| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Brief Communication |
Correspondence: 1Corresponding Author: Jens P. Teifke, Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Südufer 10, D-17493 Greifswald-Insel Riems, Germany, e-mail: jens.teifke{at}fli.bund.de
 |
Abstract |
|---|
 TOPSources and manufacturers Abstract References |
|---|
Key Words: Biopsy • classical swine fever virus • diagnosis • hog cholera • pig • RNA • skin
Classical swine fever (CSF, hog cholera) is a highly contagious and frequently fatal viral disease of domestic pigs and wild boars (feral pigs). The causative agent is a small enveloped virus with a 12.5 kb single-stranded RNA genome of positive polarity. CSF virus (CSFV) belongs to the genus Pestivirus in the family Flaviviridae.22
CSF currently has a worldwide distribution and sporadic outbreaks have commonly been recorded in the European Union (EU) during the last decade, not only in domestic pigs but also in wild boar.9,12,14 The latter have often been the source of CSF outbreaks in domestic pig farms in Germany.7,14 An early, rapid, highly sensitive and precise laboratory diagnosis is very important for detection of infected herds in the context of CSF control. The quality and diagnostic reliability of the detection method used for determination of infected animals is therefore crucial. According to the Diagnostic Manual of the EU1 and the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: Mammals, Birds and Bees of the World Organization for Animal Health (OIE)15 the virological methods used for confirmation of CSF are 1) virus isolation in susceptible cell cultures (which still is the "gold standard"), 2) immunohistochemical detection of virus antigen by fluorescent antibody test (FAT) on cryosections of tissue samples, 3) antigen ELISA, and (4) reverse transcription-polymerase chain reaction (RT-PCR) for detection of viral RNA.
The primary replication of CSFV takes place in the tonsils after natural oronasal infection. From here virus spreads to the mandibular and retropharyngeal lymph nodes via peripheral blood to bone marrow, visceral lymph nodes, and lymphatic tissues in spleen, kidneys, and small intestine (Peyer's patches).13,18,19 The virus replicates in a broad range of tissues but affects primarily the immune system, resulting in a generalized leukopenia. It was demonstrated that skin epithelial cells are also targeted by CSFV in the early period after experimental infection.3 Regarding infection of the skin, the presence and replication of related pestiviruses is proven in ruminants, as shown for bovine viral diarrhea virus (BVDV) in cattle or border disease virus (BDV) in sheep.2,17,21 Therefore, the objective of this study was to evaluate the skin as an additional sample for CSF diagnosis.
To this end, skin biopsy specimens (8-mm-diameter biopsy punch)a from wild boars (n = 6) and domestic pigs (n = 2), which were infected oronasally with 1-ml whole blood of the highly virulent CSFV strain Koslov (106 TCID50/ml), were investigated on day 4 and 7 postinfection (wild boar no. 73 and domestic pig no. 64) and 9 days postinfection (DPI) when clinical signs were present and domestic pig no. 64 died (Table 1). Subsequently, 5 domestic pigs were challenged oronasally with 1-ml cell culture supernatant of the CSFV field isolate "935" (genotype 2.3 Uelzen) derived from wild boar shot in Lower Saxony. The infectious dose contained 106.25 TCID50. The skin samples were collected in a series on different DPI and on the day of death (details see Table 2). For all animals, skin punch biopsies were taken from the dorsolateral region of the neck near the base of the ear.
|
|
For detection of CSFV RNA, a real-time RT-PCR (rtRT-PCR) protocol established for routine intra vitam diagnosis and validated in the National Reference Laboratory for CSF was used as described earlier.5,8 Briefly, RNA was extracted from homogenized skin biopsies using RNeasy® Mini Kitd according to the manufacturer's instructions and modified by addition of an internal RNA control after the lysis. For rtRT-PCR, the commercially available QuantiTect Probe RT-PCR Kitd was used. Per single well 3.25 µl RNase-free water, 12.5 µl 2x QuantiTectTM Probe RT-PCR master mix, 0.25 µl QuantiTectTM Probe RT mix, 2 µl Enhanced Green Fluorescent Protein (EGFP) mix (limit 5), and 2 µl CSF mix 1 were pooled. The EGFP mix and the CSF mix represent primer–probe mixes with the following sequences: CSF mix 1: CSF100F (100 pmol/µl) 5'-ATG CCC AYA GTA GGA CTA GCA-3', CSF192R (100 pmol/µl) 5'-CTA CTG ACG ACT GTC CTG TAC-3', CSF Probe 1 (249 pmol/µl) 5'-FAM-TGG CGA GCT CCC TGG GTG GTC TAA GT-TAMRA-3'; EGFP mix 1: EGFP1-F (100 pmol/µl) 5'-GAC CAC TAC CAG CAG AAC AC-3', EGFP2-R (100 pmol/µl) 5'-GAA CTC CAG CAG GAC CAT G-3', EGFP Probe 1(282 pmol/µl) 5'-HEX-AGC ACC CAG TCC GCC CTG AGC A-BHQ1-3'. Finally, 5 µl RNA template of the sample were added. The rtRT-PCR was carried out in the iCycler Mx3000P® QPCR System.e A sample was assessed as positive when a threshold cycle (ct) value of less than 40 was recorded.
