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Detection of the African
Swine Fever Genomic DNA on Dried Pig Blood Filter Paper
M. Guy-Gonzague*
F. Roger†
D. Rousset‡
T. Randriamparany‡
C. Crucière*
*AFSSA-LCRV, 22,
rue Pierre Curie, BP 67, 94703
†CIRAD-EMVT, Campus
International de Baillarguet, TA30/G, 34398 Montpellier Cedex 5,
‡Institut Pasteur
de Madagascar BP 1274, 101
KEY WORDS: African swine fever virus, PCR, filter paper
ABSTRACT
Polymerase chain reaction (PCR) is one of the best techniques
to detect hemadsorbing, non-hemadsorbing, and/or noncytopathic African
swine fever (ASF) strains. In some circumstances, the collection, storage,
and transport of biologic samples to an appropriate laboratory are not
easy. For this purpose, the blood sampling on filter paper, a method
widely used for the research of genomes by PCR, might be useful. This
study has shown that ASFV DNA remains detectable by PCR after dry storage
of blood on filter paper for at least 3 months at 30˚C. Thus, the
PCR was applied to filter paper blood samples from 62 pigs collected
in Malagasy areas infected by ASFV. The ASFV DNA was amplified for 34
of 62 dried blood samples. The results were similar to the previous
ones obtained with fresh samples collected at the beginning of the epizootic.
Introduction
African swine fever is a highly contagious disease. It causes high mortality and is epidemic in many African countries. It is an important disease in domestic pigs produced by an icosahedral double-stranded DNA virus classified into the Asfarviridae family, genus Asfivirus.1
A positive diagnosis requires either the detection of the virus or the demonstration of the presence of specific antivirus antibodies. In countries where the virus is established, the antibody detection can be used as a diagnosis test but, when pigs are infected with a highly virulent African swine fever virus (ASFV) strain, they have little or no detectable anti-ASFV immunoglobulines.2 The standard methods to identify ASFV are the hemadsorption test and the observation of the cytopathic effect. However, the existence of non-hemadsorbing and noncytopathic virus strains has been reported3,4 and, in this case, the presence of the ASFV genome can be identified by the polymerase chain reaction (PCR) assay.4,5 This PCR proved to be a practical, sensitive, and specific technique for the ASFV DNA detection in clinical samples of infected pigs, particularly when samples are unsuitable for virus isolation because of their deterioration.6 In the countries where the transport and the cold storage are a problem, dried samples can be mailed to the diagnostic laboratory without special environmental conditions. Such kinds of samples have been widely used for the research of viral DNA7 and RNA,8 bacteria,9 parasites, proviral DNA sequences,10 antibodies, or genetic analysis.11 The ASFV has a remarkable stability in blood.12 We have studied here the detection of AFSV DNA by PCR from pig whole blood dried on filter paper.
For this purpose, we have carried out a first experiment to validate the technique with contaminated blood. We have studied the detection of ASFV genome after the storage of dried blood spots at 30˚C for 3 months. Then, we have performed PCR from blood dried on filter paper of Malagasy pigs living in ASFV-infected areas.
Material and methods
Specimens
The whole blood samples were obtained from a pig inoculated with African swine fever field strain. This non-hemadsorbing, noncytopathic virus strain was isolated from Malagasy pig in 1998.4 The filter paper (Schleicher and Schuell) was cut into 50 mm ¥ 5 mm rectangles, spotted with 30 µL of whole blood sample serially diluted to 1:10-5 in serum from an uninfected pig, dried at room temperature, and stored in a closed bottle at 30˚C until use.
The clinical specimens were obtained from Malagasy pigs
of infected areas.
DNA Extraction From Fresh Whole Blood
The
DNA extraction was carried out as described by the O.I.E. manual6 from
blood samples serially diluted to 1:10-5 in serum from an uninfected
pig. Briefly, a 20% homogenate in phosphate-buffered saline (PBS) was
made from blood samples by grinding with a pestle (potter de Thomas,
Merck) followed by centrifugal clarification at 1000 g for 5 minutes.
The supernatant was boiled for 10 minutes and centrifuged at 13,000
¥ g for 5 minutes. Ten microliters
of the supernatant was used in the PCR test.
DNA Extraction From Dried Blood
Thirty microliters of blood were absorbed on filter paper. The blood was eluted from a stained portion of each filter in 120 µL of PBS at room temperature for 30 minutes. After centrifugation at 1000 ¥ g for 5 minutes, the supernatant was boiled for 10 minutes and centrifuged for 5 minutes at 10,000 ¥ g. Ten microliters of the supernatant were subjected to PCR.
Polymerase Chain Reaction
A DNA fragment of 278 pb from a highly conserved region of the genome coding for the p73 protein was amplified with the following primers:
Primer 1–5’
-ATGGATACCGAGGGAATAGC-3’
Primer 2–5’
-CTTACCGATGAAAATGATAC-3’
PCR was carried out
as described by the Commission of European Communities13 except that
there were one cycle at 94˚C for 1 minute, 55˚C for 30 seconds,
and 72˚C for 30 seconds; 38 cycles at 94˚C for 30 seconds,
55˚C for 30 seconds, and 72˚C for 30 seconds; and a last cycle
at 94˚C for 30 seconds, 55˚C for 30 seconds, and 72˚C
for 5 minutes.
