Practical Diagnostic Applications in Veterinary Microbiology Flashcards
How big should tissue block specimen be? How are abscesses collected?
4 x 4 x 2 cm
scrape the abscess wall + pus
How are exudates collected? How are swabs kept uncontaminated?
(joint fluids, endocardial fluid) - aseptically by sterile syringe
use swab-transport systems, like Culturettes
How should all tissues, organs, and pus be maintained during shipment/transport? What is a major exception?
refrigerator temperature
feces
Why isn’t feces kept at refrigerator temperature for shipment/transport? How should it be preserved?
pH drops, which can kill bacteria like Salmonella
placed in trasnport media, like Cary-Blair 10% suspension, and transported at room temperature
Why is aseptic technique so important for collecting specimen?
many pathogenic bacteria are fastidious and slow-growing, so contamination can make the interpretation of results much more difficult and fail to isolate the true etiologic agent
What is the best way to collect specimen from animals that have been dead for an unknown period of time? Why?
bone marrow or rib
enteric bacteria rapidly disseminate throughout tissue after death (especially coliforms and clostridia)
Which samples are not suitable for anaerobic cultures? Why?
samples contaminated with normal flora, such as intestinal contents, oropharyngeal swabs or washings, and skin
contaminated sites will ass contain anaerobic bacteria
- pus and fluids should be collected with a needle and syringe and have air expelled
How should anaerobic cultures be shipped? Anaerobic swabs? Large pieces of tissue? Small pieces of tissue?
if they cannot reach the lab within 30 mins, they should be transported in an anaerobic transport system
within commercial anaerobic transport swabs and maintained at 4 degrees C
(6 x 6 x6 cm) - will usually hold an anaerobic environment, but should be kept at 4 degrees C and processes as soon as possible
within anaerobic transport bag until processing can be done
What 3 things does the success of culture for anaerobes depend on?
- samples taken from a living or recently dead animal
- excluding air from samples in transit and maintaining at 4 degrees C
- cultures under anaerobic conditions as soon as possible
How should specimens be collected in mastitis cases?
- good collection technique, since milk samples can be easily contaminated by organisms outside the udder
- collection into sterile narrow-necked bottles (no nonsterile containers or “whirl-bags”
- CULTURE
In what 3 ways are UTI specimen collected? Which one is least likely to result in contamination?
- cystocentesis*
- catheter
- voided
How should urine samples be maintained if they are unable to be cultured immediately? When must they be cultured?
refrigerated
within 6 hours, even if kept refrigerated
On what media are urine samples inoculated on? How is this done?
blood agar and MacConkey agar
- 10 µl from cystocentesis/catheter; 1 µl voided
- calibrated loop inoculates urine into the plates
- draw loop down the center of the plate from top to bottom
- streak back and forth through initial streak
- 24 hrs incubation and colony count
What is the bacterial burden of most UTIs? When collected from a cystocentesis?
10^5 CFU/mL
> 10^3 CFU/mL
What are the most common bacteria isolated from the urinary tract? what other 3 can be found?
enterics, like E. coli, enterococci, and Proteus
- Pseudomonas
- Staphylococci
- yeasts
anaerobes and filamentous fungi are rare
What are the main bacteria and fungi known to cause abortion?
- Campylobacter spp.
- Brucella spp.
- Leptospira spp.
- H. somnus
- Mycoplasmas
- Listeria spp.
- A. pyogenes
- Aspergillus spp.
- Zygomycetes
What samples should be collected for isolation following an abortion?
both maternal and fetal samples
- fresh and fixed for histopathology is ubiquitoius organisms like Aspergillus fumigatus is suspected
What 4 fetal samples are preferred for uterine disease?
- placenta plus cotyledons (fixed and fresh)
- uterine discharge (fresh)
- fetal stomach contents (fresh)
- fetal lung, liver, kidney, and spleen (fixed and fresh)
What are the 2 preferred ways to collect Leptospira samples if it is suspected to be the cause of the abortion?
- midstream urine - centrifuged and darkfield microscopy immediately, or dilute in cultured media
- clotted blood samples for serology at the time of abortion
What 2 specimens should be collected in suspected infertility cases? What should examinations cover in cattle and mares?
- uterine discharge or vaginal mucus
- preputial washings* or semen
CATTLE - Campylobacter fetus and opportunistic aerobes (Haemophilus somnus)
MARE - coliforms (Klebsiella), other opportunistic aerobes (Streptococci), and fungi (Taylorella equigenitalis)
How should the skin be prepped for blood cultures? What are the 2 reasons that we typically collect 3 blood samples approximately an hour apart?
shaved and aseptically prepped with antiseptics, like betadine and alcohol
- animal may not always be septicemic and only have periodic shedding
- confirm any isolate obtained from one bottle is not a contaminate
Normally blood samples are inoculated into both aerobic and anaerobic bottles. Which type of bacteria most commonly are found in the blood? How do adult and pediatric bottles compare?
aerobic (less than 1% of blood isolates anaerobic)
ADULT = 5-10 mL/bottle
PEDIATRIC = 1.5 mL/bottle (used in small animals)
What are blood cultures examined for?
turbidity and Gram-stained
- subcultured on chocolate agar
no turbidity = subculture at 48 hours and 7 days before discarding
How does the lysis/centrifugation system work for blood samples? What are the pros and cons to this method?
