Lohse, Louise

Nielsen, Jens

Rasmussen, Thomas Bruun

Uttenthal, Åse

 To evaluate the diagnostic potential of meat juice for early detection of Classical swine fever virus (CSFV), meat juice and serum samples from pigs experimentally infected with different strains of CSFV were compared for virus load. From all samples, viral RNA was extracted by automated procedure before real-time reverse transcription polymerase chain reaction analysis was performed. Viral RNA was detected in meat juice, but at a lower level than in corresponding serum. Sensitivity was calculated to 91% and specificity to 97%. Disagreements between meat juice and serum results were found when samples originated from pigs infected with low virulence CSFV strains and/or when samples were collected within the first days after infection. In conclusion, while not the first choice for sample material for CSFV diagnosis, meat juice may constitute a useful alternative for herd-based studies or when blood and/or target organ material is not available. Strain virulence and time points for sample collection after infection are factors of importance for diagnostic success. </jats:p

Louise Lohse

Åse Uttenthal

Thomas Bruun Rasmussen

Jens Nielsen

Crossref

Journal of Veterinary Diagnostic Investigation

Diagnostic value of meat juice in early detection of <i>Classical swine fever virus</i> infection

To evaluate the diagnostic potential of meat juice for early detection of Classical swine fever virus (CSFV), meat juice and serum samples from pigs experimentally infected with different strains of CSFV were compared for virus load. From all samples, viral RNA was extracted by automated procedure before real-time reverse transcription polymerase chain reaction analysis was performed. Viral RNA was detected in meat juice, but at a lower level than in corresponding serum. Sensitivity was calculated to 91% and specificity to 97%. Disagreements between meat juice and serum results were found when samples originated from pigs infected with low virulence CSFV strains and/or when samples were collected within the first days after infection. In conclusion, while not the first choice for sample material for CSFV diagnosis, meat juice may constitute a useful alternative for herd-based studies or when blood and/or target organ material is not available. Strain virulence and time points for sample collection after infection are factors of importance for diagnostic success