A 10-µL portion of
the amplified product was resolved by electrophoresis in a 2% agarose
gel at 100 V for 1 hour. The band was visualized by staining with ethidium
bromide.
Results
PCR Detection of ASFV DNA From Fresh Whole Blood
An essay was done to establish the optimal dilutions of DNA still amplified by PCR. The PCR was positive until the 10-3 dilution (Fig. 1).
PCR Detection of ASFV DNA in Dried Blood Filter Paper
The study was carried out from 24 hours to 3 months. Until
3 months the DNA was still amplified until the10-3 dilution, but at
3 months the 10-3 dilution band was weaker (Fig. 2).
Use of Dried Blood Specimens for ASF Diagnosis of Malagasy Pigs
Sixty-two dried blood specimens from pigs collected in Malagasy areas infected by ASFV were mailed in France. They were tested by PCR amplification. The ASFV DNA was amplified for 34 of 62 dried blood samples (Table 1).
Discussion
In our study, the ASFV DNA has remained detectable by PCR after dry storage of the blood on filter paper for at least 3 months at 30˚C. These findings are consistent with those of other studies showing the stability of the genomic material after several weeks of storage of the filter paper: for at least 7 weeks14 to 25 weeks at 25˚C.15
The sensitivity of
the filter paper PCR method was comparable to the one with PCR with
fresh whole blood. This study was carried out with proportional quantities
of blood. This was in accordance with other data.7,14
The samples were collected from an area where we have demonstrated previously the circulation of the virus among the Malagasy pig population.4 In this study from 1998, 22 of 24 (91.6%) pigs and 5 of 8 (62.5%) pigs from the Antananarivo and Toamasina regions, respectively, were virus carriers. In the present study, the samples were collected in 2000, and 5 of 6 (83.3%) pigs and 29 of 56 (51.7%) pigs from the Antananarivo and Tomasina regions, respectively, were virus-positive. These results are similar if we consider that the percentages are calculated on a relatively low number of pigs because, since 1998, numerous pigs were slaughtered to eradicate the epizootic.
The use of dried blood filters is attractive because it
requires only a minimum volume (30 µL) of blood, the samples are easy
to collect on filter paper and to mail without risk of an epizootic
by spread of contaminated blood. These results provide evidence that
it is possible to undertake a virologic survey for ASFV in areas with
low technical infrastructure and without the need to maintain the specimens
at cold temperatures.
ACKNOWLEDGMENTS
The authors thank Dr. Labib BAKKALI-KASSIMI (AFSSA/Lerpaz,
France) for helpful suggestions.
References
1. Pringle CR: Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999. Arch Virol 144:2065–2070, 1999.
2. Mebus CA: African swine fever. Adv Virus Res 35:251–269, 1988.
3. Pini A: Isolation and segregation of non-haemadsorbing strains of African swine fever virus. Vet Rec 99:479–480, 1976.
4. Gonzague M, Roger F, Bastos A, et al: Isolation of a non-haemadsorbing, non-cytopathic strain of African swine fever virus in Madagascar. Epidemiol Infect 126:453–459, 2001.
5. Steiger Y, Ackermann M, Mettraux C, Kihm U: Rapid and biologically safe diagnosis of African swine fever virus infection by using polymerase chain reaction. J Clin Microbiol 30:1–8, 1992.
6. Manual of Standards Diagnostic Tests and Vaccines. In: O.I.d.E.W.o.f.a., ed. Health, 4th ed, part2; 2000.
7. Wang CY, Giambrone JJ, Smith BF: Detection of duck hepatitis B virus DNA on filter paper by PCR and SYBR green dye-based quantitative PCR. J Clin Microbiol 40:2584–2590, 2002.
8. Vilcek S, Strojny L, Durkovic B, Rossmanith W, Paton D: Storage of bovine viral diarrhoea virus samples on filter paper and detection of viral RNA by a RT-PCR method. J Virol Methods 92:19–22, 2001.
9. Saulnier P, Andremont A: Detection of genes in feces by booster polymerase chain reaction. J Clin Microbiol 30:2080–2083, 1992.
10. Solomon SS, Solomon S, Rodriguez II, et al: Dried blood spots (DBS): A valuable tool for HIV surveillance in developing/tropical countries. Int J STD AIDS 13:25–28, 2002.
11. Pitcovski J, Shmueli E, Krispel S, Levi N: Storage of viruses on filter paper for genetic analysis. J Virol Methods 83:21–26, 1999.
12. Plowright W, Parker J: The stability of African swine fever virus with particular reference to heat and pH inactivation. Arch Gesamte Virusforsch 21:383–402, 1967.
13. Anonymous: Diagnostic procedures for the confirmation and differential diagnosis or African swine fever. Commission of the European Communities—Directorate-general for Agriculture, vol VI/1796/95–EN (PVET/EN/3160); 1995.