- blood is added to a tube containing an agent to lyse the RBCs
- tube is centrifuged
- pellet of bacteria is resuspended and inoculated to agar
PROS: more sensitive
CONS: isolated more contaminants than culture bottles (ideal to use both systems)
How is Johne’s Disease specimen typically cultured? Which specimen are taken from live and dead animals?
culturing may not be available, but specimens can be examined by acid-fast staining smears + PCR
LIVE = scraping of rectal mucosa to detect advanced cases
DEAD = ileocecal valve or other affected areas of the alimentary tract, mesenteric LN
What are the 3 prime candidates for bacterial gastroenteritis (diarrhea/dysentery)? What are 3 other possible agents?
- Campylobacter
- Salmonella
- Yersinia
- Aeromonas
- yeasts
- agents forming toxins if foods: S. aureus, Clostridium, Bacillus, Shigella)
Which bacteria is typically only significant in neonatal diarrhea/dysentery?
enterotoxigenic E. coli
- bloody diarrhea and hemolytic anemia in humans
What strain of E. coli is significant in adult cattle? How can it be screened on culture?
E. coly O157:H7
failure to ferment sorbitol on sorbitol-MacConkey agar
What specimens can be taken from live and dead animals showing diarrhea/dysentery?
LIVE = feces
DEAD = mesenteric LN, liver, intestinal contents
What 2 bacteria may be associated with proliferative hemorrhagic enteropathy caused by intestinal adenomatosis? Where are these bacteria found?
- Campylobacter hyointestinalis*
- C. sputorum
organisms found within ileal cells - isolation from feces or live animal is unlikely
What bacteria causes swine dysentery? How is it diagnosed?
Brachyspira (Serpulina) hyodysenteriae
- microscopy
- culture: filter supernatant through small filters and inoculate filtrate onto blood agar, incubate 2-7 days under anaerobic conditions = clear hemolytic film
What is the etiologic agent of proliferative enteropathy? What are 4 common signs? How is it cultured?
Lawsonia intracelularis
- acute bloody diarrhea
- anemia
- vomiting
- necrotic enteritis and ileitis
INTRACELLULAR - grown on mammalian cell cultures and positive for acid-fast stains
- best diagnosis is by PCR of feces
How are smears from tuberculosis stained? What specimens are taken?
acid-fast
lesions - fresh and fixed in formalin for histopathology
Where are Mycoplasma and Ureaplasmas found? Why is disease due to these bacteria greatly underdiagnosed?
- respiratory tract
- urogenital tract
do not grow on conventional media - need special mycoplasma media and CO2
What kind of pathogen is Chlamydiaceae? How are they cultured? Diagnosed?
OBLIGATE intracellular
- tissue culture
- serology
- ELISA
- fluorescent antibody
- PCR
How are systemic fungal infections diagnosed?
submit tissue containing lesions both fresh and fixed in formalin for culture and histopathology
How are dermatophyte infections diagnosed? What is the most rapid way?
- plucked hairs (NOT cut)
- skin scrapes (just until blood appears)
- deliver in strong paper envelops to keep dry; inoculated to DTM and turn the medium red
Wood’s lamp for some Microsporum species only and identification of hyphae in skin of hair following incubation with 10% KOH
What specimens are collected for Strptothricosis and Dermatophiliasis? How are smears stained?
scab material plucked together with hairs from skin lesions
Methylene blue
+ culture upon request
What is the fluorescent antibody test most commonly used to diagnose? What 2 specimens are most commonly collected?
malignant edema and blackleg organisms: C. chauvoei, C. novyi. C. septicum
- bone marrow in an unopened rib
- block of affected muscle
(C. novyi and C. septicum are rapid post-mortem invaders, take samples only from recently dead animals)
Other than malignant edema and blackleg organisms, what 3 species can also be diagnosed with fluorescent antibody technique?
- Chlamydia
- Leptospira
- Brucella
What are the main advantages and disadvantages to latex agglutination tests and enzyme immunoassays?
PRO: rapid results, antigen detection means the organism does not need to be viable
CON: may not be more sensitive than cultures
What is PCR?
amplification of DNA in a sample so that the DNA can be detected by a probe or examined by electrophoresis
How is PCR done?
- sample extract, primers, Taq polymerase, dNTPs, and other reagents are added to thermal cycler
- temperature is raised to 94 degrees C to dissociate the DNA
- temperature is lowered to 54 degrees C to anneal DNA with the primers
- temperature is raised to 72 degrees to extend new DNA
- repeated for about 30 cycles and amplified logarithmically
How is PCR used for diagnostic purposes? How is a true positive determined?
rtPCR
cycle threshold value <36
Are whirl-bags suitable for liquid specimen?
NO
- use leak-proof, screw cap containers
Why are histopathology and serology commonly correlated with microbiology results?
HP: indicates whether actual invasion occurs or if the organism merely contaminants
S: indicate whether infection occurred
Including an adequate clinical history important for lab diagnosis of microbial disease. What should be included?
- breed
- age
- vaccinations
- number of animals of the same species on premesis
- number of sick animals
- symptoms
- location of lesions
- duration of problem
- suspected condition
- treatments
- samples submitted
What are some examples of growth media used? Identification media?
GROWTH = blood agar, MacConkey agar, thioglycolate broth
IDENTIFICATION = triple sugar iron agar, urea agar, indole, catalase, coagulase, oxidase, motility
Gram-positive bacteria:
Gram-negative glucose fermentors:
Oxidase-positive fermentors:
Gram-negative glucose oxidizers vs. nonsaccharolytic:
