425576

XXXXXX10.1177/1040638711425576Kase
mpimolporn et al.Rapid immunochromatographic test strip for detection of Rabies virus

Journal of Veterinary Diagnostic Investigation

23(6) 11971201
2011 The Author(s)
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DOI: 10.1177/1040638711425576
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Evaluation of a rapid
immunochromatographic test
strip for detection of Rabies virus
in dog saliva samples
Songsri Kasempimolporn,1 Wachiraporn Saengseesom,
Samrerng Huadsakul, Supatsorn Boonchang, Visith Sitprija

Abstract. An immunochromatographic test strip for Rabies virus was evaluated with dog saliva samples. The test was
initially validated against 237 dogs of known infection status, and then evaluated in the field with 1,290 live dogs. By validation
of paired salivabrain specimens obtained from dogs at necropsy, the saliva strip test was 94.4% specific and 93.0% sensitive
when compared to the gold standard fluorescent antibody test (FAT) on brain smears. The sensitivity and specificity of a nested
polymerase chain reaction (nPCR) assay using saliva were 100% compared to the FAT results. The performance of strip test
with field saliva samples from street dogs had a specificity of 98.7% in comparison to nPCR as the reference method. As the
strip test kit can potentially be used outside the laboratory and be applicable as an on-site testing assay, it represents a powerful
screening tool for epidemiological surveys and disease control. The test could be useful for the surveillance of rabies in dogs
and, in particular, be used to monitor the success of rabies control programs.
Key words: Dog saliva; immunochromatographic test strip; Rabies virus.

Rabies, the most remarkable infectious disease of the central

nervous system with the highest known case-fatality rate,
remains a threat in Asia and Africa.24 In Thailand, where dogs
are the animals most frequently reported as rabid, the disease
is endemic and wide spread. Cases of rabies in domestic animals other than dogs have been traceable to, or believed to
have been at the origin of exposure to, rabid dogs.11 In a sampling survey conducted by the Bureau of National Statistics
and the Bangkok Metropolitan Administration (BMA) in
1999, it was estimated that 633,814 dogs inhabit Bangkok.
Additionally, an estimation of the total pet dog population
was reported to be 823,504 in 2006. This number is thought
to have risen steadily, and the management of these dogs has
become a serious rabies control problem in the capital.
According to a World Health Organization recommendation, at least 70% of the dog population must be continuously
vaccinated to control rabies transmission.25 Despite an intensive citywide rabies vaccination campaign, only 62% of the
dogs investigated had received adequate immunization,12
which indicates that at least 38% of the dogs were not vaccinated for rabies. Past attempts to conduct a mass dog vaccination have been limited to Bangkok City and have not been
successful. Current rabies control programs include the elimination of the infected dog, but the risk of human rabies still
exists. Dog biterelated rabies cases in human beings have
accounted for 23 (96%) of the 24 deaths reported in 2009
of which 8 cases occurred in Bangkok (Ministry of Public

Health annual report). Low vaccination coverage and ineffective management of stray dogs are the most likely reasons
for the programs lack of success.
Reports of apparently healthy dogs that intermittently shed
Rabies virus in saliva have been reported.5 Experimentally
infected dogs were reported to have virus in the saliva up to
13 days before overt signs of rabies were observed.6 Field
observations suggest that a dog may remain healthy and shed
Rabies virus for longer than the expected 10-day observation
period prior to clinical development of rabies.21-23 Moreover,
a dog may recover from rabies with the potential for intermittent virus shedding.14 It cannot be predicted in which dogs
rabies infection will persist, and it may lead to infection of an
unexpected, untreated bite victim. The offending dogs usually escape, which can result in a potential spread of infection. Effective dog rabies control strategies not only serve to
reduce human deaths but also can reduce the overall costs
associated with rabies prevention. The surveillance of Rabies
virus infection in dogs would help to prevent its spread to
other animals.
From the Queen Saovabha Memorial Institute, World Health
Orgaization Collaborating Center for Research on Rabies, Thai Red Cross
Society, Bangkok, Thailand.
1
Corresponding Author: Songsri Kasempimolporn, Queen Saovabha
Memorial Institute, Thai Red Cross Society, 1871 Rama IV Road,
Bangkok 10330, Thailand. songsri.k@redcross.or.th

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Kasempimolporn et al.

