Illustration by Felecia Paras

CE Article #1

Evaluating Polymerase
Chain ReactionBased Tests
for Infectious Pathogens*
Julia Paxson, DVM, PhD
Tufts University Cummings School of Veterinary Medicine

ABSTRACT: Polymerase chain reaction (PCR)based diagnostic tests can allow rapid and sensitive
detection of equine infectious pathogens. However, PCR is not without limitations and is an
inappropriate diagnostic tool for some diseases.This review analyzes the advantages and limitations
of PCR-based diagnostic tests for several important equine infectious pathogens and suggests
questions for practitioners to consider when choosing a commercial PCR test for clinical diagnosis
of a particular infectious pathogen.

he polymerase chain reaction (PCR)

exponentially amplifies selected DNA
sequences1 and has become an increasingly popular tool for pathogen detection. 2
PCR-based pathogen detection is rapid, can
detect much smaller quantities of pathogen
than many other tests, is independent of host
response, can distinguish vaccination f rom
pathogen infection, and is important in identifying pathogens such as viruses and rickettsiae
that are otherwise not easily isolated. 35 Although PCR-based tests have great potential in
pathogen detection, clinicians should consider
the limitations of PCR testing in general as
well as in working with particular pathogens.
Correct and informed interpretation of test
results is critical to the successful use of PCRbased diagnostics. Clinicians should carefully question the laboratory concerning
standard sample handling procedures and testing protocols

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*A companion article titled Polymerase Chain Reaction Test Interpretation appeared in the May
2008 issue.

308

because variations in DNA extraction protocols,

PCR techniques, and reaction monitoring can alter
the validity of the test. False-negative and falsepositive test results can occur secondary to inhibition of the reaction,6,7 sample handling errors, and
the limitations of laboratory techniques. In addition, it is important to recognize that test results
reflect only the presence or absence of pathogen
genetic material in a sample. PCR pathogen tests
rely on the detection of pathogen genetic material
(DNA or RNA). A negative PCR test result will
be generated if no genetic material is present in the
sample of an infected and clinically affected individualfor example, the apparent lack of Sarcocystis neurona DNA in the cerebrospinal fluid (CSF)
of horses with equine protozoal myeloencephalitis
(EPM).8 Conversely, the presence of genetic material does not guarantee the presence of live organisms; a horse with a positive PCR test result for
Salmonella spp may not be actively shedding viable
organisms.9 Although PCR-based testing is a
potentially powerful tool, because of its possible
limitations and the misinterpretation of results, it
should be independently evaluated for each
pathogen of interest (Table 1).
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Table 1. PCR Testing Results for a Variety of Infectious Equine Pathogens

PCR/Standarda Outcome
Pathogen

Salmonella
spp

Study

PCR Test
9

ssPCR(hisJ)
Cohen et al
14
icPCR(ompC)
Amavisit et al
Kurowski et al15 rPCR(spaQ)

Lawsonia spp Lavoie et al17

ssPCR

+/+

+/-

-/+

Total

Conclusions

11
2
80

60
42
4

0
0
0

110
96
230

PCR is sensitive in detecting this organism.

Monitor fecal inhibition using internal controls.
DNA from nonviable organisms can result in
false-positive PCR results. Confirm results
with culture.

NA

29

PCR is prone to fecal inhibition. Use internal

controls.

Clostridium
difficile and
Clostridium
perfringens

Magdesian et al26 ssPCR

Herholz et al22 ssPCR

36
74
0
NA NA NA

130
NA

Sensitive for identification of pathogenic

strains.

Streptococcus
equi

Newton et al38

nPCR

37

16

70

PCR is sensitive in detecting this organism.

Identifies more animals that test positive than
does culture.

Rhodococcus
equi

Sellon et al34

NA

53

PCR is generally sensitive in detecting this

organism.
PCR tests distinguish pathogenic and
nonpathogenic strains.

EHV1

Varrasso et al5

nPCR(glH)

20

71

PCR is sensitive in detecting this organism.