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Downloaded from orbit.dtu.dk on: Dec 18, 2017Diagnostic value of meat juice in early detection of classical swine fever infectionLohse, Louise; Uttenthal, Åse; Rasmussen, Thomas Bruun; Nielsen, JensPublished in:Journal of Veterinary Diagnostic InvestigationLink to article, DOI:10.1177/1040638711416855Publication date:2011Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Lohse, L., Uttenthal, Å., Rasmussen, T. B., & Nielsen, J. (2011). Diagnostic value of meat juice in early detectionof classical swine fever infection. Journal of Veterinary Diagnostic Investigation, 23(5), 1005-1008. DOI:10.1177/1040638711416855 http://vdi.sagepub.com/InvestigationJournal of Veterinary Diagnostic http://vdi.sagepub.com/content/23/5/1005The online version of this article can be found at: DOI: 10.1177/1040638711416855 2011 23: 1005J VET Diagn InvestLouise Lohse, Åse Uttenthal, Thomas Bruun Rasmussen and Jens Nielsen infectionClassical swine fever virusDiagnostic value of meat juice in early detection of   Published by: http://www.sagepublications.comOn behalf of:   Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. can be found at:Journal of Veterinary Diagnostic InvestigationAdditional services and information for       http://vdi.sagepub.com/cgi/alertsEmail Alerts:   http://vdi.sagepub.com/subscriptionsSubscriptions:   http://www.sagepub.com/journalsReprints.navReprints:   http://www.sagepub.com/journalsPermissions.navPermissions:   What is This? - Sep 9, 2011Version of Record >>  at Tech Knowl Center - Inst 5500 on September 21, 2011vdi.sagepub.comDownloaded from Journal of Veterinary Diagnostic Investigation23(5) 1005 –1008© 2011 The Author(s)Reprints and permission: sagepub.com/journalsPermissions.navDOI: 10.1177/1040638711416855http://jvdi.sagepub.comClassical swine fever (CSF) is a serious, highly contagious disease in swine. The causative agent is Classical swine fever virus (CSFV), which is a positive-stranded RNA virus belonging to the Pestivirus genus in the Flaviviridae fam-ily.10 Although many countries in Europe are free from the disease, sporadic or massive new outbreaks are still occa-sionally detected.3,4,7,11 Clinical signs of CSF are not pathog-nomonic, as confirmation of this disease is usually based on laboratory diagnosis. Thus, the availability of blood and/or tissue material is essential. Tissue samples from target organs of CSFV, including tonsils, spleen, kidney, ileum, and lymph nodes, constitute the optimal choice for virus detection (2002, Classical swine fever diagnostic manual, document number C 381, eur-lex.europa.eu 32002D0106 EN; OIE: 2010, Classical swine fever (hog cholera), www.oie.int/eng/normes/mmanual/a_summry.htm). In situations under less optimal conditions, such sample material may not be avail-able. Therefore, the use of alternative materials may be justified.Skeletal muscle tissue can be easily collected for labora-tory examination from abattoir carcasses, wild boars killed in road accidents, and imported meat products. From this material, meat juice can be extracted for further diagnostic analysis. Previous studies have performed pathogen and/or antibody analyses of meat juice for laboratory detection of several infections including salmonellosis, Aujeszky disease, porcine reproductive and respiratory syndrome, trichinello-sis,1,6,8,9 and CSF.5,14 However, the existence of numerous CSFV strains of different virulence make the diagnostic approach for CSFV infection less straightforward, as virus distribution may differ with virulence.2 This fact could influ-ence the suitability of meat juice as a potential diagnostic sample material.In the present study, the diagnostic potential of meat juice for detection of CSFV RNA was tested by examination of skeletal muscle tissue samples from pigs experimentally infected with 7 CSFV strains of different genotypes and viru-lence. The performance of meat juice was compared with contemporary analyses of serum collected from the same animal at the same time points. All material originated from CSFV infection experiments in pigs performed at the National Veterinary Institute (Lindholm, Kalvehave, Denmark) in accordance with the requirements of the Danish Animal Experiments Inspectorate.For evaluation, a real-time reverse transcription poly-merase chain reaction (RT-PCR) assay method in an auto-mated RNA extraction system was used on sampled material from a total of 85 pigs. All samples were collected from pigs at time of euthanasia, which varied from day post-inoculation (dpi) 5–28. For pigs infected with CSFV-Bergen, CSFV-Glentorf, CSFV-Romania, CSFV-Israel, and mock-inoculated control pigs, the experimental design included sequential 416855 XXXXXX10.1177/1040638711416855Lohse et al.Early detection of Classical swine fever virus infectionFrom the National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark. 1Corresponding Author: Louise Lohse, Technical University of Denmark, National Veterinary Institute, Lindholm, DK-4771 Kalvehave, Denmark. loloh@vet.dtu.dkDiagnostic value of meat juice in  early detection of Classical swine  fever virus infectionLouise Lohse,1 Åse Uttenthal, Thomas Bruun Rasmussen, Jens NielsenAbstract. To evaluate the diagnostic potential of meat juice for early detection of Classical swine fever virus (CSFV), meat juice and serum samples from pigs experimentally infected with different strains of CSFV were compared for virus load. From all samples, viral RNA was extracted by automated procedure before real-time reverse transcription polymerase chain reaction analysis was performed. Viral RNA was detected in meat juice, but at a lower level than in corresponding serum. Sensitivity was calculated to 91% and specificity to 97%. Disagreements between meat juice and serum results were found when samples originated from pigs infected with low virulence CSFV strains and/or when samples were collected within the first days after infection. In conclusion, while not the first choice for sample material for CSFV diagnosis, meat juice may constitute a useful alternative for herd-based studies or when blood and/or target organ material is not available. Strain virulence and time points for sample collection after infection are factors of importance for diagnostic success.Key words: Classical swine fever virus; meat juice; reverse transcription polymerase chain reaction; swine. at Tech Knowl Center - Inst 5500 on September 21, 2011vdi.sagepub.comDownloaded from 1006 Lohse et al.killings in groups of 3–4 on dpi 5, 10, and 21/22, respectively, except for individual pigs, which had to be euthanized at an earlier time point for animal welfare reasons. After collection, the sampled materials were prepared for further examination as follows. Serum was obtained by centrifugation of blood collected in plain tubes with no anticoagulant at 237 g for 10 min and then stored at –40°C until use. Meat juice was obtained from samples of the quadriceps muscle. Thus, mus-cle tissue was cut into pieces of approximately 2 cm × 2 cm × 2 cm, put into plastic bags, frozen at –20°C, and followed by gentle thawing at 5°C. Through this process, the released meat juice was collected without further preparation into cryotubes and stored at –40°C until use. For each analysis, 100 µl of sample material (serum or meat juice) was used.The RNA was extracted with a commercial nucleic acid isolation kit,a according to the manufacturer’s instructions using an automated robot.a After RNA extraction, the material was stored at –80°C in the sample cartridge until PCR was carried out. Real time RT-PCR was performed as previously described12 using the primers CSFV6 and CSFV7 together with a CSFV-specific TaqMan probe (5′-FAM-CCCTGG GTGGTCTAAGTCCTGAGTACAG-TAMRA-3′). The reac-tions were cycled,b and, to the fluorescence data obtained, a threshold cycle (Ct) value was assigned to each sample as a measure for the virus quantity. The relative quantity of viral RNA (the viral load) in each sample was expressed directly as Ct values without recalculating the genome equivalents.Data obtained from the serum and meat juice analyses of pigs inoculated with the different CSFV strains were com-pared. Based on clinical records from the performed experi-ments, data sets from individual strains were designated into low, moderate, or high virulence (Table 1). To study the pos-sible association between serum and meat juice, a Spearman rank correlation was calculated on the total data set of inoculated animals, and the correlation coefficient (rs) was determined.The Ct values of serum and meat juice from individual pigs were depicted with relation to infection day in the experiment and illustrated in Figure 1A–C. The results indi-cate that CSFV RNA is detected in meat juice, yet in a lower quantity than in serum. Furthermore, throughout the entire experimental period, negative samples of meat juice as well as serum will occur in a group of pigs infected with a CSFV strain of low virulence (Fig. 1A).All sample results were assigned to either positive (Ct value < 40) or negative (Ct value ≥ 40) value (Table 1), and by comparison, meat juice sensitivity was calculated to 91% and specificity to 97% of serum. The coefficient of concordance for the 2 tested materials was 93%, indicating that a strong correlation exists. Six pairs of samples were in disagreement; in 5 cases, serum tested positive for CSFV RNA while nega-tive in meat juice, and in 1 case, the relationship was reversed. Four of the disagreements were related to strains in the cate-gory of low virulence: 1 sample from a CSFV-Glentorf–infected pig (dpi 5) and 3 samples from CSFV-Bergen–infected pigs (dpi 5/10). Two disagreements were related to the cate-gory of high virulence; both samples were collected from CSFV-Romania–infected pigs (dpi 5). All 13 mock-infected control pigs tested negative in serum as well as in meat juice. The calculated correlation coefficient (rs) was = 0.89, with a 95% confidence interval of (0.82; 0.93) and a P value < 0.0001, which indicates that there is quite a strong positive correlation between the 2 sample materials.In order to address situations where blood and/or target organ material for CSFV diagnosis is not available, the poten-tial of meat juice as diagnostic substrate was evaluated. Previous studies on meat juice and muscular tissue reflect dif-ferent results and diverging opinions regarding the diagnostic Table 1. Diagnostic value of meat juice in early detection of classical swine fever infection: virulence and genotype information of the 7 Classical swine fever virus (CSFV) strains used and results after real-time reverse transcription polymerase chain reaction (RT-PCR) examination of serum and meat juice samples.*Sample material Meat juice SerumVirulence designation Strain Strain GenotypeNo. of pigs examined dpiRT-PCR positiveRT-PCR negativeRT-PCR positiveRT-PCR negativeLow CSFV-Glentorf CSF0911 1.1 10  5–21 2 8 3 7 CSFV-Bergen CSF0906 2.2 10  5–21 4 6 5 5Moderate CSFV-Paderborn CSF0277 2.1 13 15–24 13 0 13 0 CSFV-Eystrup CSF0910 1.1 10  8–28 5 5 5 5High CSFV-Romania CSF1019 2.3 10  5–18 8 2 10 0 CSFV-Kozlov CSF0382 1.1  9 6–8 9 0 9 0 CSFV-Israel CSF1047 2.1 10  5–11 10 0 10 0Control Mock inoculated NR NR 13  5–28 0 13 0 13Total 85 51 34 55 30*dpi = day post-infection; NR = not relevant. Strain ID and genotype designation is obtained from the CSFV database from The European Central Reference Laboratory. RT-PCR-positive and -negative results indicate threshold cycle <40 and ≥40, respectively. at Tech Knowl Center - Inst 5500 on September 21, 2011vdi.sagepub.comDownloaded from  Early detection of Classical swine fever virus infection 1007value of this material.5,13,14 Based on the present study with CSFV strains of different virulence, the levels of CSFV RNA in meat juice and corresponding serum samples were com-pared. By comparison of data from all inoculated pigs, an overall sensitivity of 91% for detection of CSFV in meat juice was calculated. The results obtained in the current study sup-port previous work,5 which concluded that meat juice is most suitable as substrate for diagnosis of CSFV from pigs infected with strains of high or moderate virulence. In low virulence CSFV infections, virus detection in meat juice as well as in other nonlymphatic organs can be problematic due to low virus load and sparse tissue distribution in the infected pig. Therefore, for low virulent strains, demonstration of serum antibodies several weeks after infection may be of diagnostic value. Furthermore, the results suggest that time point after infection has an important effect in sample collection, as some virus strains, even when classified as highly virulent, will not be disseminated to non–virus-targeted organs within the body of the pig until the end of the first week after infection. Such a problem was encountered in pigs inoculated with CSFV-Romania, where demonstration of viral RNA in meat juice only was possible in 1 out of 3 pigs at dpi 5. Corresponding serum samples were all positive for viral RNA, indicating ongoing viremia. The finding does not correspond with con-clusions from a Swiss study,13 which could not demonstrate any influence of time point of infection for sample collection for detection of CSFV RNA. However, the results from the Swiss and the present study are not directly comparable as the presented data are based on homogenized muscle tissue and meat juice, respectively. In a previous Danish study,14 a com-parison of virus detection in meat juice and corresponding homogenized quadriceps muscle tissue revealed lower Ct val-ues in meat juice than in corresponding muscle tissue samples, indicating that a higher RT-PCR sensitivity for the former sample material exists. The finding is consistent with the Swiss study13 that could not recommend muscular tissue as diagnos-tic material for CSFV, regardless of strain virulence, and found that only 58% of the tested samples were CSFV positive by RT-PCR compared with parallel examination of target organs. It is speculated whether the lower sensitivity of muscle tissue samples compared with meat