• Classical culture
• Molecular or PCR
• Antigen detection surveys

Histology/Immunohistology

Molecular probes

Electron microscopy

ELISA (Enzyme-linked immunoessay)

FAT (Flourescent Antibody Test)

CF (Complement Fixation)

HA (D & I) (Hemoaggluttination Assay - direct and indirect)

1. How are cultures transported?

2. What type of systems are used to identify?

3. Is testing in house, commercial kit, sent out?

4. What is the QC for the lab?

5. Is the lab member of NACLS or other accreditation program?

All purpuse

Differential

Selective

Biochemical

Serologic

Molecular

This is a common isolation media - non selective

Differentiates for hemolytic action

• Selective for gram neg
• Not all gram neg will grow
• Differential for lactose fermentation - pink (if yes), colorless (if non-fermeter)

• Sugar (esp. lactose)
• H2S Production
• Growth/precipitation of bile salts
• Inhibition of Gram positive and some Gram neg species

• Differentiate for Gram + Bacteria
• Staph aureus (ferments it - yellow color)
• Coagulase neg Staph species are red
• Timing is critical

Phenylethyl Alcohol Agar

• Discourages growth of Gram - Bacteria
• Used to free Gram + organisms to grow w/o contaminatio
• Proteus is a common gram - contaminant
• Staphilococci and Streptococci will grow

• Slow growing microbes have no chance
• Fastidious microbes
• Non-cultivable microbes
• New molecular techniques (PCR) are more accurate
• Antigen capture essays

1- May require different media

2 - Morphology and microscopic examination often needed

• Hard to lomit access to air
• Biopsy of FNA are preferred sample medium
• Anaerobic swabs are least desirable
• Check with the lab (some prefer samples at RT)

1. Isolation on cell lines

2. ID in clinical materia via electron microscopy

3. Detection of viral antigens

4. Detection of virus-specific DNA/RNA

5. Characteristic cellular pathology

6. Antibody response

• Submit it in virus medium
• at 4 dC
• Buffered at physiologic pH
• Protein buffer such as BSA is often used
• Use antibiotics to inhibit bacterial growth

Blood

Tissues

Swabs (be careful, some inhibit PCR)

• Current infections vs. exposure
• Time interval for seroconversion (paired sera and 4 fold rise)
• IgG=Chronic dz; IgM=Acute dz.
• Screening tool

• Negative status of a group of animals (ex: HW test)
• Vaccine efficacy

1. Collection site

2. Must represent active disease process

3. Source of contamination must be controlled

4. Live, recently-dead, in-contact (list)

Blood

CSF

Joint Fluid

Bladder Urine

SQ

(Surgical preparation required)

Skin

Mucosa

(try to minimize contamination)

Fine needle aspirates,

Biopsies,

Blood

Skin scrapings,

Swabs of various secretions.

Joint fluids,

CSF,

Abcesses

(joint fluid may need to be collected into a syringe with some heparin in it)

1. Collect into a syringe

2. Cap tightly

3. Refrigerate

4. Transport on ice

• Collect sx biopsy aceptically
• Transport on ice in sterile container
• For small samples add 2 drops of sterile saline to keep moist
• If suspecting anaerobic organizm, transport in proper media (Portacult TM tubes from BD Biosciences)

• collect volume proportionate to the size of the patient
• inject asceptically into Wampole Insolator TM blood culture tube or any other culture tube
• if patient is on antimicrobilas, list them
• transport on ice by mail immediately
• if not on antimicrobilas, may hold blood at RT for up to 8 hours while other samples are drawn

• asceptically vented with needle for aerobes (remove needle before transport)
• leave unvented for anaerobic culture

• Submit 5-10 grams
• in sterile screw-cap specimen container
• avoid the use of swabs
• refregirate and transport specimen on ice

• do not adequately protect anaerobic or microaerophillic organismas
• may not have sufficient amount of sample

• aspirates and biopsies are always preferrable
• collect swab samples from ears, nose, throat, skin, genitalia;
• clean and disinfect surface prior to collection
• gen. purpuse swabs may contain substances that will inhibit growth of some microbes
• use appropriate transport media (ask the lab)
• when using systems containing liquid transport medium, crush the ampule to moisten swab.