In areas where dog rabies is endemic, all dog-bite victims

should seek medical attention immediately, and administration of rabies post-exposure prophylaxis after being bitten
should not be delayed. A biting dog should be euthanized
whenever possible and its fresh brain tissue tested for rabies
by the fluorescent antibody test (FAT).4Although sophisticated and expensive, nucleic acid amplification methods can
be more sensitive than FAT with brain tissue samples that are
too decomposed for direct examination7 and can detect viral
RNA in samples, such as saliva and cerebrospinal fluid (CSF),
which are unsuitable for FAT testing.3 Serological testing of
serum is not usually helpful in dogs with previous rabies vaccination history. Recently developed molecular techniques
permit the diagnosis of rabies in live animals by looking for
evidence of Rabies virus infection in saliva and CSF.
However, Rabies virus can be detected in a much higher frequency antemortem in saliva than in CSF.20 Molecular methods have detected evidence of Rabies virus successfully in
human cases where conventional methods could not be
applied.2 Reverse transcription polymerase chain reaction
(RT-PCR) of RNA extracted from saliva was the most reliable
diagnostic test.13,16 However, nested (n)PCR was required in
almost all cases to compensate for the often extremely limited
amount of RNA in antemortem samples. A more convenient
and affordable diagnostic test should be developed that can be
used outside the laboratory and be applicable as an on-site
test. Recently, a rapid immunochromatographic test strip was
developed and evaluated using clinical samples, particularly
brain tissue.8,15 In the present study, a test strip capable of
detecting Rabies virus was conducted on large numbers of
dog saliva samples, and its performance evaluated by comparing with standard methods.
A total of 237 dog saliva samples were collected from dog
carcasses submitted to the rabies diagnostic unit at Queen
Saovabha Memorial Institute, the Thai Red Cross Society.
Most of these carcasses were suspected rabid dogs. Saliva
collection was performed as described elsewhere.10 From the
same dogs, brain tissue specimens (medulla and hippocampus) were collected and submitted for routine FAT. For FAT
staining, fluorescein isothiocyanateconjugated anti-rabies
antibody prepared in rabbitsa was used. An additional 1,290
saliva samples were obtained from apparently healthy dogs
seen for surgical sterilization at the Rabies Control Subdivision
of the Veterinary Public Health Division, BMA. All of the
dogs were randomly captured from streets and public places
of Bangkok. Sample collection was done with the dog under
deep barbiturate anesthesia. All saliva samples were stored at
20C until used.
Total RNA was extracted from saliva specimens using a
chemical solution.bThe RT-PCR was performed in 2 g of
extracted RNA following a one-step process using a commercial kit.c Subsequently, the primary amplified product
was subjected to a second-round PCR. All amplifications
were carried out on a thermocycler,d using a denaturation
temperature of 94C, an annealing temperature of 60C, and

