Download Effect of temperature on the detection of porcine epidemic diarrhea

Brief Communications
237
J Vet Diagn Invest 16:237–239 (2004)
Effect of temperature on the detection of porcine epidemic diarrhea virus and
transmissible gastroenteritis virus in fecal samples by reverse
transcription–polymerase chain reaction
Kwonil Jung, Chanhee Chae1
Abstract. The effect of storage temperature was determined for the detection of porcine epidemic diarrhea
virus (PEDV) and transmissible gastroenteritis virus (TGEV) in fecal samples from experimentally and naturally
infected pigs by multiplex reverse transcription–polymerase chain reaction (RT-PCR). To examine the effect of
storage temperature on the ability to detect PEDV and TGEV RNA by multiplex RT-PCR, fecal samples were
stored for different temperatures (4, 21, 36, and 45 C) before extracting viral RNA. The PEDV and TGEV
nucleic acids in fecal samples were stable up to 3 days at 4 C. The PEDV and TGEV nucleic acids were
consistently detected in fecal samples up to 60 hours at 21 C and 24 hours only at 36 and 45 C. Thereafter,
the number of positive signals declined. Positive signals were detected in fecal samples stored at 4 C by 240
hours. The PEDV and TGEV nucleic acids were consistently detected in fecal samples up to 60 hours at 21 C
and 24 hours only at 36 and 45 C. The results of this study suggested that storage temperature has significant
effect on the detection of PEDV and TGEV nucleic acids from fecal samples by multiplex RT-PCR.
Porcine epidemic diarrhea virus (PEDV) and transmissible
gastroenteritis virus (TGEV) are 2 highly contagious enteric
viruses affecting mainly neonatal pigs. The PEDV and
TGEV infections are considered difficult to distinguish clinically and histopathologically.1,4,6,8,9 Rapid differential diagnosis and treatment are crucial to reduce mortality and morbidity from PEDV- and TGEV-induced enteritis in piglets.
A definitive diagnosis of porcine epidemic diarrhea and
transmissible gastroenteritis requires isolation of PEDV and
TGEV from intestinal contents. However, there are inherent
difficulties in isolating PEDV in cell culture.2,5 In recent
years, there has been much progress in the development of
molecular techniques, such as reverse transcription–polymerase chain reaction (RT-PCR), for the differential diagnosis of PEDV and TGEV from fecal samples.3,7,10 Collection of fecal samples does not require euthanasia of sick
animals for necropsy or shipping of whole-intestine specimens through courier services, as is currently done. In 2002,
more than 1,200 fecal samples were submitted to the Department of Veterinary Pathology at Seoul National University in Seoul to diagnose PEDV and TGEV (C. Chae, personal communication).
Ideally, clinical fecal samples collected for the detection
of PEDV and TGEV must be kept cool and transported to
the diagnostic laboratory as quickly as possible. This is often
a problem especially if farms are located a long distance
from a specialized diagnostic laboratory. Harsh environmental conditions during shipment of fecal samples could degrade viral RNA and result in failure of its detection. The
objective of this study was to evaluate the effect of various
temperatures on the stability of PEDV and TGEV nucleic
acids from fecal samples for multiplex RT-PCR assay.
Twenty colostrum-deprived, 3-day-old pigs were randomFrom the Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul
National University, Seoul, Republic of Korea.
1Corresponding author.
ly divided into 4 groups of 5 pigs each. Each group was
randomly assigned to 1 of 4 treatments. The 4 treatments
included oral inoculation with cell culture containing 1 of 2
viruses, PEDV strain SNUVR9714965 or TGEV strain
SNUVR980473,4 with both PEDV and TGEV, or with uninfected cell culture medium. Pigs in each of the 3 groups
were inoculated orally with 3 ml of tissue culture fluid containing 104 50% tissue culture infective doses (TCID50)/ml
of PEDV, 3 ml of tissue culture fluid containing 104 TCID50/
ml of TGEV, or 3 ml of tissue culture fluid mixture containing 104 TCID50/ml of PEDV and TGEV (1:1). Five control
pigs were exposed in the same manner to uninfected cell
culture supernatant. All pigs from each group were euthanized and necropsied at 36 hours postinoculation. The methods were approved previously by the Seoul National University, Institutional Animal Care and Use Committee.