• also good for fastidious resp. pathogens and anaerobes,
• Commercial systems: Amies semi-colid transport medium

• Place wash fluid in sterile tube
• Recovery rate for fastidious organisms is higher than with nasal swabs or oropharyngeal swabs
• Transport and lab setup ASAP as saline not a good medium
• For mail-in samples, refrigeration and use of suitible transport medium is recommended

• record when and how urine was collected
• collect into sterile container
• cystosentesis is preferable (catheter, too)
• transport specimen on ice
• urine is a good growth medium
• refregirate if not tested >1 hour post collection
• process no later than 72 hours post collection

• use uterine swab
• use guarded swabs for collection
• place a swab in an appropriate medium
• culture results from pre-breeding vaginal swabs must be interpreted with caution
• beta-hemolytic streptococcus spp and Mycoplasma spp are commonlyt recovered in pre-breeding vaginal specimens of healthy, reproductively sound dogs.

• Nx should be done as soon as possible
• least amt of change
• >6 hrs post mortem = can have much contamination
• if uncontaminated, survival of pathogens in tissues is usually good
• short-term refrigeration and freezing are acceptable for many organisms
• some organisms do not survive freezing well (Rabies)

• Fastideous respiratory pathogens
• Zygomycetous fungi
• Pithium insidiosa
• Rabies virus

• List DDX or reasons fos submitting
• use freshest sample possible
• ideal sample size 2-4 cm3 of parenchimal tissue, centerd on the margin of lesion
• to clear contaminants, rinse with saline, sterile water or 70% ethyl alcohol to minimize subsequent sample contamination

• ligated intestine 8-10 cm in length
• not intestinal contents
• Sites: duodenum. ileum, jejunum, colon) may depend on pathogen being sought.

• spleen
• heart
• blood
• bone marrow

• civil: $250-27,500 per violation per day
• criminal (intentional) $500k or 5 year in prison.

• open
• delay
• refuse to ship

• biological toxins
• infectious substances
• diagnostic specimens
• biochemical waste
• cultures
• genetically modified organisms

• formalin
• alcohol
• dry ice
• fixative solutions

• clinic accession number
• patient identification information
• medical record number
• sourse of sample
• date and time of sample collection
• name of the person collecting the sample
• related clinical history
• contact info

• vaccines (prevent/ameliorate)
• anti-microbilas (static/cidal)
• husbandry and epidemiology (decrease risks)

• Maternal-derived antibodies neutralize vaccine virus (active immunity is in >98% of puppies if last vaccine series are done 14-16 wks of age)
• Vaccine is poorly immunogenic (manufacturing errors/viral strain passage error),
• Post-manufacturing error (shipment at high or too cold temp)

• May be similar to each other
• head shaking
• rubbing affected ear on the floor
• rotating head toward affected side
• ear is usually painful with discharge and inflam.

• specimen not labeled
• mislabeled
• prolonged transport
• improper or leaky container
• specimen is not suitible for request
• duplicate specimens except for blood)
• no paperwork attached

• Bacteria and fungi (not primary culprits)
• overgrowth of normal flora (Staph and Malassezia)
• Check relative numbers (increase means overgrowth)
• OE results from primary inflammatory process
• inflammatory changes lead to proliferation and establishment of infection

Blood and fecal cultures are collected improperly

Organisms are shed in "waves" or "showers"

Facial nerve paralysis

Horner's syndrome (miosis, ptosis, enophthalmos, protrusion of the nictatans)

• more pronounced head tilt toward the affected side
• may circle and fall toward the affected side
• generalized incoordination: may cause difficulty in walking and rising
• spontaneous, horizontal to rotary eye movement
• rarely may ascend vestibulochochlear and facial nerve to the brain
• This stage would lead to meningitis, brain stem absess and death.

• Acute or chronic inflammation of the epithelium of the external ear canal
• variably charecterized by erytheme, edema, increased sebum or serumen, desquamation of epithelium

• microbial infection is usually a secondary factor
• microbial growth exacerbates inflammatory rxn
• identification of contributing internal and external factor is key to successful treatment

parasites (Otodectes in cats), demodex, etc.