an extension temperature of 72C. Two sets of primers were

used to amplify 1,473base pair (bp; 5-GTAACACCCC
TACAATGGATGC-3, at position 57-78, and 5-CAAAGAT
CTTGCTCATGTTTGG-3, at position 508-1529) and
524-bp (5-GACATGTCCGGAAGACTGG-3, at position
319-337, and 5-GTATTGCCTCTCTAGCGGTG-3 at position 823-842) fragments of Rabies virus nucleoprotein gene,
for the first- and second-round PCR, respectively. The nPCR
products were electrophoresed in 1.2% agarose gels and stained
by ethidium bromide.
The test strip was constructed on the principles of immunochromatography using a combination of purified polyclonal antibody (pAb) and monoclonal antibody (mAb). The
pAb was raised in horses, which were regular donors for
equine rabies immunoglobulin (Ig)G production at the Thai
Red Cross Horse Farm.19 Such horses received a series of
purified Vero (African green monkey kidney epithelial) cell
rabies vaccine.e The IgG was fractionated out from the whole
serum by the salting-out method with saturated ammonium
sulfate.17 Horse anti-rabies IgG was conjugated to colloidal
gold particles (diameter: 20 nm)f and sprayed on a glass fiber
padg at 10 l/cm using a dispensing system.h Mouse mAb
was produced according to a standard method.1 Briefly,
somatic cell hybrids between mouse myeloma cells and
spleen cells derived from BALB/c mice immunized with a
purified chick embryo cell rabies vaccinei were cloned. One
hybridoma clone producing mAb RM25 specific to Rabies
virus glycoprotein was selected to grow in protein-free
medium.j Purified anti-rabies mAb RM25 was micro-sprayed
with an automated dispenserk onto a nitrocellulose membranel
at 1.5 l/cm in the test zone. Rabbit anti-horse IgGm was
micro-sprayed on the same nitrocellulose membrane at 1.5
l/cm in the control zone. The membrane was dried at 40C
overnight. The test strip was assembled and housed in plastic
cassettesn and stored in a desiccated plastic bag.
One hundred microliters of saliva was added to the sample hole of the test strip. The results were available within 10
min. Positive saliva sample was signified by the appearance
of 2 redpurple bands, one at the test line and one at the control line, respectively. The sample was considered negative if
only one band appeared in the control zone of the strip. The
test was not valid if the control line did not appear.
The strip test was initially performed with the fixed CVS11 strain of Rabies virus from the culture supernatant of
infected BHK (baby hamster kidney)-21 cells and saliva
samples from dogs that were previously known to be positive or negative for Rabies virus infection. The detection
limit of the strip test was determined using CVS-11 strain
culture supernatant possessing an infectious titer of 104.67
LD50/0.03 ml. The test detected approximately 10-3.31-fold
diluted CVS-11 (101.36 LD50/0.03 ml). The PCR detection
limit in the same assay was 100.46 LD50/0.03 ml, which was
lower than that of the strip test (data not shown).
Saliva samples from 237 dog carcasses, which were previously known to be positive or negative for Rabies virus

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Rapid immunochromatographic test strip for detection of Rabies virus

Table 1. Evaluation of a rapid immunochromatographic

test strip for detection of Rabies virus in dog saliva samples:
performance of strip test, fluorescent antibody test, and nested
polymerase chain reaction assay in detecting Rabies virus in dog
carcasses.*
FAT (brain
smears)
Strip test
+

Total

PCR (saliva)

53
4
57

10
170
180

53
4
57

10
170
180

*FAT = fluorescent antibody test; PCR = polymerase chain reaction; + =

positive; = negative.
Number of saliva samples.

Table 2. Evaluation of a rapid immunochromatographic

test strip for detection of Rabies virus in dog saliva samples:
comparison of strip test to nested polymerase chain reaction assay
for detecting Rabies virus in field saliva samples.*
PCR
Strip test

Total

0
0
0

17
1,273
1,290

*PCR = polymerase chain reaction; + = positive; = negative.

Number of saliva samples.

infection by FAT at necropsy, were examined for the presence of Rabies virus by PCR and test strips. Each specimen
was coded and tested blindly. The results obtained by FAT
using brain and strip test using saliva specimens from the
same dog for Rabies virus antigen testing were as follows.
Of 237 specimens tested, 53 were concordantly positive and
170 were concordantly negative. The remaining 14 samples
showed discordant results. Four were positive by FAT but
negative by strip test. The other 10 samples were negative by
FAT but positive by strip test (Table 1). The sensitivity and
specificity of the strip test relative to the standard FAT were
93.0% and 94.4%, respectively, whereas the sensitivity and
specificity of the PCR using saliva were 100% compared to
the FAT on brain smears (Table 1).
The performance of strip test was further evaluated with
field saliva samples from stray dogs by using PCR as the
reference method. Most samples were largely expected to
be negative. Among 1,290 samples, 17 produced a positive
result on the strips but all were negative on analysis with the
PCR. The occurrence rate of false-positive reactions was
1.3%, which equates to a specificity of 98.7% (Table 2).
It is a fact that the load of infective virus in saliva is lower
than that in brain. Virus may be absent or present at very low
levels in saliva. The findings in the current study showed that

