Practical Applications in Veterinary Microbiology

Question 1

COLLECTION OF SPECIMENS
* Tissue blocks (approx. __ x __ x __ ___)
* Abscesses - scraping of abscess ____ or ___ itself plus ____.
* Exudates (?) - collect aseptically by _____ syringe.
* Swab collection: use a _____-transport system, e.g. _________

Answer 1

4, 4, 2, cm, wall, wall, pus, joint fluids, endocardial fluid, etc., sterile, swab, Culturette

Question 2

All tissues, organs, pus, must be maintained ____ (_________ temperature)
during shipment or transport. What is the exception to this?

Answer 2

cold, refrigerator

An exception is feces.

Question 3

When feces is refrigerated the _____ drops, which may
kill some bacteria, such as _________. Feces should be placed in transport media (such as ?), and can then be transported at ____ temperature.

Answer 3

pH, Salmonella, Cary-Blair, 10% suspension, room

Question 4

The use of ______ technique in collecting specimens cannot be overemphasized. Many pathogenic bacteria are fastidious and slow
growing. ____________ of specimens makes interpretation of results
difficult and may result in failure to isolate the ____ etiologic agent

Answer 4

aseptic, Contamination, true

Question 5

Enteric bacteria rapidly ________ throughout the tissues after death.
Therefore, collection of specimens from an animal that has been dead for
an ________ period of time are often difficult to interpret. However, bone ______ or a ____ from dead animals may yield the causative organism.

Answer 5

disseminate, unknown, marrow, rib

Consider anything unknown to be potentially zoonotic.

Question 6

Anything that will not grow routinely on an aerobic culture, you will have to ask for an ___________ culture.
Culture can be carried out for organisms such as ?

Answer 6

anaerobic

Clostridia (form spores and are aerotolerant),
Fusobacterium, Bacteroides, Peptostreptococcus (very sensitive to air), and many others.

Question 7

Samples contaminated with normal flora, such as intestinal contents,
oropharyngeal swabs or washings, and skin, are not suitable because ___________ sites all contain ________ bacteria. _______
conditions must be maintained. Therefore, ___ and ____ should be
collected with a needle and syringe and the ____ expelled.

Answer 7

contaminated, anaerobic, Anaerobic, pus, fluids, air

Question 8

If samples cannot reach the laboratory within 30 min., they should be transported in ?

Answer 8

an anaerobic transport system.

Question 9

If swabs must be used they should be submitted in commercial ________ transport swabs and maintained at ___°C, if not processed ___________.

Answer 9

anaerobic, 4, immediately

Question 10

Large pieces of tissue (___ X __ X __ ___) will usually maintain an ______
environment, but should be kept at __°C and processed ______. Smaller pieces of tissue (or other specimens) should be
maintained in an ______ transport ____ until processing can be done

Answer 10

6, 6, 6, cm, anaerobic, 4, ASAP, anaerobic, bag

Question 11

The success of culture for anaerobes is heavily dependent on
* 1. Samples taken from a _____ or ______ dead animal.
* 2. Excluding ____ from the samples in _____, and maintaining at __°C.
* 3. Culture under _______ conditions ________.

Answer 11

living, recently, air, transit, 4, anaerobic, ASAP

Question 12

In the case of Mastitis
* Culture - milk samples can easily be contaminated by organisms from _________
the udder, so it is essential that a good collection technique is used (* _____ few
mls of milk expressed and the sample collected from _______ the udder*).
* The collection should be made into ____ _________-necked bottles. Milk samples should not be submitted in _______ containers or “_____-_____”.

Answer 12

outside, first, within, sterile, narrow, nonsterile, whirl-bags

Never submit liquid samples in containers that can leak.

Question 13

Urinary Tract Infections
* Specimens (usually urine) can be collected by ___________, _______, or ______.
Of these methods _________ is least likely to result in contamination of the specimen

Answer 13

cystocentesis, catheter, voided, cystocentesis

Question 14

There are strict rules to follow regarding urine specimens. All specimens should
be _____________ if they cannot be cultured immediately. The specimen must be
cultured __________ ____ _____, even if kept at __________ temperature.

