Veterinary Microbiology 174 (2014) 607–608
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Letter to the Editor Alternative sampling strategies for passive classical and African swine fever surveillance in wild boar – Extension towards African swine fever virus antibody detection To the Editor, We recently reported on the use of blood swab samples for passive classical and African swine fever (CSF and ASF) surveillance in wild boar (Petrov et al., in press). Upon availability of the article online, we were asked by national and international colleagues whether this approach would be suitable also for antibody detection. While antibody detection might not be the primary focus of diagnostic investigations in fallen animals, we think that an approach that would allow both pathogen and antibody detection in one easy-to-collect sample matrix, combined with simple shipment, and long-term storage, would be optimal under field conditions. For this reason, we tried to rapidly answer this question on a limited set of sero-positive and sero-negative blood samples from animal experiments. Given the current epidemiological situation of ASF in the European Union wild boar population (cases in several Eastern Member States with a tendency to spread, see OIE WAHID), we feel that the audience of Veterinary Microbiology would benefit from a brief addendum to the above-mentioned article. For this reason, we report on the outcome of our initial studies here, while further validation is still in progress. 1. Study design and outcome A total of 42 porcine EDTA blood samples were employed to test applicability of swab fragments for antibody detection. The expected status of the blood sample was related to the corresponding serum sample of the same animal and sampling day. The result of the p72 antibody ELISA (Ingezim PPA Compac, Ingenasa, Madrid, Spain) was used as a reference. Genotube swabs (Prionics, Zurich, Switzerland), were dipped into the respective blood sample and left to dry for at least 12 h at room temperature. Thereafter, diamond-shaped fragments (app. 5 mm lateral length) were cut with sterile scissors and transferred to the ELISA system. To test samples close to the ‘‘worst-case-scenario’’, 30 samples were included that http://dx.doi.org/10.1016/j.vetmic.2014.09.018 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
had been stored for more than 21 months at 4 8C. This set comprised 12 samples from sero-negative animals, and 18 samples from sero-positive animals. The latter had been immunized twice with an inactivated preparation of genotype II ASFV Armenia08 (Blome et al., 2014). Samples were included from days 28 to 41 post immunization. The second set of samples comprised animals that had been inoculated with ASFV OURT88/3 (genotype I, non-hemadsorbing). These samples (n = 10) had been taken 29 days post inoculation and were stored approximately 1 month at 4 8C. Also here, negative animals (n = 2) were included. To compare the performance with dried blood on filter papers as foreseen in the ELISA protocol (see below), we tested 14 samples also on this matrix (the second set of samples and two long-term storage samples, see Table 1). The commercially available ID Screen1 African Swine Fever Indirect antibody ELISA (ID.vet, Grabels, France) allows a protocol for dried blood on filter papers. We used this protocol to test the swab fragments. The original protocol foresees the use of two filter paper punches with a diameter of 6 mm. We replaced them with two of the abovementioned Genotube fragments and performed all subsequent steps according to the manufacturer’s instructions. Based on the above-mentioned set of samples, we could clearly demonstrate that antibody detection is possible also from Genotube swabs (see Table 1). Fourty out of 42 samples were in complete agreement with the serological status, and an additional sample that had a doubtful status was detected positive. Only one doubtful sample gave a negative result. Comparison of dried blood on filter paper and on Genotube swabs gave similar results (see Table 1), also in terms of raw data values (data not shown). No false positive reactions occurred, even with samples stored for several months (see Table 1). Despite the fact, that further validation is clearly needed and ongoing, these initial results are most promising and could prompt the inclusion of antibody detection from swabs in the field. Due to the very high virulence of the ASFV strains currently circulating in Eastern Europe (Gabriel et al., 2012; Blome et al., 2013), antibody detection is still a rather rare finding. However, to obtain a full picture of the epidemiological situation, and to fulfil all legal requirements (e.g. Commission Decision 2003/422/EC), the search for antibodies is mandatory. Another important issue would be to isolate the causative virus strains for further characterization. In this respect,
Letter to the Editor / Veterinary Microbiology 174 (2014) 607–608
608
Table 1 EDTA blood sample details and results. The status of the sample was defined by a p72 antibody ELISA (Ingezim PPA Compac, Ingenasa) of the corresponding serum sample. The storage time is depicted in month (M). DPI = days post inoculation; neg = negative according to the test criteria; dbt = doubtful according to the test criteria; pos = positive according to the test criteria; nd = not done; inact. = inactivated. Genotube