To examine the effect of storage temperature on the ability
to detect PEDV and TGEV RNA by multiplex RT-PCR, fecal samples were stored at different temperatures before extracting viral RNA. At intervals (12, 24, 36, 48, 60, 72, 84,
96, 108, 120, 132, 144, 156, 168, 180, 192, 204, 216, 228,
and 240 hours), a representative fecal sample from each pig
was used to extract viral RNA and then RT-PCR was performed as described previously.3,7,10 The multiplex RT-PCR
amplified a 412-base pair (bp) region from the membrane
protein gene of PEDV and a 612-bp region from the nucleocapsid protein gene of TGEV.10 Vero and swine testicular
cells were used to isolate PEDV and TGEV from fecal samples as described previously.3,7 A total of 480 fecal samples
(2 fecal samples per storage time and temperature) were randomly selected for virus isolation.
An additional 10 fecal samples from each pig naturally
infected with either PEDV or TGEV were also used in this
investigation. Cases were selected on the basis of virus isolation, clinical signs, histopathological lesions, and in situ
hybridization, as described previously.3,7
To determine whether prolonged storage of fecal samples
at different temperatures affected the functionality of the
Downloaded from vdi.sagepub.com by guest on January 21, 2015
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/1
0/0
0/0
0/0
1/1
0/0
0/0
0/0
3/0
0/0
0/0
0/0
0/2
0/0
0/0
0/0
1/1
0/0
0/0
0/0
4/0
0/0
0/0
0/0
0/2
0/0
0/0
0/0
1/1
0/0
0/0
0/0
4/0
0/0
0/0
0/0
0/2
0/0
0/0
0/0
1/1
0/0
0/0
0/0
3/0
2/0
0/0
0/0
0/2
0/0
0/0
0/0
1/1
0/0
0/0
0/0
4/0
2/0
0/0
0/0
0/2
0/1
0/0
0/0
2/2
1/0
0/0
0/0
4/0
3/0
0/0
0/0
0/3
0/2
0/0
0/0
2/2
1/1
0/0
0/0
4/0
4/0
0/0
0/0
0/3
0/3
0/0
0/0
2/2
2/2
0/0
0/0
5/0
3/0
0/0
0/0
0/3
0/3
0/0
0/0
2/2
2/2
2/0
0/0
5/0
3/0
0/0
0/0
0/3
0/3
0/2
0/0
3/2
2/2
3/3
0/0
5/0
4/0
1/0
0/0
0/3
0/3
0/3
0/0
4/2
3/3
3/3
0/0
5/0
4/0
2/0
0/0
0/5
0/3
0/4
0/0
4/3
4/4
4/4
0/0
5/0
4/0
2/0
0/0
0/5
0/4
0/4
0/0
5/4
4/4
4/4
0/0
5/0
5/0
3/0
0/0
0/5
0/4
0/4
0/0
5/5
5/3
4/4
0/0
Both viruses
TGEV
PEDV
Downloaded from vdi.sagepub.com by guest on January 21, 2015
* Storage time (hr).
† Number of samples positive for PEDV by multiplex RT-PCR.
‡ Number of samples positive for TGEV by multiplex RT-PCR.
5/0
5/0
3/0
0/0
0/5
0/5
0/4
0/0
5/5
5/5
4/4
0/0
5/0
5/0
3/0
0/0
0/5
0/5
0/4
0/0
5/5
5/5
4/4
0/0
5/0
5/0
4/0
0/0
0/5
0/5
0/5
0/0
5/5
5/5
4/5
1/0
5/0
5/0
5/0
2/0
0/5
0/5
0/5
0/2
5/5
5/5
5/5
2/2
5†/0‡
5/0
5/0
5/0
0/5
0/5
0/5
0/5
5/5
5/5
5/5
5/5
4
21
36
45
4
21
36
45
4
21
36
45
228
216
204
192
180
168
156
144
132
120
Number of samples
108
96
84
72
60
48
36
24
12*
Temperature
(C)
multiplex RT-PCR, 300 fecal samples from 15 pigs experimentally inoculated with PEDV, TGEV, and both viruses
were examined after storage for 12–240 hours at temperatures from 4 to 45 C. Each specific primer pair for PEDV
and TGEV yielded an RT-PCR product of the expected size
from RNA extracted from fecal samples (Fig. 1). One positive sample per storage time and temperature was randomly
selected for sequence analysis. RT-PCR products of each virus were sequenced, and their identity was confirmed as
PEDV and TGEV (data not shown).
