (most common in UK).
Life cycle - All hookworms demonstrate a direct life cycle - eggs passed in the faeces, once ingested develop within 2-3 weeks to adult worms.
Ancylostoma spp. larvae are capable of penetrating skin and thus making their way to the intestine.
Control – prompt removal of faeces and disposal will help to prevent larvae developing on grass, however regular worming to control Toxocara spp. will usually also control hookworms.
Whipworms.
Eggs are passed in the faeces of infected dogs and can lead to considerable and persistent contamination of the environment.
First stage larva protected by the egg shell and can survive in the environment for years.
Dogs are infected when they eat eggs containing infective larvae - pre-patent period is 2 - 3 months and infected dogs may continue to shed eggs for up to a year.
Control - depends on removing dogs where possible from the contaminated environment and repeated anthelmintic treatment (regular worming to control Toxocara spp. will also control hookworms so long as a treatment with an appropriate spectrum of activity is selected.)
Prompt removal of faeces and disposal will help to prevent eggs developing in the environment.
Paedogenesis
production of many new individuals from a single larval form
chemical treatments for nematodes
Anthelmintics - various applied via oral, pour-ons, spot-ons,
chemical treatments for cesodes
Oral praziquantel or double dose pyrantel applied orally
chemical treatments for trematodes
Triclabendazole,
Closantel, Nitroxynil,
Albendazole, Oxyclozanide
applied oraly
chemical treatments for protozoa
Sulphonamides applied orally
control of Ectoparasites in large animals
Dipping Showers Weekly bathing with specific product Spot ons and Pour ons Injectables Isolate affected animals Remove eggs – comb, insecticide Dress lesions with appropriate insecticide Tailing, dagging Endoparasite control Remove manure from grazing and housing areas to remove insects Manure heap fermentation Temperature kills eggs and larvae
Remove breeding sites such as stagnant water or remove animals from breeding area
E.g. Tick survival dependent on tick’s requirement for water
Must have relative humidity greater than 90%
Common in wetter, marshy areas
E.g. Mosquitoes eggs laid in standing water
Larvae live in standing water
Insecticide or repellent impregnated ear tags, tail bands and halters
Screens to prevent insect access to housing
Electrocution grids to kill insects within housing
Fans to affect flight
Wash and treat all bedding, grooming tools, equipment, rugs,
Or leave unused for 3 weeks
lice control:
Cattle - in cattle, a range of pour-on or spot-on synthetic pyrethroids, e.g. permethrin, are available for louse control, also pour-on and injectable macrocyclic lactones (MLs), e.g. ivermectin also commonly used.
Most insecticides registered for use on cattle are not active against louse eggs and so a second treatment may be required.
The timing and frequency of treatments depends on individual circumstances, however in many cases treatment in late autumn or early winter will give adequate control of cattle lice, often when cattle are housed for the winter.
Sheep - treatment of chewing lice in sheep is by organophosphate (OP) dip or by topical synthetic pyrethroids.
Pour-on products should be avoided in fully fleeced sheep, as this results in a less effective treatment and increases the risk of resistance.
Trematode-liver fluke
Fasciola hepatica (cattle)
Dicrocelium dendriticum (sheep)
Body system affected- alimentary: liver
Highly pathogenic
Acute-Sudden death or dullness, anaemia, dyspnoea, ascites and abdominal pain
Subacute-Rapid weight loss, anaemia, submandibular oedema and ascites in some cases.
Drugs (resistance seen)
Move onto lower risk pasture
infective strategy Galba truncatula snail
-Wet pasture
Trematode: stomach fluke ‘Rumen fluke (helminth)
Paramphistomium Calicophoron daubneyi
Body system affected- alimentary: rumen
Life cycle-similar to liver fluke, however once in the definitive host:
After excysting in the small intestine, the tiny immature rumen fluke migrate ‘upstream’ and settle in the rumen and reticulum, where they mature and lay eggs.
Infective strategy: Galba truncatula snail
Pathogenesis: generic
Diagnostic methods:
faecal egg count (FEC) by sedimentation,
PM,
meat inspection
Treatment options-advice not to treat, limited drugs available
Emerging issues: only identified in the UK last 20 years,
Nematodes: Trichostrongyles (helminths)
Ostertagia ostertagi (abomasal), Telorsagia cercumsincta (abomasal), Haemonchus spp (abomasal), Dictyocaulus spp (respiratory &alimentary), Nematodirus spp (small intestine), Trichostrongylus spp
Life cycle-direct
Infective strategy –need infective pastures, critical temperatures, periparturient rise in production, hypobiosis
Pathogenicity varies
genera and species,
numbers of nematodes
age (maturity)
nutritional status
body condition
Diagnostic methods: FWEC but will give false negative in early stages of disease due to lack of mature egg producing parasites.
Treatment options-drugs, grazing management control,
lung worm vaccine
Nematodes: Strongyles
heminths
Cyathostomins, Chabertia (colon), Oesophagostomum (colon)
Body system affected- alimentary
Life cycle-simple, non migratory
Pathogenesis-no clinical significance unless concurrent disease
Diagnostic methods-FWEC
Treatment options-drugs
Nematodes: Ascarids
helminths
Ascaris suum, Trichinella spp,
Body system affected- small intestine, liver, lung
Life cycle-direct
Infective strategy-infective L3 and arrested development.
Migratory or non-migratory
Pathogenesis-Milk spot liver in pigs
Diagnostic methods-PM, meat inspection
Treatment options-drugs, hygiene
Emerging issues-increasing numbers of outdoor sows so more difficulty in eliminating them from a premises.
Protozoa: Sporozoan
Coccidia (Eimeria spp)
Causes Diarrhoea in calves and lambs
alimentary-small intestine in sheep, caecum and colon in cattle
Life cycle:direct, highly host specific
Causes diarrhoea in calves under a year old and lambs 1-2months old
-each host can be infected by a number of different Eimeria spp
Reside in the small intestine in sheep,
Caesium and colon in cattle
-Infective strategy:lambs infected by chronically infected adults which shed oocysts, the lambs then produce large numbers of oocysts to infect others. The sporulated infective oocyst contains 4 sporocysts each containing 2 sporozoites (8sporozoites/sporulated oocyst)
Pathogenesis: 11 species infect sheep but only 3 are pathogenic, 13 infect cattle but only 2 are pathogenic
Diagnostic methods: Faecal worm egg counts for coccidial oocysts
Treatment options:
Drugs to treat and prevention
Environmental action
Feed off the floor Improve hygiene
Lower stress levels
Low numbers in FWEC can be normal ‘background’ levels, only concerned when get high numbers over 5000 per gram
In feed medication
Oral drenches
Need to get some exposure to gain immunity
Protozoa: Sporozoan
Cryptosporidium parvum
Causes diarrhoea in young calves and lambs
Body system:small intestine
Life cycle:direct
Infective strategy:
Pathogenesis: as the oocysts grow they disrupt the brush border, decreasing absorption of fluids from the lumen
Causes diarrhoea in young calves and lambs, 2-4weeks old
it develops at the junction between the micro-villous brush border and the cytoplasm gut epithelial cells. It produces very small (~4mm) sporulated oocysts which either initiate another cycle of asexual reproduction in the same host or are shed from the body via the faeces
Diagnostic methods:
SNAP test for crypto,
faecal smear after Giemsa stain;
Identification of organism in stained gut sections of post mortem
Treatment options:
Drugs to treat and prevention
Environmental action
Control other diarrhoea pathogens
Colostrum management
Emerging issues:important cause of food poisoning in humans from contaminated water sources or meat. High risk pathogen for immunosuppressed people
As well as causing disease in its own right, Crypto is an opportunistic pathogen and loves to tag onto the back of rotavirus infections when the intestinal lumen is already damaged, further damaging the intestine causing much more severe diarrhoea
Hygiene, cleaning, drying and disinfecting equipment used to feed and house young
Good colostrum management
Protozoa: Sporozoan Iospora spp (very similar to coccidiosis)
Usually non pathogenic but in high levels Causes enteritis in piglets
affects the small intestine causing diarrhoea, dehydration, and loss of electrolytes, perhaps death
Life cycle:direct
Treatment options:
Drugs to treat and prevention
Environmental hygiene
confinement-raised, one to three week old, nursing piglets and is less frequent and severe in older recently weaned piglets.