For detection of virus antigen by direct FAT, skin biopsies were snap-frozen in n-heptane. After washing at room-temperature with phosphate-buffered saline (PBS) for 5 minutes, sections were incubated for 60 minutes with 1:50 diluted fluorescein-isothiocyanate (FITC)-labeled anti-CSF monoclonal antibody BioXf mixed at a ratio of 3:1 with 0.005% Evans blueg to reduce background autofluorescence. For three-color-immunofluorescence, polyclonal rabbit anti-human von Willebrand factorh (1:500 in PBS) and the anti-CSF BioX were applied. Indocarbocyanin (Cy3)-conjugated goat anti-rabbit antibodyi (1:1,000 in PBS) was used as secondary antibody. To stain nuclear chromatin, TO-PRO-3 iodidej (1:2,000 in PBS) was added during secondary antibody incubation. To preserve fluorescence, sections were sealed in glycerol buffer containing 1,4-diazobicyclo(2,2,2)-octane (DABCO)g 25 mg/ml and analyzed with a confocal laser-scanning microscope (LSM510).
As positive control for virus isolation, rtRT-PCR and FAT lymphatic tissues from a domestic pig, experimentally infected with CSFV "Alfort 187" and accredited-as-positive reference material were used.20 Skin samples from healthy animals, which were tested free as from pestivirus and related antibodies, served as negative controls.
Sera were tested for antibodies to CSFV by means of a virus neutralization test as indirect neutralizing peroxidase-linked antibody assay as earlier described.11 In an outbreak scenario, it is not only necessary to use highly sensitive, specific, and fast diagnostic methods but also tissues that are representative targets for CSFV and easy to sample. The investigation of skin punch biopsies derived from animals challenged with the Koslov strain revealed that at 4 DPI the rtRT-PCR was already positive for 2 tested animals (ct 28 and 35.8), and 1 wild boar of these was also positive by virus isolation. Six wild boars and 1 domestic pig were tested positive by rtRT-PCR at 9 DPI. In contrast, CSFV was detected in the skin of only 3 wild boars by virus isolation. Remarkably, FAT failed to detect CSFV antigen until this time-point. Although all animals showed clinical signs specific for CSF, distinct skin lesions were not present until 9 DPI. At necropsy 11 DPI, all animals had lesions characteristic for CSF (Table 1).
As CSFV genotype 2.3 Uelzen has been the source of several outbreaks of CSF in domestic pigs and wild boars in Germany during the last 2 decades, skin biopsy specimens were also evaluated for this virus type. In principle, the results are consistent with the observations in the group infected with CSFV Koslov but the course of disease and detection of CSFV was slightly delayed (Table 2). The first virologically positive results were obtained at 7 DPI. The domestic pigs were scored RNA positive compared to 3 pigs that were evaluated positive by virus isolation. All animals were diagnosed positive at 9 DPI in both tests. In contrast, immunostaining on cryosections of skin revealed later detection of CSFV antigen at 11 DPI (1 of 5 pigs, animal no. 22). Staining was present in the vascular plexus of the upper dermis, within spindle shaped mesenchymal cells, and at this time point in a small number of basal keratinocytes only (Fig. 1A). At 9 DPI after infection with CSFV Koslov (Fig. 1B) and in a similar extent at 18 DPI after infection with CSFV genotype 2.3 Uelzen, viral antigen was detected as brilliant homogeneous green fluorescence in the cytoplasm of numerous keratinocytes, cells of the inner and outer root sheath epithelium, hair bulb, vascular endothelium, and intravascular leukocytes. By detection of von Willebrand factor, confocal laser-scanning microscopy showed unambiguously that endothelial cells were infected (Fig. 1C, 1D). This is consistent with histopathological findings that showed endothelial cell swelling and degeneration, and microthrombi in dermal capillaries in this location (data not shown). It is plausible that infection of the epidermis takes place via singular keratinocytes in close proximity of infected capillaries in the upper dermis. In the skin of 4 animals, CSFV antigen was detectable between 15 and 22 DPI. However, animal no. 25 showed no immunostaining, which is in contrast to rtRT-PCR and virus isolation. Except for pig no. 21 all other animals developed a low viremia at 4 DPI. Three days later, the content of CSFV in the blood of the infected domestic pigs reached values between 101.75 and 104.0 TCID50/ml (data not shown). Notably, virus isolation was negative in 2 pigs at 15 DPI (pigs nos. 24 and 25) and at 16 DPI (pig no. 25), which had tested positive for CSFV and RNA before. Probably, the moderate neutralizing antibody titers (40 ND50) detected at 15 DPI in these 2 animals were the reason for this negative outcome of virus isolation.
|
In conclusion, the diagnostic investigations of skin punch biopsies by virus isolation, rtRT-PCR, and FAT strongly suggest that skin tissue is a suitable target for detection of CSFV infected animals. The ease of collection antemortem compared to blood sampling makes cutaneous punching an interesting alternative for intra vitam CSF diagnostics. As the diagnostic sensitivity of the CSFV rtRT-PCR assay was higher than that of virus isolation and immunostaining of cryosections, RNA detection by rtRT-PCR should be used for diagnosis of CSF based on skin biopsies. Depending on the virulence of CSFV and the stage of the viremia, a reliable positive diagnosis usually cannot be expected within the first days after infection but is consistent towards the end of the first week postinfection.
|
Acknowledgments |
|---|
|
Sources and manufacturers |
|---|
|
TOPSources and manufacturersAbstractReferences |
|---|
a. Tierärztebedarf Lehnecke, Schortens, Germany.
b. Collection of Cell Lines in Veterinary Medicine, FLI, Riems Island, Germany.
c. Dr. B. Köllner, FLI, Riems Island, Germany.
e. Stratagene, Heidelberg, Germany.
f. Bio-X Diagnostics, Jemelle, Belgium.
g. Sigma-Aldrich, Taufkirchen, Germany.
h. DakoCytomation, Hamburg, Germany.
j. Molecular Probes, Invitrogen, Karlsruhe, Germany.
|
References |
|---|
|
TOPSources and manufacturersAbstractReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