1199

saliva samples could be used as an alternative to brain specimens for Rabies virus testing, but results might depend on
the stage of disease at the time of sample collection. Saliva
collection during the late stage yields more positive results.18
The present work would suggest that at least the PCR and
FAT in the case of 237 established cases performed equally
well. The reason for such a result might be that the dogs were
probably euthanized late in the course of the disease. Virus is
well distributed in many parts of the body of dogs (including
salivary glands) during the final stage of infection. However,
in this condition, the strip test presented lower sensitivity
compared to the PCR. The detection limit of PCR (100.46
LD50/0.03 ml) is approximately 8-fold more sensitive than
the strip test (101.36LD50/0.03 ml).
Relative to FAT, the sensitivity of the strip test was 93.0%,
and indicates that approximately 7% false negatives should
be expected. False-negative results identified by the strip test
may be explained by the fact that the load of virus in saliva
samples was below the detection limit of the strip. False negatives may be a consequence of technical limitations when
saliva is the preferred clinical sample. Given the fact that misdiagnosis of rabies-shedding dogs by strip test may occur,
careful interpretation is required in cases where Rabies virus
infection is suspected. The test can be limited in terms of the
amount of antigen that can be detected. The testing of serial
saliva taken at different time intervals may be necessary. The
quantum of virus and the duration of virus shedding in saliva
are of paramount importance for rabies diagnosis. Any doubtful or negative results need to be confirmed by animal history
or other laboratory techniques. Samples taken for antemortem diagnosis cannot definitively rule out rabies. If a suspicion of rabies persists despite negative findings, repeated
sampling may be necessary. Additionally, performance validation carried out on saliva samples demonstrated that falsepositive results were generated with the test strip. It is possible
that some components in saliva, such as a small number of
immune active and epithelial cells, small amounts of IgG,
digestive enzymes, and a broad spectrum of bacteria of the
normal flora9 can affect the specificity of the test. The results
of the field evaluation with stray dogs might suggest that the
diagnostic specificity of strip test is possibly a little better
than the initial validation suggested (98.7% vs. 94.4%) when
compared to PCR.
Although PCR demonstrated a better performance when
compared to strip test in the diagnosis of infected dogs, both
the technological expertise necessary to perform saliva strip
tests and the requirement for specialized laboratory equipment are minimal compared to PCR. From an epidemiological point of view, a rapid test is the fundamental basis for
surveillance and control of every major infectious disease
that allows interventions to be implemented in real time.
Nested PCR takes at least 7 hr to perform, which can not be
done practically in the field, whereas strip test could be carried out at the sites where the specimen is collected and the
results can be read directly by the naked eye within 10 min.

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Kasempimolporn et al.

Such a test can help veterinarians, public health professionals, and others concerned with rabies prevention and control
in the area to decide whether prompt euthanasia of the dogs
or having them caged and observed for clinical signs is indicated. Alternatively, samples that are classified as positive by
strip test can be forwarded to the local laboratory, where confirmatory diagnosis could be performed. The strip test has
a significant potential use for diagnosing rabies under field
conditions where rabies diagnosis is unavailable for the
moment and when brain material is unavailable for testing.
However, even strip test has sensitivity limits, and neither
conventional postmortem FAT nor PCR can rule out a diagnosis of rabies unless samples are taken appropriately, forwarded to the examining laboratory in good condition, and
tests are controlled rigorously. If used appropriately, the
saliva strip test can be a valuable adjunct to traditional methods for rabies antemortem diagnosis. As cited above, Rabies
virus may be present in the saliva of infected dogs for some
considerable period of time before the onset of clinical signs.
The diagnosis of rabies in a living dog by strip test may provide a positive diagnosis. This may be beneficial for dog
rabies control. However, a negative result should not be interpreted as indicating a rabies-negative animal. The results of
the current study demonstrate that the test strip works for
Rabies virus detection in dog saliva. The strip test can offer
speed, simplicity, and reasonable sensitivity and has a great
potential for field use.
Acknowledgements
The authors thank Pacific Biotech Co., Ltd. for technical assistance.

Sources and manufacturers

a. TRC, Thai Red Cross Society, Thailand.
b. TRIzol-LS, Gibco-BRL, Gaithersburg, MD.
c. SuperScript III with Platinum Taq, Invitrogen, Carlsbad, CA.
d. MWG-Biotech, Germany.
e. Verorab, Sanofi Pasteur, France.
f. Heron Diagnostics, Thailand.
g. Rapid 24, Whatman, Fairfield, NJ.
h. Imagene, Hanover, NH.
i. Rabipur, International AG, Basel, Switzerland.
j. CD Hybridoma Medium AGT, Gibco-BRL, Gaithersburg, MD.
k. BioDot, Irvine, CA.
l. Prima60, Whatman, Fairfield, NJ.
m. Sigma-Aldrich, St. Louis, MO.
n. Mcmould Entico, Thailand.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.