FB

Tumor

Hypersensitivity (atopy, food s., contact dermatitis)

keratinization disorders hypothyroidism, autoimmune disease, juvenile cellulitis, irritants)

• Congenital or environmental
• Conformation
• Masses
• Overuse of astringents
• FBs

Breeds that have conformation predisposition for otitis?

• Droopy ears (cocker spaniel, Basset)
• Stenotic ear canal (Shar pei, English bulldogs)
• Excessive hair growth (Schnauzer)

Polyps

Squamous and basal cell carcinomas

other tumors

• Bacteria: Staph pseudointermedius, beta-hemolytic Streptococcus, Pseudomonas auriginosa, Proteus mirabilis
• Yeasts: Massezia pachydermatis, Candida, Microsporum canis

Sedation or anesthesia may be required

Look for progressive pathologic changes

Permits identification of: deep otic FBs, impected debris, low grade infections with Otodectes cyanotis, ruptured or abnormal tympanic membrane

Use cotton-tipped applicator and roll on glass slide

heat fix for microbes, oil for mites

stain with a 3 step quick stain or Modified Wright's

Examine under a microscope

Look first under low power, then - high power

• take a sample before otoscopy is complete
• before any cleaning solutions are used
• before ointments are applied
• take a sample into a sterile culturette (for culture and sensitivity)
• Sites to sample: horizontal canal (most infections arise here); middle ear (if tympanic membrane is ruptured)

1. depends on the stage of the dz

2. if no concurrent OE, give broad spectrum anbx for >14 days

3. With OE, Consider doing meringotomy, aspirate, flush, instill anbx

4. Avoid aminoglycosides if close to the brain

5. Choose anbxs nased on MIC

6. Treat for 10-14 days

7. If yeast is present, give topical andtifungal systemic only if recurrent infections or no drainage from the ear canal

chronic otitis

if neutrophils or rod-shaped bacteria are seen on a smear

if tx with topical antimicrobials fails

All cases of otitis media

1. OM-WITH INTACT TYMPANUM, USUALLY RESPONDS WELL TO SYSTEMIC ABXS

2. If chronic OE, and if tympanum ruptures, success is reduced.

Pseudomonas

-Frustrating and difficult to treat because treatment may take >2 months, supporative exhudate will be seen, severe epithelial ulceration, pain, edema of the canal.

• Multifaceted approach
• ID primary cause and manage it
• Remove exhudate and clean and dry the canal
• ID and treat OM
• Select appr. abx from the results of culture and MIC
• Use at an effective dose for app. amt of time
• Treat both topically and systemically

• Eyelids
• Cilia
• Blink reflex
• Intact conjunctiva and cornea
• Precorneal tear film
• Tight junctions of endothelial cells and epithelial cells
• (Blood:aqueous and blood:retinal barrier)
• Nonspecidic antimicrobials in tears

Lysozyme

Lactoferrin

Antimicrobial peptides

-acts on a glycan chain in the peptidoglycan of bacterial cell wall

- decreases in lysozyme constantly correlate with increase in eye infection

Whad does lactoferrin do?

- binds Iron (Fe)

-improves function of NK cells

-decreases formation of C3 convertase

- cationic substances present in tears, conjunctiva, cornea

- broad spectrum natural abx

- prompt cell signaling to activate host immune response

• Numbers and morphology of bacteria and yeast, fungal hyphae
• WBC?
• Evidence of phagocytosis of organisms
• Acantholytis or neoplastic cells

• ID primary issue and eliminate vias tx
• Clean out the ear and dry
• Topical therapy, unless ear canal is occulded in (OM/OE)
• Topical meds will be inactivated by exhudate
• Detergents and DSS are not to be used if tympanic membrane is ruptured

-Serumenolytic solution (Carbamide peroxide or DDS=Dioctyl sodium sulfosuccinate);

- Antibacterial cleansing solutions (Chlorhexidine Aline Plus Povidone iodine, TrisEDTA)

• antibacterial agents plus corticosteroids to reduce exudation, pain, swelling
• decrease glandular secretions
• use least patent corticosteroid that will reduce inflammation
• if patient has a history of recurrent bacterial otitis with otodectes infestation, use topical products such as Tresaderm with antibacterial and antiparasitic agents