14. Noda S, Eizuru Y, Minamishima Y, Ikenoue T, Mori N: Detection of human T-cell lymphotropic virus type 1 infection by the polymerase chain reaction using dried blood specimens on filter papers. J Virol Methods 43:111–122, 1993.
15.
De Swart RL, Nur Y, Abdallah A, et al: Combination of reverse
transcriptase PCR analysis and immunoglobulin M detection on filter
paper blood samples allows diagnostic and epidemiological studies of
measles. J Clin Microbiol 39:270–273, 2001.
Figure
1. Specific
band amplified from ASFV DNA from fresh whole blood. M, marker; 1 to
6:1:10-5 blood dilutions; 7, control.
M 1 2 3 4 5 6 7 M
320 pb
242 pb
278 pb
Figure 2. Specific bands amplified
from ASFV DNA in dried blood filter paper stored 24 hours (A); 1, 2,
and 3 weeks (B, C, and D); and 3 months (E) at1 :10-3 dilutions
(1–4).
278
pb
Table
1. Detection
of ASFV Genomic DNA by PCR Dfter Dry Storage, on Filter Papers, of Malagasy
Pig Blood Samples Collected From Different Areas
Nos. Areas Origine PCR Positive/Total
1 Antananarivo Ferme Ecole Tombotsoa Positive
2 Antananarivo Ferme Ecole Tombotsoa Positive 5/6
3 Antananarivo Ferme Ecole Tombotsoa Negative
4 Antananarivo Ferme Ecole Tombotsoa Positive
5 Antananarivo Ferme Ecole Tombotsoa Positive
6 Antananarivo Ferme Ecole Tombotsoa Positive
7 Toamasina Abattoir Andodabe Negative
8 Toamasina Abattoir Andodabe Positive
9 Toamasina Abattoir Andodabe Positive 3/5
10 Toamasina Abattoir Andodabe Negative
11 Toamasina Abattoir Andodabe Positive
12 Toamasina Abattoir Brickaville Positive 1/1
13 Toamasina Abattoir Fénérive-Est Positive
14 Toamasina Abattoir Fénérive-Est Positive
15 Toamasina Abattoir Fénérive-Est Negative 3/6
16 Toamasina Abattoir Fénérive-Est Negative
17 Toamasina Abattoir Fénérive-Est Positive
18 Toamasina Abattoir Fénérive-Est Negative
19 Toamasina Abattoir Ivoloina Toamasina Negative
20 Toamasina Abattoir Ivoloina Toamasina Negative
21 Toamasina Abattoir Ivoloina Toamasina Negative
22 Toamasina Abattoir Ivoloina Toamasina Positive
23 Toamasina Abattoir Ivoloina Toamasina Positive
24 Toamasina Abattoir Ivoloina Toamasina Positive 6/11
25 Toamasina Abattoir Ivoloina Toamasina Positive
26 Toamasina Abattoir Ivoloina Toamasina Positive
27 Toamasina Abattoir Ivoloina Toamasina Positive
28 Toamasina Abattoir Ivoloina Toamasina Negative
29 Toamasina Abattoir Ivoloina Toamasina Negative
30 Toamasina Abattoir Mahanoro Negative
31 Toamasina Abattoir Mahanoro Positive
32 Toamasina Abattoir Mahanoro Negative
33 Toamasina Abattoir Mahanoro Positive
34 Toamasina Abattoir Mahanoro Positive
35 Toamasina Abattoir Mahanoro Negative
36 Toamasina Abattoir Mahanoro Negative 8/13
37 Toamasina Abattoir Mahanoro Positive
38 Toamasina Abattoir Mahanoro Positive
39 Toamasina Abattoir Mahanoro Positive
40 Toamasina Abattoir Mahanoro Positive
41 Toamasina Abattoir Mahanoro Positive
42 Toamasina Abattoir Mahanoro Negative
43 Toamasina Abattoir Tamatave Positive
44 Toamasina Abattoir Tamatave Positive
45 Toamasina Abattoir Tamatave Negative
46 Toamasina Abattoir Tamatave Negative
47 Toamasina Abattoir Tamatave Positive
48 Toamasina Abattoir Tamatave Positive 7/13
49 Toamasina Abattoir Tamatave Negative
50 Toamasina Abattoir Tamatave Negative
51 Toamasina Abattoir Tamatave Negative
52 Toamasina Abattoir Tamatave Negative
53 Toamasina Abattoir Tamatave Positive
54 Toamasina Abattoir Tamatave Positive
55 Toamasina Abattoir Tamatave Positive
56 Toamasina Abattoir Vatomandry Negative
57 Toamasina Abattoir Vatomandry Positive
58 Toamasina Abattoir Vatomandry Negative
59 Toamasina Abattoir Vatomandry Negative 1/7
60 Toamasina Abattoir Vatomandry Negative
61 Toamasina Abattoir Vatomandry Negative
62 Toamasina Abattoir Vatomandry Negative
ISSN# 1542-2666