Answer 14

refrigerated, within 6 hrs, refrigerator

Question 15

In private practice, the specimen should be inoculated to a _____ agar and ________ agar plate as follows: ___ μl (for cystocentesis and catheter specimens) or ___ μl (voided specimens) from a calibrated loop should be used to inoculate urine to the plates. The loop should be drawn down the _____ of the plate from ___ to _____, and then the loop should be streaked ____ and ___
through the ____ streak. After ___ hrs incubation, the colonies are counted and
the number of ____/ml is estimated. Most urinary tract infections will have a
bacterial burden of _____ than ____ CFU/ml. However, from a cystocentesis, any
number _____ CFU/ml is significant. If in doubt, the culture should be ______.

Answer 15

blood, MacConkey, 10, 1, center, top, bottom, back, forth, initial, 24, CFU, greater, 10^5, >10^3, repeated

Question 16

The most common bacteria isolated from the urinary tract are ?

Answer 16

enterics (e.g. E. coli, enterococci, Proteus), as well as Pseudomonas, staphylococci, and occasionally yeasts. Anaerobes and filamentous fungi are rare.

Question 17

Answer 17

Sterile loop and go through area a few times, streaking away from it. Do that 4 times.
Semi quantitative result
1-4, recorded in quadarant 1, etc.

Question 18

Answer 18

Question 19

Uterine Infections
* Abortions - the investigation should be comprehensive for all bacteria and fungi known to cause abortion in a particular animal species.
* Examples of bacterium that cause abortions are:
- hard to grow
- culture not very useful for most abortions unless ______ has been infected.
- fungal abortion –> __________is very evident
* Both maternal and fetal samples should be submitted when possible.

____ and __________-fixed samples for histopathology are essential if
ubiquitous organisms, such as Aspergillus fumigatus are to be identified.

Answer 19

Campylobacter sp., Brucella sp., Leptospira sp., H. somni, mycoplasmas, Listeria sp., A. pyogenes, Aspergillus sp.,
Zygomycetes and others.

fetus, histopath, Fresh, formalin

Question 20

Preferred samples (fetus)
* ______ plus _________ (fixed and fresh) or uterine ______ (fresh).
** Fetal _______ contents (fresh), plus fetal lung, liver, kidney, and spleen (fixed and fresh).
* Midstream urine for ________: This sample needs to be centrifuged and examined by _____ microscopy immediately,
diluted and cultured in appropriate media, and/or blood collected for serology. Contact reference lab for details.
* Clotted blood samples for serology, at the time of abortion and 2 weeks later, should be sent to your Regional Laboratory, particularly for difficult to isolate agents such as Leptospira.

Answer 20

Placenta, cotyledons, discharge, stomach, leptospirosis, darkfield

Question 21

Infertility
* Specimens:
* Uterine ______ or vaginal _____.
* ______ washings (_______) or ____. Submit urine (as above) if leptospirosis is suspected.
* Unless otherwise directed, examinations should cover:
* Cattle: Campylobacter fetus, and opportunistic aerobes (e.g. Haemophilus
somnus). A special transport medium is recommended for C. fetus.
Culture should be carried out within 4-6 hours of collection.
*** Mares: Coliforms (especially Klebsiella), other opportunist aerobes (such
as streptococci, fungi, and Taylorella equigenitalis).
* The laboratory should be notified if specimens are to be collected and
submitted for an infertility investigation. This will allow:
* Special _____ media to be prepared.
* Advice on the use of transport media for ?

Answer 21

discharge, mucus, Preputial, preferred, semen, selective, C. fetus.

often times happens in mares, often due to streptococci, klebsiella sometimes
go in with protected brush and ?

Question 22

Blood Cultures
* The skin needs to be _________ and ______ prepped with antiseptic, such as ______
and ______. Once prepped, the skin should not be touched with the fingers.
** ____ blood samples should be collected, approximately __ hour apart*. The reason for this is two-fold: 1) the animal may not always be _____ and may only _____ be
shedding organisms into the blood; 2) to confirm any isolate obtained from ___ bottle is not a contaminant.
* Normally samples will be inoculated into aerobic and anaerobic bottles. However, lessthan 1% of blood isolates are anaerobic
and many labs have dispensed with doing
routine anaerobic cultures. There are two size blood culture bottles: adult and pediatric. Adult size requires 5-10 mls/bottle; the pediatric size requires about 1.5 ml/bottle. The pediatric size is useful for small animals.
* The bottles are examined for turbidity and gram-stained and cultured onto chocolate
agar. If there is no turbidity, blind subcultures are done at 48 hours and 7 days before
being discarded.
* Alternatively, lysis/centrifugation systems may be used. In this system the blood is added
to a tube containing an agent to lyse the red blood cells. The tube is then centrifuged
and the pellet of bacteria is resuspended and inoculated on to agar for culture. This
system has shown to be more sensitive, but isolates more contaminants, than culture
bottles. The ideal is to use both systems

Answer 22

shaved, aseptically, betadine, alcohol, Three, 1, septicemic, periodically, one

Question 23

Johne’s Disease
* Cultural techniques may not be available, but specimens can be examined by the ____-_____ staining of smears. See PCR under “Non- culture diagnostic methods”.
* Specimens: Live animal - Scraping of ______ mucosa (detects ______ cases)
* Dead animal - _____ valve or other affected areas of ______ tract, ______ lymph nodes.

Answer 23

acid-fast, rectal, advanced, ileocecal, alimentary, mesenteric

Question 24

Diarrhea/Dysentery
* _______, ______, and possibly _____ should be considered prime candidates for bacterial ______.
* Other possible agents are _____, yeasts, and agents that form toxins in foods
(e.g. S. aureus, Clostridium, and Bacillus; Shigella may cause gastroenteritis only in
primates.
* Enterotoxigenic __.___ is probably only of significance in neonates. However, E. coli
0157:H7 (causes bloody diarrhea and hemolytic uremia syndrome in humans) may be significant in adult cattle. The 0157:H7 strain can be screened for by failure to ferment sorbitol on sorbitol-MacConkey agar.

Answer 24

Campylobacter, Salmonella, Yersinia, gastroenteritis, Aeromonas, E. coli

Question 25

Question 26

Normally do not find _______ outside of the primates

Answer 26

Shigella

Question 27

Avian species –> typically have gram ___ bacteria

Answer 27

+

Question 28

Intestinal diseases in pigs
* Campylobacter hyointestinalis, and C. sputorum sp. mucosalis may cause intestinal adenomatosis, which may be associated with proliferative hemorrhagic enteropathy, and necrotic and regional ileitis. The etiologic role of C. sputorum subsp. mucosalis in this syndrome is questionable, and is more firmly established for C. hyointestinalis. The organisms are within the ileal cells and so isolation from feces or a live animal is unlikely.
* Specimens: _______ dead or _____ pig. Intestine containing ______ lesions
(usually _____ small intestine and ____ large intestine), and/or mucosal _____.
* Submit some material fixed for histopathology and some fresh for culture.

• Swine dysentery – Brachyspira (Serpulina) (Treponema) hyodysenteriae. This disease
  can usually be diagnosed by microscopy, but culture is possible. Filter supernatant of
  fecal material through 0.8 μm, 0.45 μm, and then 0.2 μm filters and inoculate filtrate to
  blood agar. Incubate 2-7 days under anaerobic conditions. Bacteria will form a clear hemolytic film. Examine by darkfield microscopy for spirochetes.
• Specimens: _____ from a live animal, or _____ and ______ from a dead animal.

Answer 28

Recently, killed, hemorrhagic, terminal, proximal, scrapings, Feces, intestine, contents

Question 29

Continued
–> Proliferative enteropathy
* The etiologic agent of this disease is Lawsonia intracellularis. The
symptoms are acute bloody diarrhea and anemia, usually in mature
animals. Vomiting may also be present. Necrotic enteritis and ileitis is also
common.
* This organism is an * ________ _____ pathogen* and cannot be _____
in bacteriology labs. It has been grown in ________ cell cultures. It is
positive in the modified acid-fast stain. The best method for diagnosis is ___ of feces or _______. Less sensitive tests are FA and ELISA for detection of antibodies.