Available as part of a respiratory panel.
Best results are from nasopharyngeal swabs.

EHV4

Varrasso et al5

nPCR(glH)

Best results are from nasopharyngeal swabs.

Influenza A

Quinlivan et al42 qRT-PCR(mat)

27

171

Best results are from nasopharyngeal swabs.

EIAV

Nagarajan and
Simard48

nPCR(gag)

81

122

PCR is sensitive in detecting this organism

but not yet accepted internationally.

West Nile
virus

Johnson et al51

RT-nPCR(E)

10

73

PCR is unreliable in detecting this organism.

Viremia does not correlate with clinical signs.

Potomac
horse fever

Mott et al3

nPCR

17

27

Suggest conducting paired IFA titers and

PCR testing.

Anaplasma spp Bullock et al54

nPCR(16s)

NA

375

PCR is sensitive in detecting this organism

compared with paired IFA titers.

Sarcocystis spp Miller and

Bernard8

ssPCR

116

259

PCR tests are unreliable in detecting this

organism due to the lack of pathogen in the
CSF.

a
Standard indicates
b

alternative diagnostic method (e.g., culture, viral isolation, parasite visualization, Coggins test for EIAV).
A sampling of commercial availability. The PCR methods used and the reported sensitivities and specificities may vary by laboratory: 1 =
Colorado State University; 2 = University of Illinois; 3 = Iowa State University; 4 = Kansas State University; 5 = University of Minnesota; 6 =
University of Missouri; 7 = Indiana Animal Disease Diagnostic Lab at Purdue University; 8 = University of Georgia; 9 = North Carolina State
University; 10 = Dynagenics Veterinary Diagnostics; 11 = University of California, Davis; 12 = Cornell University; 13 = Equine Biodiagnostics,
University of Kentucky.

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Polymerase Chain ReactionBased Tests CE

Availabilityb

Indications

Screening test for inpatients

Individual diagnosis
Environmental testing

Yesfecal
Laboratories: 1, 2, 4, 6,
7, 8, 11

Individual diagnosis
Alternative to culture of this
fastidious pathogen

Yesfecal
Laboratories: 3, 6, 7, 11, 12

Use for identification of

pathogenic strains

Yesfecal
Laboratories: 1, 2, 3, 5, 7

Individual diagnosis
Yesnasal or TTA
Screening for inapparent carriers Laboratories: 8, 11
Used for early individual
YesTTA, nasal
diagnosis
Laboratories: 8, 11
Also useful for monitoring on
endemic farms
Useful for early diagnosis

Yesfetal tissue, nasal

Laboratories: 1, 11, 12

Useful for early diagnosis

Yesnasal
Laboratories: 1, 11

Useful for early diagnosis

Yesnasal
Laboratories: 1, 3, 5, 7, 11

Rapid screening method for

carrier animals

No

Postmortem only
Not recommended as an
antemortem diagnostic tool

Yesbrain
Laboratories: 1, 2, 5, 10, 11

Useful for clinical diagnosis

Yesbuffy
Laboratories: 10, 11

Useful for clinical diagnosis

Yesbuffy
Laboratories: 9, 10, 11

Not recommended as an
YesCSF
antemortem diagnostic tool at Laboratories: 13
this time
EHV = equine herpesvirus; EIAV = equine infectious anemia virus;
icPCR = internal-control PCR; IFA = immunofluorescence assay;
mPCR = multiplex PCR; NA = not available; nPCR = nested PCR;
qRT-PCR = quantitative RT-PCR; rPCR = real-time PCR; RT-nPCR
= reverse-transcription nested PCR; RT-PCR = reverse-transcription
PCR; ssPCR = single-step PCR; TTA = transtracheal aspirate.

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SALMONELLA SPECIES
Salmonella infection is a cause of potentially fatal enteric
disease in horses and is associated with potentially high
mortality rates of 42% to 44% related to virulent
strains.10,11 Active fecal shedding in the general horse
population is roughly 0.8%, as determined by single
fecal culture.12 However, fecal shedding within susceptible populations, such as horses with gastrointestinal or
respiratory disease, appears to be increased.13 Monitoring sources of infection, especially asymptomatic carrier
animals that shed the organisms in their feces, is of particular interest to large veterinary hospitals prone to
nosocomial salmonellosis outbreaks.10 PCR testing has
been investigated as a method of identifying carrier animals, following outbreaks, assessing sources of infection,
and assessing the effectiveness of disinfection methods.
The reliability of fecal PCR-based Salmonella testing
has been addressed by targeting a variety of genes associated with pathogenic Salmonella spp and by a variety
of DNA extraction techniques designed to reduce inhibition caused by urea and bilirubin compounds in feces.
The inhibition of PCR is monitored by including positive internal controls. In one study, PCR targeting of the
Salmonella ompC gene (encoding an outer membrane protein) and construction of an appropriate targeted internal
control enabled direct monitoring of inhibition in each
PCR.14 In this clinical study, all samples that tested positive on culture also tested positive on a single-step PCR
test. In addition, the authors noted a large number of
samples that tested positive on PCR testing and negative
on culture. 14 Real-time PCR has also been used to
increase the specificity of the reaction by including a
sequence-specific fluorogenic probe in addition to the
standard PCR primer pair. A study using real-time PCR
to target the spaQ gene (a Salmonella pathogenicity gene
that may be deleted in nonpathogenic environmental Salmonella strains) demonstrated that PCR testing was more
sensitive than culture for detecting pathogenic Salmonella
spp in a clinical setting.15
Results of multiple environmental sample testing
studies suggest that PCR test methods tend to be consistently more sensitive than culture for assessing possible sources of Salmonella infection. This may be
attributable to the fact that bacterial growth in culture
can be inhibited by the presence of disinfectants and
that PCR testing detects viable and nonviable organisms.16 Many teaching hospitals now routinely use PCR
testing to detect possible environmental contamination,
with the understanding that samples that test positive
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on PCR testing may reflect the presence of nonviable

organisms but that samples that test negative on PCR
testing reflect a high probability that the environment
has been effectively decontaminated.9

LAWSONIA INTRACELLULARIS
Although bacterial culture has been the gold standard
for detecting Lawsonia intracellularis, culture of this fastidious intracellular bacterium is difficult and serial
serologic testing is often used as an alternative diagnostic. Initial PCR testing of clinical samples was unreliable because of the degree of inhibition caused by fecal
by-products.17 In addition, serial PCR testing indicates
that animals test negative within 4 days of initiating
antibiotic treatment but can be seropositive for up to 6
months.18 Sample dilution, nested PCR, and the more
recent inclusion of internal mimics to detect inhibition
have led to greater success in detecting L. intracellularis

for the tcdB gene, which has been shown to be present

in all human toxigenic strains of C. difficile.10,23 Using
internal controls to monitor possible fecal inhibition,
this real-time PCR test in humans reportedly has a sensitivity of 100% and a specificity of 94%.23
C. perfringens isolates are commonly classified as types
A through E based on toxin production. In addition,
type A isolates produce C. perfringens enterotoxin, and
types A and C have been shown to produce 2 toxin
(Cpb2). Because some strains of C. perfringens can be
commensal in the equine large intestine, differentiation
of pathogenic from nonpathogenic strains is crucial in
making a diagnosis. Some studies have identified pathogenic C. perfringens through amplification of the enterotoxin gene.25 However, recent studies indicate that the
presence of 2 toxin without the enterotoxin is sufficient
to cause gastrointestinal disease in horses.22,27 Furthermore, studies indicate that exposure of some pathogenic

PCR-based diagnostic tests are useful for rapid and early detection of select equine pathogens.
in piglets. In these studies, inhibition (as assessed by
failure to amplify the mimic) was observed in roughly
10% of cases, even with the use of diluted samples and
nested PCR testing, but the inclusion of internal mimics
to detect inhibition appears to have good sensitivity (24
of 24 piglets that tested positive on histology also tested
positive on nested PCR testing).19,20

CLOSTRIDIUM DIFFICILE AND

CLOSTRIDIUM PERFRINGENS
Clostridial colitis can be associated with antibiotic
administration in horses.21 PCR testing is used for rapid
identification of toxigenic strains of Clostridium difficile
and Clostridium perfringens.21,22 Enteropathogenicity of
C. difficile strains in humans and horses has been associated with the presence of enterotoxin A (TcdA) and
cytotoxin B (TcdB),23,24 and in horses, culture of C. difficile does not correlate to the detection of pathogenic
toxins.25 Toxigenic strains can be identified with commercial enzyme-linked fluorescent immunoassays for
detecting TcdA. However, the sensitivity of these assays
appears to be lower than the sensitivity of PCR assays
for identifying the tcdA gene in clinical samples.26 In
addition, recent reports of enteropathogenic strains that
test negative for TcdA and positive for TcdB in humans
have prompted the development of a real-time PCR test
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C. perfringens strains to the antibiotic gentamicin can

induce expression of the cpb2 gene, furthering the association of C. perfringens overgrowth with antibiotic use
in horses.28 These results indicate the need for PCR
testing of multiple possible toxin genes to most accurately identify possible pathogenic C. perfringens strains.

NEORICKETTSIA RISTICII
Potomac horse fever (equine monocytic ehrlichiosis) is
caused by the rickettsial bacterium Neorickettsia risticii
and is associated with colitis in horses.29 The disease can
be diagnosed by a variety of methods, including bacterial culture, paired immunofluorescence assay (IFA)
titers,30 and either peripheral blood mononuclear cell or
fecal PCR testing.3 An IFA cannot distinguish between
past or present infection and vaccination and is associated with a high rate of false-positive results.30 Compared with bacterial culture and IFA, the nested PCR
test appears to detect the presence of the bacterium earlier in experimentally infected animals. However, when
the nested PCR test was used in naturally infected
horses, only 81% of animals that tested positive on culture also tested positive on peripheral blood mononuclear cell PCR testing, which may be attributable to
poor recovery of Neorickettsia DNA from clinical specimens.3 More recently, a real-time PCR assay for blood
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or fecal samples demonstrated good specificity as well as

sensitivity comparable with that of nested PCR, but it
was not as sensitive as culture.31

RHODOCOCCUS EQUI
Rhodococcus equi is associated with the development of
severe pyogranulomatous pneumonia and ulcerative
enteritis in foals younger than 6 months and is traditionally diagnosed by culture and phenotypic analysis. However, bacterial culture of transtracheal aspirate (TTA)
samples can be inhibited by previous antimicrobial use
and can be confounded by the presence of relatively nonpathogenic environmental R. equi strains.32 Compared
with bacterial culture from TTA samples, PCR testing
appears to be more sensitive.33,34 Creation of a multiplex
assay to simultaneously target the Rhodococcus
sppspecific choE and vapA genes as well as a 16s ribosomal internal control demonstrated high specificity and
sensitivity, enabling differentiation of virulent and environmental strains as well as providing appropriate internal controls.33 More recently, real-time PCR assays have
been developed to target the vapA gene with even greater
sensitivity than standard PCR testing, enabling detection
of much smaller quantities of the bacterium.3537 Internal

amplification controls based on the choE gene have also

been included to monitor possible reaction inhibition.37
Real-time PCR studies have not yet documented success
of these techniques in a clinical setting.

STREPTOCOCCUS EQUI SUBSP EQUI

Streptococcus equi subsp equi is highly contagious and
commonly associated with barn-wide outbreaks of
strangles that can be hard to control, with healthy carriers emerging in more than 50% of strangles outbreaks.38
Culture of either nasopharyngeal or guttural pouch
swabs can be unrewarding during the early clinical
phase, and studies using nested PCR to target the S.
equi subsp equi M protein gene indicate that PCR testing is more sensitive than culture.38 However, clinicians
must recognize that PCR may be detecting amplification of DNA from nonviable organisms and, therefore,
the sample may not be from an infective source. Small
numbers of false-negative PCR test results have been
attributed to inhibition secondary to excessive suppurative material in the samples.38 A multiplex PCR assay
has also been established to rapidly differentiate
between S. equi subsp zooepidemicus and S. equi subsp
equi isolates based on the newly described superanti-

Polymerase Chain ReactionBased Tests CE

genic toxins SeeH and SeeI, which are present in S. equi

subsp equi, but not in S. equi subsp zooepidemicus.39

EQUINE RESPIRATORY VIRUSES

Equine influenza A2, equine herpesvirus (EHV) 1, and
EHV4 infections are considered to be some of the most
important contagious respiratory diseases in horses.40,41
Reverse-transcriptase PCR (RT-PCR) testing has
demonstrated greater sensitivity than has virus isolation
from nasal swab samples, which may be particularly
important given the difficulties in isolating some field
strains of the influenza A virus subtypes.42 Multiplex PCR identification and differentiation of EHV1
and EHV4 also appear to be sensitive.5 The development of a real-time
PCR-based test to quantify mRNA
expression (and thereby distinguish
between latent and active infection)
is promising.36 Because respiratory
virus infections are common, work
has been done to develop a comprehensive panel of commercial PCR
tests that can detect EHV1, EHV2,
EHV4, EHV5, equine adenovirus 1,
equine adenovirus 2, equine arteritis
virus, and equine rhinitis A virus by
using a combination of single-step
PCR, nested PCR, and RT-PCR
tests. 43 Most recently, a real-time
PCR assay for EHV1 was developed
and has shown good sensitivity and
specificity in a laboratory setting,
although few data for positive clinical samples are available.44
EQUINE INFECTIOUS
ANEMIA VIRUS
Equine infectious anemia virus (EIAV)
is an RNA virus of the family Retroviridae. Animals infected with EIAV
have an acute febrile response that
can be fatal, followed by an inapparent carrier stage.45 During the asymptomatic phase, carrier animals have
traditionally been detected by use of
an agar gel immunodeficiency assay,
a Coggins test, 46 or a competitive
ELISA.47 The principal drawback of
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315

these diagnostic tests is their reliance on the presence of

specific antibodies that can be absent in early infection.
Nested PCR testing for proviral DNA appears to detect
more EIAV-positive animals earlier in the course of
infection and is as specific as the agar gel immunodeficiency assay.48

EQUINE ENCEPHALITIS VIRUSES

Eastern, Western, and Venezuelan equine encephalitis
viruses and West Nile virus are RNA viruses that cause
infectious encephalitis in horses.49 In patients infected

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with these viruses, detection of the viral antigen or viral

nucleic acid in serum is possible only if blood is collected
during the viremic phase, which lasts 3 to 5 days and
may not correlate with the development of clinical
signs.50 Exposure to the virus can be confirmed serologically using an ELISA. Definitive postmortem diagnosis
via cell culture isolation from equine brain tissue can be
difficult, possibly because of low levels of viral replication
within brain tissue. 51 Several PCR tests, including
nested5052 and real-time RT-PCR,4 have been developed
for postmortem diagnosis using equine brain tissue. Both
nested and real-time RT-PCR have demonstrated a high
sensitivity in detecting the virus in brain tissue compared
with the sensitivity of culture.4 However, the same PCR
techniques are not reliable when used for antemortem
detection of West Nile virus in serum or CSF of clinically ill animals. Viremia may occur before the develop-

itations of the immunoblot tests. The test appears to be

of little use in antemortem diagnosis because it has a
high rate of negative results in possibly infected animals
(e.g., one study demonstrated 116 results of CSF that
tested positive on Western blot testing but negative on
PCR testing).8 S. neurona merozoites are usually found
intracellularly or within the parenchyma of the spinal
cord and brain, and parasite antigen and DNA are rarely
found in CSF.8 However, PCR testing has been found to
be useful in postmortem identification of parasites from
neuronal tissue.36 In the absence of parasite DNA, falsenegative PCR test results are likely. Unlike immunoblot
tests that yield a dichotomous result (positive or negative), a recently developed indirect fluorescent antibody
test enables more precise quantification of both serum
and CSF antibody levels. A recent study demonstrated
correlation between serum and CSF indirect fluorescent

The validity of each PCR-based diagnostic test depends on the application,

the PCR protocol that is used, and the correct interpretation of results.
ment of clinical signs,51 and serology is currently the only
clinically useful method of antemortem diagnosis of
West Nile encephalitis in horses.

ANAPLASMA PHAGOCYTOPHILUM
Anaplasma phagocytophilum (formerly Ehrlichia equi)
causes tick-borne vasculitis in horses.53 A diagnosis is
traditionally made by visualization of morulae in granulocytes or retrospectively based on an indirect IFA of
paired serum samples. Studies using nested PCR amplification of the 16s rRNA gene from peripheral blood
mononuclear cells suggest that PCR is a sensitive
method of detection that can detect the presence of the
organism several days before morulae can be identified
with light microscopy.54,55
SARCOCYSTIS NEURONA
The neurologic disease EPM can develop when S. neurona protozoa invade the central nervous system. A diagnosis is traditionally based on a CSF immunoblot
because it is believed that the presence of host antibodies
in the CSF (in an uncontaminated sample with minimal
bloodbrain barrier damage) is most closely correlated
with clinical disease.24 A PCR test to detect S. neurona in
CSF has been developed in an attempt to overcome limCOMPENDIUM EQUINE

antibody test antibody detection, and the authors suggest

that, in most cases, CSF or serum antibody quantification may be sufficient for determining the probability of
infection.56

CONCLUSION
PCR-based diagnostic tests have become increasingly
popular in equine medicine for the rapid and accurate
detection of infectious pathogens. PCR-based tests can
be extremely sensitive diagnostic tools, allowing pathogen detection earlier in the course of the disease (e.g.,
infection with A. phagocytophila54 or EIAV48). However,
because fragmented nucleic acids from nonviable cells
or viruses can be readily amplified using PCR, PCRbased tests cannot identify animals that harbor nonviable pathogen and are no longer contagious (e.g., horses
with Salmonella infection44). False-negative PCR test
results from infected animals can occur when the tested
sample does not contain pathogen genetic material (e.g.,
lack of S. neurona schizonts in CSF samples,24 lack of
encephalitis virus particles in the serum of infected animals that have passed the short-lived viremic phase,50,51
lack of the targeted virulence genes in some pathogenic
strains of R. equi34). Correct interpretation of PCR test
results and a thorough knowledge of the protocol used
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are critical in evaluating PCR-based tests. False-positive

PCR test results from uninfected animals are possible if
contaminating DNA is amplified; therefore, PCR test
results should be monitored by including controls, and
false-positive results should be limited by careful sample
collection and good laboratory practices. False-negative
PCR results from infected animals can result from low
DNA copy number, poor primer performance and the
presence of inhibitory substances in the sample (e.g.,
urea, heme), and prior antimicrobial use. Better DNA
extraction techniques, the inclusion of appropriate controls, and techniques such as nested PCR have been
used to monitor PCR test success and reduce potential
problems.3
PCR-based diagnostic tests appear to be useful in
rapidly detecting Salmonella spp,14 and new developments in PCR testing for other fecal pathogens (e.g.,
Lawsonia and Clostridium spp) have increased the value
of these tests.26 PCR-based tests are now routinely used
for rapid and early detection of S. equi subsp equi,38 R.
equi,33 N. risticii,3 A. phagocytophila,54 and equine respiratory viruses.42 Although international regulations still
stipulate use of the traditional agar gel immunodeficiency assay or Coggins test, PCR-based tests for EIAV
are also promising.48 PCR-based antemortem diagnosis
of EPM8 and viral encephalitis51 from CSF does not
appear to be reliable.
Because of the variety of factors that can influence the
validity of PCR results, it is important to understand the
possible limitations of the test in general and in regard
to the particular pathogen of interest. Table 1 summarizes the use of PCR tests in detecting a variety of infectious equine pathogens. When used appropriately, the
many variations on the basic PCR protocol can greatly
enhance the clinical usefulness of the particular test.
Because different PCR tests are available, practitioners
should discuss with their laboratory which protocol to
use for the pathogen of interest and how to best interpret the results from a given PCR test.