PEDV nucleic acid was consistently detected in fecal samples up to 132 hours at 4 C, 72 hours at 21 C, 24 hours at
36 C, and 12 hours at 5 C. Thereafter, the number of positive
signals declined. Positive signals were detected in fecal samples stored at 4 C by 216 hours, 21 C by 180 hours, 36 C by
108 hours, and 45 C by 24 hours. TGEV nucleic acid was
consistently detected in fecal samples up to 96 hours at 4 C,
60 hours at 21 C, 36 hours at 36 C, and 12 hours at 45 C.
Thereafter, the number of positive signals declined. Positive
signals were detected in fecal samples stored at 4 C by 228
hours, 21 C by 168 hours, 36 C by 120 hours, and 45 C by
24 hours. In the pigs from coinfected group, PEDV and TGEV
were detected simulatenously in fecal samples up to 72 hours
at 4 C. Thereafter, the number of positive signals declined. By
240 hours, no positive signals were detected in fecal samples
stored at 4 C (Table 1). PEDV and TGEV were isolated from
fecal samples from pigs experimentally inoculated with PEDV
and TGEV and both viruses. Neither PEDV nor TGEV was
isolated from fecal samples that were found negative by multiplex RT-PCR. No positive signal for PEDV and TGEV was
detected in fecal samples from control pigs. Therefore,
PEDV and TGEV were not recovered from any control pig.
In the pigs naturally infected with either PEDV or TGEV,
PEDV nucleic acid was consistently detected in fecal samples up to 108 hours at 4 C, 48 hours at 21 C, 12 hours at
36 C, and 12 hours at 5 C. Thereafter, the number of positive
signals declined. Positive signals were detected in fecal samples stored at 4 C by 228 hours, 21 C by 168 hours, 36 C
by 96 hours, and 45 C by 24 hours. TGEV nucleic acid was
consistently detected in fecal samples up to 84 hours at 4 C,
48 hours at 21 C, 36 hours at 36 C, and 12 hours at 45 C.
Virus
Figure 1. Agarose gel electrophoresis of multiplex RT-PCR–
amplified PEDV and TGEV complementary DNA products. From
left to right: M, 100-bp DNA ladder; lane 1, positive control for
PEDV and TGEV; lane 2, negative control; lane 3, fecal sample
from experimentally PEDV- and TGEV-coinfected pigs stored for
12 hours at 45 C; lane 4, fecal sample from experimentally PEDVand TGEV-coinfected pigs stored for 48 hours at 45 C.
240
Brief Communications
Table 1. Effect of storage time at different temperatures on the detection of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in fecal samples
from experimentally infected pigs by multiplex reverse transcription–polymerase chain reaction (RT-PCR).
238
Brief Communications
Thereafter, the number of positive signals declined. Positive
signals were detected in fecal samples stored at 4 C by 204
hours, 21 C by 132 hours, 36 C by 84 hours, and 45 C by
24 hours. PEDV and TGEV were isolated from fecal samples from pigs naturally infected with either PEDV or TGEV.
Neither PEDV nor TGEV was isolated from fecal samples
that were found negative by multiplex RT-PCR.
This study has important implications for the diagnosis of
PEDV and TGEV in the fecal samples. An important practical point is the effect of transportation temperature of the
specimens over long distances on the performance of the RTPCR assay. Fecal sample integrity is a key factor for successful RT-PCR assays for accurate diagnosis. Storage temperature of clinical specimens is known to influence the stability of viral RNA.11 The degradation of viral RNA at routine storage temperature could result in the failure of its
detection. Based on RT-PCR assay, PEDV and TGEV nucleic acids were stable in fecal samples up to 3 days at 21
C. Stability would be presumably even higher at 4 C. It must
be emphasized that care should be taken to guarantee the
temperature during transport and prevent exposure of the
samples to severe temperature fluctuations.