Protozoa: Sporozoan
Sarcocyst
Causes abortion and meat condemnation in sheep and cattle
Causes abortion & congenital abnormalities in humans
Body system affected- alimentary and reproductive
Life cycle: indirect, specific pairings of intermediate and definitive hosts
Over 250 types: Species-specific prey-predator life cycles
After ingestion of sporocysts by a suitable intermediate host, sporozoites are liberated and initiate development of schizonts in vascular endothelia of mesenteric arterioles and lymph nodes. A second generation of endothelial schizonts is produced in capillaries from several organs. Merozoites released from these schizonts invade the muscle fibers and develop into the typical sarcocysts
Natural infections are usually asymptomatic.
No effective treatments
prevent ingestion of prey carcasses or raw tissues by omnivorous or carnivorous animals
Some drugs are used abroad as a prevention but are not licensed in the UK
Effective cooking or freezing kills the parasite in meat.
Protozoa: Sporozoan
Toxoplasma gondii
Body system affected- alimentary and reproductive
Life cycle: indirect, facultative heteroxenous
Infective strategy: capable of developing in almost any cell type
Clinical signs:
Barren to tup,
abortion,
mummification
stillbirth (occasionally one live lamb born with a dead lamb)
birth of weak lambs
White focal necrosis on the placenta
Definitive host is the adolescent naive cat who is infected by eating infected rodents, produce oocysts in their faeces before becoming immune.
Toxoplasmosis is caused by toxoplasma oocysts picked up from feed or hay, or off pasture that has been contaminated by cat faeces.
These oocysts are very resilient and can survive for very long periods in feed or on pasture
Once a ewe has been infected, she soon becomes immune and is unlikely to show signs of the disease in subsequent years. It is only when an infection is picked up for the first time during pregnancy that problems occur. The stage at which an infection is picked up during pregnancy will determine the outcome as there is an approximate six week lag period between infection and onset of clinical signs: First 60 days – foetus absorbed and the ewe appears barren. 60 – 120 days – abortion in late pregnancy with mummified foetuses, stillbirths or weak and sickly lambs that often die.
There are five main syndromes of abortion in sheep –
Barren to tup,
abortion,
mummification (particularly common with toxoplasmosis),
stillbirth (occasionally one live lamb born with a dead lamb)
birth of weak lambs which fail to suckle properly and often succumb to disease in young life.
It is however common to have many or all of these syndromes on a farm at the same time. A clinical sign which is characteristic of toxoplasmosis abortion is the development of small white areas in the cotyledons (buttons) of the placenta. These are caused by focal necrosis (death of cells) in areas of the placenta due to damage caused by multiplication of the Toxoplasma organism.
Protozoa: Sporozoan
Neospora caninum
Body system affected- alimentary and reproductive
Life cycle: indirect – canids definitive host
Infective strategy – cattle ingest infected canid faeces
Diagnostic methods:
PM calf
Maternal antibodies
Treatment options: none, test and cull or don’t breed replacement heifers!