• Tuberculosis
• Smears are be stained by the _____-______ method and examined. Culture and
  identification requires a reference laboratory.
• Specimens: Lesions, fresh and some fixed in formalin for histopathology.

Answer 29

obligate, intracellular, cultured, mammalian, PCR, histopathology, acid-fast

Question 30

For tuberculosis, use an acid fast stain

Question 31

Mycoplasma
* Mycoplasmas and ureaplasmas are found as commensals of the
respiratory and urogenital tract, respectively, of all animals.
* When animals are stressed or immunosuppressed, these organisms
can often cause diseases such as pneumonia, reproductive failure,
arthritis, and mastitis.
* Disease due to these bacteria is probably greatly ______ diagnosed
because they do not grow on _______ media. These organisms can be cultivated on special mycoplasma media in _____. However, any one medium will not grow all species of mycoplasmas. _____ is
required to speciate them, and is done at national reference labs.

Answer 31

under, conventional, CO2, Antisera

Question 32

Chlamydia
* There are nine spp. in the genus Chlamydia. They are:
* Chlamydia trachomatis, C. pneumoniae, (humans); C. psittaci (zoonotic/avian);
C. abortus (abortion isolates); C. felis (feline pneumonitis); C. caviae (guinea pig
inclusion conjunctivitis); C. pecorum; C. suis (pigs); C. muridarum (mice).
* It must be remembered that Chlamydiaceae are * _________ _______
pathogens*. Therefore, they are not culturable on culture _______, but must be
cultured in _____ culture. Therefore, specimens must be submitted to a
virology lab for culture. Alternatively, a less sensitive but specific diagnosis can
be made by serology, or antigen detection by ELISA or fluorescent antibody. A
technique that is likely more sensitive and specific than culture is PCR.

Answer 32

OBLIGATE, intracellular, medium, tissue

Question 33

Anthrax
* Still present in ?
* If anthrax is suspected the State or Federal (APHIS) Reference
Laboratory should be contacted regarding specimens.

Answer 33

anthrax alley (Mid-west; Texas-Dakotas)

Question 34

Fungal Infections
* Systemic infections - submit tissue containing lesions both ____ and fixed
in ________ for culture and histopathology, respectively.
* Dermatophytes - submit hairs ______ (not cut) from the lesions and edges
of lesions. Also send skin ________ from the _____ of the lesions, scrape
until blood _____ appears. These skin scrapings could be submitted with the _______ blade used for collection. The hairs and skin scrapings arrive drier and less contaminated if sent in a strong ______ envelope. Hairs are inoculated to _____; dermatophytes are _____ and turn the medium ____.
* More rapid methods include fluorescence of hair under a _____ lamp (some Microsporum species only), and identification of ______ in skin or
hair following incubation on a slice with ___% _____.

Answer 34

fresh, formalin, plucked, scrapings, edge, just, scalpel, paper, DTM, white, red, Wood’s, hyphae, 10% KOH

if you put too many contaminants in DTM, they will overgrow

Question 35

Streptothricosis or Dermatophiliasis
* Specimens: _____ material, plucked together with _____, from the skin _____.
* _______ ____-stained smears are usually sufficient for a conclusive diagnosis. Culture will only be carried out on request.

Answer 35

Scab, hairs, lesions, Methylene, blue

Question 36

Nonculture Diagnostic Methods
* Fluorescent Antibody (F.A.) Technique :
* FA may be used for diagnosis of a variety of diseases including, but not
limited to:
* Malignant Edema and Blackleg organisms: C. chauvoei, C. novyi and C. septicum.
* Specimens: Bone ______ in an _____ rib, Block of affected _____.
* C. novyi and C. septicum are rapid post-mortem invaders. Therefore,
specimens should be taken from a recently dead animal or otherwise the
results must be interpreted with great caution.
* Chlamydia, Leptospira and Brucella diagnosis can also be made by F.A. directly
from the appropriate specimens (e.g. tissues, impression smears, or swab
smears).