A multiplex RT-PCR is naturally less sensitive than individual RT-PCR or nested PCR. In this study, all fecal samples that were negative by multiplex PCR were also tested
by individual RT-PCR. Five samples that were negative by
multiplex RT-PCR were positive by individual RT-PCR (data
not shown). It is recommended to retest the negative fecal
samples by individual RT-PCR if viral infection was suspected. No attempt was made to check with porcine respiratory coronavirus (PRCV). The PRCV evolved from TGEV
when a spontaneous deletion occurred near the N-terminus
of the spike gene.12 Because the target gene for TGEV in
multiplex RT-PCR was N protein, the multiplex RT-PCR
used in this study could detect the nucleic acid of PRCV.
Therefore, some fecal samples from naturally infected pigs
may be positive because of the presence of PRCV.
It is not always possible to transport fecal samples to a
diagnostic laboratory in ,1 day, and prolonged delays between collection of specimens and RT-PCR assay in the laboratory, as well as potential exposure to extreme environmental temperatures during shipping, may have deleterious
effects on the identification of PEDV and TGEV. Because
239
freezing is commonly recommended when an extended delayed is expected, this study suggests that it would be better
if samples were stored at subzero.
Acknowledgement. This work was supported by contract
research funds of the Research Institute for Veterinary Science from the College of Veterinary Medicine, Seoul National University and BK21, Republic of Korea.
References
1. Chae C, Kim O, Choi C, et al.: 2000, Prevalence of porcine
epidemic diarrhoea virus and transmissible gastroenteritis virus
infection in Korean pigs. Vet Rec 147:606–608.
2. Hofmann M, Wyler R: 1988, Propagation of the virus of porcine
epidemic diarrhea in cell culture. J Clin Microbiol 26:2235–2239.
3. Kim B, Chae C: 2001, In situ hybridization for the detection of
transmissible gastroenteritis virus in pigs and comparison with
other methods. Can J Vet Res 65:33–37.
4. Kim B, Chae C: 2002, Experimental infection of piglets with
transmissible gastroenteritis virus: a comparison of three strains
(Korean, Purdue and Miller). J Comp Pathol 126:30–37.
5. Kim O, Chae C: 1999, Application of reverse transcription polymerase chain reaction to detect porcine epidemic diarrhea virus
in Vero cell culture. J Vet Diagn Invest 11:537–538.
6. Kim O, Chae C: 2000, In situ hybridization for the detection and
localization of porcine epidemic diarrhea virus in the intestinal
tissues from naturally infected piglets. Vet Pathol 37:62–67.
7. Kim O, Chae C: 2002, Comparison of reverse transcriptionpolymerase chain reaction, immunohistochemistry, and in situ
hybridization for the detection of porcine epidemic diarrhea virus in pigs. Can J Vet Res 66:112–116.
8. Kim O, Chae C: 2003, Experimental infection of piglets with a
Korean strain of porcine epidemic diarrhoea virus. J Comp
Pathol 129:55–60.
9. Kim O, Chae C, Kweon C-H: 1999, Monoclonal antibody-based
immunohistochemical detection of porcine epidemic diarrhea virus antigen in formalin-fixed, paraffin-embedded intestinal tissues. J Vet Diagn Invest 11:458–462.
10. Kim O, Choi C, Kim B, Chae C: 2000, Detection and differentiation of porcine epidemic diarrhoea virus and transmissible
gastroenteritis virus in clinical samples by multiplex RT-PCR.
Vet Rec 146:637–640.
11. Laude H, Van Reeth K, Pensaert M: 1993, Porcine respiratory
coronavirus: molecular features and virus-host interactions. Vet
Res 24:125–150.
12. Lee D-H, Li L, Andrus L, Prince AM: 2002, Stabilized viral
nucleic acids in plasma as an alternative shipping method for
NAT. Transfusion 42:409–413.
Downloaded from vdi.sagepub.com by guest on January 21, 2015