ACKNOWLEDGMENTS
The author thanks Drs. Melissa Mazan, Mary Rose Paradis, Lois Wetmore,
Daniela Bedenice, and Rose Nolen-Walston for their comments.

4.

5.
6.
7.

8.

9.

10.

11.

12.

13.

14.
15.

16.

17.

18.

19.

20.

21.

22.

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ARTICLE #1 CE TEST

CE

This article qualifies for 2 contact hours of continuing

education credit from the Auburn University College of
Veterinary Medicine. Subscribers may take individual
CE tests or sign up for our annual CE program.
Those who wish to apply this credit to fulfill state relicensure
requirements should consult their respective state
authorities regarding the applicability of this program.
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scores at CompendiumEquine.com.

1. Which statement regarding PCR is incorrect?

a. PCR is the most sensitive diagnostic tool for all
equine infectious pathogens.
b. False-negative and false-positive PCR test results can
occur.
c. Pathogen genetic material (DNA or RNA) must be
present in the tested sample.
d. Positive PCR test results can occur in animals without active infection.
2. PCR testing is not a valid diagnostic modality for
a. confirming active infection.
b. screening horse populations for asymptomatic carrier animals that shed a pathogen in their feces.
c. detecting infection of fastidious pathogens that are
hard to culture or otherwise isolate.
d. confirming infection with a virulent (rather than environmental) strain of bacteria.
3. To confirm active Salmonella infection in a horse
with a positive PCR test result, a clinician should
a. obtain a positive culture result.
b. obtain positive antibody titer results.

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CE Polymerase Chain ReactionBased Tests

c. confirm the presence of associated clinical signs.

d. confirm the presence of leukopenia.
4. PCR inhibition caused by fecal samples
a. can be monitored with internal controls.
b. is rare.
c. can be associated with prior antimicrobial use.
d. is more common in animals with diarrhea.
5. Use of PCR to monitor environmental sources of Salmonella spp
a. is not inhibited by the prior use of detergents.
b. may reflect the presence of nonviable organisms.
c. is more sensitive than culture.
d. all of the above
6. Use of PCR to detect R. equi
a. is straightforward because all Rhodococcus strains are equally pathogenic.
b. is less sensitive than bacterial culture of TTA fluid.
c. is rarely diagnostic because genetic material is rarely present in TTA or
fecal samples.
d. may have increased diagnostic value if the pathogenic vapA gene is
targeted.
7. Use of PCR to detect S. equi subsp equi
a. can be inhibited by prior use of antimicrobials.
b. always indicates the presence of active infection.
c. is more sensitive than bacterial culture.
d. is not subject to inhibition in purulent samples.
8. Which statement regarding viral disease is incorrect?
a. For some viral diseases, rising paired IFA titers with a negative PCR test
result may still indicate active infection.
b. For some respiratory viral diseases, PCR testing may be more sensitive
for testing nasal swabs than blood.
c. For viral encephalitis diseases, PCR testing is more sensitive for testing
blood than brain tissue.
d. Both RNA and DNA viruses can be detected using PCR techniques.
9. PCR-based antemortem diagnosis of encephalitis virus infection is
hampered by
a. inhibition of PCR.
b. a short viremic phase.
c. large numbers of false-positive PCR results.
d. overwhelming inflammation.
10. In diagnosing diseases such as EPM, PCR testing can be ineffective
because
a. PCR is generally inhibited in clinical samples.
b. genetic material is not usually present in the samples.
c. clinical signs of disease precede the ability of PCR to detect the
pathogen.
d. it is effective for testing CSF samples, but not blood samples.

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July/August 2008