Causes over 10% of the UK abortion. Disease and the risk of abortion can be vertically transferred transplacentally from mother to daughter
Cattle ingest infected faeces
Transmission
Dogs are definitive hosts of N caninum and are capable of shedding oocysts in feces after eating tissues of infected animals.. Neospora oocysts have an impervious shell that enables survival in soil and water for prolonged periods after canine feces have decomposed. Intermediate hosts such as cattle become infected by ingesting oocysts. Cattle do not produce oocysts and thus do not transmit infections horizontally to other cattle, but latent infection may endure permanently in their tissues and is transmitted to canids by carnivorism.
Protozoa: Sporozoan
Babesia divergens
alimentary and reproductive
Life cycle: indirect via Ixodes ricinus
Pathogenesis- lyses red blood cells
Diagnosis:
Clinical signs: a sudden fever, diarrhoea followed by constipation, red urine (caused by the haemoglobin pigment from the burst red blood cells), anaemia (with rapid pulse, fast breathing and pale membranes), milk drop, depression and weakness, and abortion of pregnant
Recent movement to pastures known to harbour ticks
Blood smears can show up the parasite
Treatment options
Mild cases may recover without treatment.
Drugs available for treatment and prevention
Vaccines available abroad
Tick control
Babesia divergens. The disease is spread between cattle by ticks (Ixodes ricinus in the UK). The babesia is injected into the bloodstream by the tick and then invades the red blood cells and begins dividing, eventually rupturing the cell. Clinical signs begin around 2 weeks after infection
Diagnosis:
clinical signs
Recent movement to pastures known to harbour ticks
Blood smears can show up the parasite
Protozoa: Flagellates
Trichomonas fetus
Body system affected- reproductive
infertility caused by embryonic death
results in repeat breeding
pyometra, endometritis, or a mummified fetus
Life cycle-direct, found in the genital tracts of cattle
Infective strategy- males transferring it
Pathogenesis: The parasite interacts with bacteria that normally reside in the intestinal tract by adhering to the intestinal epithelium of the host
Diagnostic methods-
Focus on bulls: Repeated culture (single test identify 90%–95%), PCR isolation
Swab vaginal discharge/mucus
Treatment options-drugs, herd culling. Biosecurity, vaccines
When cows are bred naturally by an infected bull, 30%–90% become infected, suggesting that strain differences exist
Protozoa: Ciliates Balantidium coli (pigs)
Body system affected- alimentary Life cycle-direct Pathogenesis-diarrhoea Diagnostic methods-FWEC Treatment options-drugs, improved hygiene Emerging issues-zoonosis Faeco oral transmission Significant human pathogen in developing countries
Which companion animal ectoparasites would require a superficial skin scrape to potentially identify?
Cheyletiella is a surface-dwelling mite, so superficial skin scrapes should be performed. No capillary ooze is necessary. Superficial scrapings can also be used to diagnose ectopic Otodectes infestation, where mites are living outside the ear canals.
Which species of animal does Dermacentor variabilis affect?
Dermacentor variabilis is a 3-host tick, targeting smaller mammals as a larva and nymph and larger mammals as an adult. Although it is normally found on dogs, this tick will readily attack larger animals, such as cattle, horses, and even humans.
Name a Trichodectes louse that affects dogs.
Trichodectes canis is a chewing louse of dogs. It is very host-specific and cannot infest any other species than the dog. It can have serious effects in puppies and older, debilitated animals. T. canis can also act as an intermediate for the tapeworm Dipylidium caninum
Name a Trichodectes louse that affects cows.
Trichodectes scalaris
Name TWO diseases that can be transmitted by Ctenocephalides species.
Ctenocephalides canis can act as intermediate hosts for parasitic worms including the double-pored tapeworm, Dipylidium caninum, and the nematode, Acanthocheilonema reconditum
mites in Rabbits
Cheyletiella parasitovorax Listrophorus gibbus Psoroptes cuniculi Demodex cuniculi Sarcoptes scabiei var cuniculi Notoedres cati var cuniculi Trombicula autumnalis
Fipronil is toxic to
rabbits