Answer 36

marrow, unopened, muscle

Question 37

Latex agglutination tests and enzyme immunoassays,
including lateral flow immunoassays
* Detection of _____ or _____, but are becoming more popular and will be
seen in veterinary medicine with more frequency.
* Besides rapid results, an advantage of antigen detection tests is that the
organisms do not need to be _____.
* A disadvantage is that currently these tests may ___ be more _____ than
culture.

Answer 37

antigen, antibody, viable, not, sensitive

Question 38

Polymerase Chain Reaction (PCR)
* **PCR involves __________ of ____ in a sample. Detection of the DNA can then be
done by a probe, or examination of the amplification product by electrophoresis
alone or by restriction enzyme fingerprinting.
* The DNA from a sample containing a very small number of organisms can be
amplified by using forward and reverse DNA primers of a region known to be specific
for the organism of interest.
* An extract of the sample, the primers, Taq polymerase, dNTPs, and other reagents
are added to a thermal cycler. The temperature is raised to 94°C to dissociate the DNA, the temperature is lowered to 54°C to anneal the DNA with the primers, and the temperature is raised to 72°C to extend (synthesize) new DNA. This cycle is
repeated automatically for about 30 cycles. The DNA is amplified logarithmically.
*** For diagnostic purposes, ______-______ PCR must be used, and a true positive
determined by the CT (cycle threshold) value. CT values __ ____ are normally positive.

Answer 38

amplification, DNA, Real-time, <36

Question 39

SUMMARY OF SPECIMEN COLLECTION AND SUBMISSION*
* Get the samples to the laboratory as ______ as possible. If they cannot be
dispatched immediately, place in a ____-box in the car or in a _______ until they can be ____. Place a ____-pack or bag of ___ with the specimens to keep them cool in _____. If the specimens require lengthy transport, ____ quickly and ship on ___ ice.
* Specimens from animals dead more than 6 hours are usually _______ for microbiology as post-mortem invasion by organisms such as ______-______ and _____ is rapid. Useful to remember that _____-_____ is often the last tissue to be invaded. An ________ ___ containing bone-marrow can be a good
specimen.
* Specimens from animals recently treated with ______ may not yield significant bacteriological results.

Answer 39

quickly, cool, refrigerator, sent, cool, ice, transit, freeze, dry, unsuitable, coli-forms, clostridia, bone marrow, unopened, rib, antibiotics

Question 40

Continued
* Pack the samples intelligently:
* Separate the different specimens. (e.g., place liver and lung in separate containers.)
** Make sure that a container will not leak in transit.
* Do not contaminate the outside of the containers. This can be a serious health hazard
to laboratory staff.
** Fluids should be placed in leak-proof, screw-cap containers.
* Whirl-bags are not acceptable for liquid specimens.
*** The containers should be sterile before the introduction of a sample for bacteriology.
** Label all specimens carefully.
** It is generally useful to correlate microbiology results with other procedures such as
histopathology and/or serology. This is particularly important in the case of
ubiquitous bacteria and fungi. Histopathology can indicate whether actual invasion of
the tissue occurred, or whether the organisms isolated were merely contaminants.

Question 41

Continued
* Serological results can indicate whether infection with the particular
microorganism has occurred. However, infection can occur without
any overt signs of disease.
* Select specimens and request examinations relevant to the problem
under investigation.
* If you have any doubts about what specimens to send, consult your
laboratory.
* Only use swabs if better samples are unobtainable: e.g. block of
tissue, mucosal scrapings, fluid that can be taken in a sterile syringe,
etc.

Question 42

Include an adequate clinical history. The history should
include:
* Species of animals which are affected
* Breed
* Age
* Vaccinations
* Number of animals of same species on premises
* Number of animals sick
* Symptoms
* Location of lesions
* Duration of problem
* Suspected condition
* Treatments
* Samples submitted

Question 43

SIMPLIFIED KEY TO IDENTIFICATION OF COMMON
VETERINARY BACTERIAL PATHOGENS
* Growth Media:
* Blood agar and MacConkey agar
* Thioglycolate broth may be included as a backup for fastidious bacteria and
anaerobes.
*
* Identification Media and Tests:
* Triple sugar iron agar
* Urea agar
* Indole (tube or spot)
* Catalase
* Coagulase
* Oxidase
* Motility

Question 44

Question 45