Funding
This work was supported by the Vejdusit Foundation under the
Royal Patronage of Her Royal Highness Princess Galyani Vadhana
Krom Luang Naradhiwas Rajanagarindra.

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16. Noah DL, Drenzek CL, Smith JS, et al.: 1998, Epidemiology
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450578

JVDXXX10.1177/1040638712450578

Erratum
Journal of Veterinary Diagnostic Investigation
24(4) 813
2012 The Author(s)
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DOI: 10.1177/1040638712450578
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Corrigendum

Stegelmeier, BL, et al.: 2010, Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats. J Vet Diagn Invest. 22: 570577

In the article Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in goats by Bryan L. Stegelmeier et al., the
published mean body weight and the means and statistics of serum biochemistries were carried out on groups of 4 animals,
not 3, as described in the Material and Methods section. The additional animal in each group was part of an auxiliary physiologic study and though the animals were dosed and treated the same, they were not necropsied and were not included in the
histologic study. To correct this oversight, the corrected weight and chemistry table (shaded cells indicate corrected numbers)
are listed below. The differences are minimal and do not alter the conclusions. In addition, reference 7 has been deleted.
Material and Methods: Fifteen, yearling, female Spanish goats weighing 29.4 3.4 kg (mean standard deviation) were
randomly divided into 5 groups with 3 animals per group.

References: Reference 7 should be deleted

Corrected Table 1. Selected mean serum biochemical data from groups of 3 goats dosed with rayless goldenrod (Isocoma pluriflora)
to obtain benzofuran ketone doses of 0, 10, 20, 40, and 60 mg/kg body weight for 7 days.*
Serum result (mean standard deviation)
Serum test (reference range)
Creatinine kinase (< 350 U/l)

Cardiac troponin-I (<0.40 U/l)

Aspartate aminotransferase (<125 U/l)

Alanine aminotransferase (<55 U/l)

Lactate dehydrogenase (<1,560 U/l)

Dose
0
10
20
40
60
0
10
20
40
60
0
10
20
40
60
0
10
20
40
60
0
10
20
40
60

Day 0
226 93
226 160
967 1233
125 18
202 93
<0.02 0.0
<0.02 0.0
<0.02 0.0
<0.02 0.0
<0.02 0.0
96 7
147 69
164 82
112 17
96 13
39 3
44 1
41 9
46 2
40 7
1,061 145
1,334 668
1,650 1,546
1,054 201
1,026 287

Day 3
107 6
118 8
306 276
117 24
202 124
<0.02 0.0
<0.02 0.0
0.17 0.26
<0.02 0.0
<0.02 0.0
91 6
104 11
284 248
102 12
115 31
37 3
42 3
57 34
44 4
44 5
1,075 62
1,050 223
2,617 2,685
1,162 130
1,277 348

Day 6

Day 7
a

73 16
206 184a
240 113a
6,699 5,329b
2,987 3,701a
<0.02 0.0
<0.02 0.0
0.05 0.03
1.98 3.39
1.38 2.31
83 2a
89 8a
293 252ab
991 184c
819 571bc
38 0a
39 2a
63 38ab
134 24a
118 84ab
875 213a
942 265a
1,185 449a
5,996 2,491b
3,623 2,924ab

66 30a
495 623ab
497 277ab
16,270 11,054b
10,433 4,326ab
<0.02 0.0
<0.02 0.0
<0.02 0.0
1.79 2.97
0.13 0.18
72 3a
97 13a
376 256a
3,277 1,556b
2,095 1,333b
43 18a
37 1a
61 25a
333 127b
267 176b
573 115a
709 182a
753 447a
9,891 3,210b
7,011 5,205a

*Different means (<0.05) between groups are indicated with superscript letters.
Estimates of normal range were determined as 2 standard deviations from mean values of control goats and pretreatment samples. These ranges are
probably laboratory and assay specific.
Cardiac troponin-I concentrations below detection limits are reported as <0.02 ng/ml.