Animal ID
DPI
Storage
Virus
Status
Result swab
Result filter
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
HS1 HS2 HS3 HS4 HS5 HS6 HS7 HS8 HS9 HS11 HS12 HS13 HS3 HS4 HS6 HS7 HS8 HS9 HS11 HS8 HS12 HS13 HS4 HS6 HS7 HS8 HS9 HS11 HS12 HS13 HS1 HS2 HS3 HS4 HS5 HS6 HS7 HS8 HS9 HS10 HS1 HS2
0 0 0 0 0 0 0 0 0 0 0 0 28 28 28 28 28 28 28 35 28 28 41 41 41 41 41 41 41 41 29 29 29 29 29 29 29 29 29 29 0 0
21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 1M 1M 1M 1M 1M 1M 1M 1M 1M 1M 1M 1M
– – – – – – – – – – – – Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 – –
neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg pos pos pos pos pos pos pos dbt dbt pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos neg neg
neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg pos pos pos pos pos pos pos neg pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos neg neg
nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd pos pos pos pos pos pos pos pos pos pos pos pos neg neg
preliminary studies showed that ASFV isolation from Genotube swabs was possible in blood monocyte derived macrophage cultures while CSFV could not be isolated (data not shown). Probably, the latter could be obtained from RNA transfection. Easy sampling and testing by using swabs for both pathogen and antibodies could facilitate this task and present a pragmatic approach also for other scenarios, e.g. for wild-life monitoring in Africa. References Blome, S., Gabriel, C., Beer, M., 2014. Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation. Vaccine 32 (June (31)), 3879–3882. Blome, S., Gabriel, C., Dietze, K., Breithaupt, A., Beer, M., 2012. High virulence of African swine fever virus caucasus isolate in European wild boars of all ages. Emerg. Infect. Dis. 18, 708. Gabriel, C., Blome, S., Malogolovkin, A., Parilov, S., Kolbasov, D., Teifke, J.P., Beer, M., 2011. Characterization of African swine fever virus caucasus isolate in European wild boars. Emerg. Infect. Dis. 17, 2342–2345.
inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact.
Petrov, A., Schotte, U., Pietschmann, J., Dra¨ger, C., Beer, M., Goller, K.V., Blome, S., 2014. Alternative sampling strategies for passive classical and African swine fever surveillance in wild boar. Vet. Microbiol., http://dx.doi.org/10.1016/j.vetmic.2014.07.030.
Sandra Blome* Katja V. Goller Anja Petrov Carolin Dra¨ger Jana Pietschmann Martin Beer Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald - Insel Riems, Germany *Corresponding
author. Tel.: +49 38351 71144; fax: +49 38351 71275 E-mail address: sandra.blome@fli.bund.de
30 August 2014
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Letter to the Editor Alternative sampling strategies for passive classical and African swine fever surveillance in wild boar – Extension towards African swine fever virus antibody detection To the Editor, We recently reported on the use of blood swab samples for passive classical and African swine fever (CSF and ASF) surveillance in wild boar (Petrov et al., in press). Upon availability of the article online, we were asked by national and international colleagues whether this approach would be suitable also for antibody detection. While antibody detection might not be the primary focus of diagnostic investigations in fallen animals, we think that an approach that would allow both pathogen and antibody detection in one easy-to-collect sample matrix, combined with simple shipment, and long-term storage, would be optimal under field conditions. For this reason, we tried to rapidly answer this question on a limited set of sero-positive and sero-negative blood samples from animal experiments. Given the current epidemiological situation of ASF in the European Union wild boar population (cases in several Eastern Member States with a tendency to spread, see OIE WAHID), we feel that the audience of Veterinary Microbiology would benefit from a brief addendum to the above-mentioned article. For this reason, we report on the outcome of our initial studies here, while further validation is still in progress. 1. Study design and outcome A total of 42 porcine EDTA blood samples were employed to test applicability of swab fragments for antibody detection. The expected status of the blood sample was related to the corresponding serum sample of the same animal and sampling day. The result of the p72 antibody ELISA (Ingezim PPA Compac, Ingenasa, Madrid, Spain) was used as a reference. Genotube swabs (Prionics, Zurich, Switzerland), were dipped into the respective blood sample and left to dry for at least 12 h at room temperature. Thereafter, diamond-shaped fragments (app. 5 mm lateral length) were cut with sterile scissors and transferred to the ELISA system. To test samples close to the ‘‘worst-case-scenario’’, 30 samples were included that http://dx.doi.org/10.1016/j.vetmic.2014.09.018 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