juice might be due to PCR inhib-iting factors released by tissue homogenization or dilution of virus content in the muscle tissue preparation process.In conclusion, the results from the present study indicate that the use of meat juice as diagnostic material for CSFV infection is justified under certain conditions (e.g., when used as material for herd-based screening). However, meat juice cannot be rec-ommended as an isolated diagnostic material for early detection of CSFV infection in individual pigs when blood and/or target organ material is available, as the diagnostic sensitivity for meat juice is lower than that for serum. Potential use of meat juice as a reliable diagnostic sample will depend on the actual strain virulence and on the relative time of sample collection in the specific disease situation.Figure 1. Threshold cycle (Ct) values obtained from examined sample material after real-time reverse transcription polymerase chain reaction procedure. Depicted Ct values are shown for individual pigs and related to day post-infection (PID). Results from the individual groups of pigs inoculated with different Classical swine fever virus strains are designated into categories of virulence (A–C), based on clinical records. ∆ = meat juice; × = serum. at Tech Knowl Center - Inst 5500 on September 21, 2011vdi.sagepub.comDownloaded from 1008 Lohse et al.AcknowledgementsThe authors thank MajBritt Eicke for excellent technical assistance.Sources and manufacturersa. MagNA Pure LC Total Nucleic Acid Isolation kit, Roche A/S, Hvidovre, Denmark.b. MX4000, Stratagene, AH-diagnostics, Aarhus, Denmark.Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.FundingThe current study was financially supported by Directorate for Food, Fisheries and Agri Business in Denmark, grant no. 2007- 776.References 1. Beck R, Gaspar A, Mihaljević Z, et al.: 2005, Evaluation of ELISA for detection of Trichinella antibodies in meat juice samples of naturally infected pigs. Vet Parasitol 132:91–95. 2. Belák K, Koenen F, Vanderhallen H, et al.: 2008, Compara-tive studies on the pathogenicity and tissue distribution of three virulence variants of classical swine fever virus, two field iso-lates and one vaccine strain, with special regard to immunohis-tochemical investigations. Acta Vet Scand 50:34. 3. Elber AR, Stegeman A, Moser H, et al.: 1999, The classical swine fever epidemic 1997–1998 in The Netherlands: descrip-tive epidemiology. Prev Vet Med 42:157–184. 4. Fritzemeier J, Teuffert J, Greiser-Wilke I, et al.: 2000, Epide-miology of classical swine fever in Germany in the 1990s. Vet Microbiol 77:29–41. 5. Kaden V, Lange E, Nagel-Kohl U, Bruer W: 2009, Meat juice as diagnostic sample for virological and serological diagnosis of clas-sical swine fever. Dtsch Tierarztl Wochenschr 116:173–179. 6. Le Potier MF, Fournier A, Houdayer C, et al.: 1998, Use of muscle exudates for detection of anti-gE antibodies to Aujesz-ky’s disease virus. Vet Rec 143:385–387. 7. Moennig V, Floegel-Niesmann G, Greiser-Wilke I: 2003, Clini-cal signs and epidemiology of classical swine fever: a review of new knowledge. Vet J 165:11–20. 8. Mortensen S, Strandbygaard B, Bøtner A, et al.: 2001, Moni-toring porcine reproductive and respiratory syndrome virus infection status in swine herds based on analysis of antibodies in meat juice. Vet Res 32:441–453. 9. Nielsen B, Ekeroth L, Bager F, Lind P: 1998, Use of muscle fluid as a source of antibodies for serologic detection of Sal-monella infection in slaughter pig herds. J Vet Diagn Invest 10:158–163. 10. Paton DJ, McGoldrick A, Greiser-Wilke I, et al.: 2000, Genetic typing of classical swine fever virus. Vet Microbiol 73: 137–157. 11. Pol F, Rossi S, Mesplède A, et al.: 2008, Two outbreaks of clas-sical swine fever in wild boar in France. Vet Rec 162:811–816. 12. Rasmussen TB, Uttenthal Å, Reimann I, et al.: 2007, Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus. J Gen Virol 88:481–486. 13. Thür B, Hofmann MA: 1998, Comparative detection of clas-sical swine fever virus in striated muscle from experimentally infected pigs by reverse transcription polymerase chain reac-tion, cell culture isolation and immunohistochemistry. J Virol Methods 74:47–56. 14. Uttenthal A, Storgaard T, Oleksiewicz MB, de Stricker K: 2003, Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA. Vet Microbiol 92:197–212. at Tech Knowl Center - Inst 5500 on September 21, 2011vdi.sagepub.comDownloaded from 

Diagnostic value of meat juice in early detection of classical swine fever infection

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Diagnostic value of meat juice in early detection of classical swine fever infection

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