had been stored for more than 21 months at 4 8C. This set comprised 12 samples from sero-negative animals, and 18 samples from sero-positive animals. The latter had been immunized twice with an inactivated preparation of genotype II ASFV Armenia08 (Blome et al., 2014). Samples were included from days 28 to 41 post immunization. The second set of samples comprised animals that had been inoculated with ASFV OURT88/3 (genotype I, non-hemadsorbing). These samples (n = 10) had been taken 29 days post inoculation and were stored approximately 1 month at 4 8C. Also here, negative animals (n = 2) were included. To compare the performance with dried blood on filter papers as foreseen in the ELISA protocol (see below), we tested 14 samples also on this matrix (the second set of samples and two long-term storage samples, see Table 1). The commercially available ID Screen1 African Swine Fever Indirect antibody ELISA (ID.vet, Grabels, France) allows a protocol for dried blood on filter papers. We used this protocol to test the swab fragments. The original protocol foresees the use of two filter paper punches with a diameter of 6 mm. We replaced them with two of the abovementioned Genotube fragments and performed all subsequent steps according to the manufacturer’s instructions. Based on the above-mentioned set of samples, we could clearly demonstrate that antibody detection is possible also from Genotube swabs (see Table 1). Fourty out of 42 samples were in complete agreement with the serological status, and an additional sample that had a doubtful status was detected positive. Only one doubtful sample gave a negative result. Comparison of dried blood on filter paper and on Genotube swabs gave similar results (see Table 1), also in terms of raw data values (data not shown). No false positive reactions occurred, even with samples stored for several months (see Table 1). Despite the fact, that further validation is clearly needed and ongoing, these initial results are most promising and could prompt the inclusion of antibody detection from swabs in the field. Due to the very high virulence of the ASFV strains currently circulating in Eastern Europe (Gabriel et al., 2012; Blome et al., 2013), antibody detection is still a rather rare finding. However, to obtain a full picture of the epidemiological situation, and to fulfil all legal requirements (e.g. Commission Decision 2003/422/EC), the search for antibodies is mandatory. Another important issue would be to isolate the causative virus strains for further characterization. In this respect,
Letter to the Editor / Veterinary Microbiology 174 (2014) 607–608
608
Table 1 EDTA blood sample details and results. The status of the sample was defined by a p72 antibody ELISA (Ingezim PPA Compac, Ingenasa) of the corresponding serum sample. The storage time is depicted in month (M). DPI = days post inoculation; neg = negative according to the test criteria; dbt = doubtful according to the test criteria; pos = positive according to the test criteria; nd = not done; inact. = inactivated. Genotube
Animal ID
DPI
Storage
Virus
Status
Result swab
Result filter
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
HS1 HS2 HS3 HS4 HS5 HS6 HS7 HS8 HS9 HS11 HS12 HS13 HS3 HS4 HS6 HS7 HS8 HS9 HS11 HS8 HS12 HS13 HS4 HS6 HS7 HS8 HS9 HS11 HS12 HS13 HS1 HS2 HS3 HS4 HS5 HS6 HS7 HS8 HS9 HS10 HS1 HS2
0 0 0 0 0 0 0 0 0 0 0 0 28 28 28 28 28 28 28 35 28 28 41 41 41 41 41 41 41 41 29 29 29 29 29 29 29 29 29 29 0 0
21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 21 M 1M 1M 1M 1M 1M 1M 1M 1M 1M 1M 1M 1M
– – – – – – – – – – – – Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 Armenia08 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 OURT88/3 – –
neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg pos pos pos pos pos pos pos dbt dbt pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos neg neg
neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg pos pos pos pos pos pos pos neg pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos neg neg
nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd pos pos pos pos pos pos pos pos pos pos pos pos neg neg
preliminary studies showed that ASFV isolation from Genotube swabs was possible in blood monocyte derived macrophage cultures while CSFV could not be isolated (data not shown). Probably, the latter could be obtained from RNA transfection. Easy sampling and testing by using swabs for both pathogen and antibodies could facilitate this task and present a pragmatic approach also for other scenarios, e.g. for wild-life monitoring in Africa. References Blome, S., Gabriel, C., Beer, M., 2014. Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation. Vaccine 32 (June (31)), 3879–3882. Blome, S., Gabriel, C., Dietze, K., Breithaupt, A., Beer, M., 2012. High virulence of African swine fever virus caucasus isolate in European wild boars of all ages. Emerg. Infect. Dis. 18, 708. Gabriel, C., Blome, S., Malogolovkin, A., Parilov, S., Kolbasov, D., Teifke, J.P., Beer, M., 2011. Characterization of African swine fever virus caucasus isolate in European wild boars. Emerg. Infect. Dis. 17, 2342–2345.
inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact. inact.
Petrov, A., Schotte, U., Pietschmann, J., Dra¨ger, C., Beer, M., Goller, K.V., Blome, S., 2014. Alternative sampling strategies for passive classical and African swine fever surveillance in wild boar. Vet. Microbiol., http://dx.doi.org/10.1016/j.vetmic.2014.07.030.
Sandra Blome* Katja V. Goller Anja Petrov Carolin Dra¨ger Jana Pietschmann Martin Beer Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald - Insel Riems, Germany *Corresponding
author. Tel.: +49 38351 71144; fax: +49 38351 71275 E-mail address: sandra.blome@fli.bund.de
30 August 2014
