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ABSTRACT: Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain "Koslov". CP7_E2alf provided clinical protection upon challenge as no severe clinical signs or mortality was observed in the vaccinated piglets. Post mortem examination revealed pathological changes associated to CSFV only in the mock-vaccinated piglets. No infectious CSFV could be isolated from the tonsils of the vaccinated piglets. Two weeks after vaccination at the time of challenge, the vaccinated piglets only, had an increase in the ELISA antibody titer. Interestingly, the maternally derived immunity in the mock-vaccinated control piglets seems to neutralize the challenge virus. Thus, the previously observed 100% mortality in naïve (negative for antibodies to CSFV) piglets infected with CSFV Koslov was reduced in the control piglets of this study to 30% for challenge at the age of 7weeks and 50% at the age of 10weeks, respectively. In conclusion, CP7_E2alf proved to be effective in preventing mortality, severe clinical signs and pathological lesions in 5 or 8weeks old piglets positive for maternal antibodies derived from sows vaccinated intramuscularly 4weeks before farrowing with one dose of C-strain vaccine.
Vaccine 08/2012; 30(45):6376-81. · 3.77 Impact Factor
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ABSTRACT: Host-virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population.
Veterinary Microbiology 04/2012; 159(3-4):327-36. · 3.33 Impact Factor
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Bernd Hoffmann,
Sandra Blome,
Paolo Bonilauri,
Jovita Fernández-Piñero,
Irene Greiser-Wilke,
Andy Haegeman,
Mats Isaksson,
Frank Koenen,
Neil LeBlanc,
Immanuel Leifer,
Marie-Frederique Le Potier,
Willie Loeffen,
Thomas Bruun Rasmussen,
Tomasz Stadejek,
Karl Ståhl,
Marylène Tignon, Ase Uttenthal,
Wim van der Poel,
Martin Beer
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ABSTRACT: The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications. In conclusion, the ring trial showed reliability of classical swine fever diagnosis on an international level and helped to optimize CSFV-specific RT-PCR diagnostics.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 09/2011; 23(5):999-1004. · 1.21 Impact Factor
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ABSTRACT: 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.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 09/2011; 23(5):1005-8. · 1.21 Impact Factor
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ABSTRACT: The severity of classical swine fever virus (CSFV) infection is believed to be determined by different factors, including the virulence of the strain as well as factors related to the host. In the present study, we infected 6- and 11-week-old pigs of unique sanitary status with CSFV strain Eystrup to elucidate the influence of age on virulence. In both age-groups, a mild clinical course correlated well with the gross-pathological findings at necropsy. The minor variations of clinical, pathological, haematological and immunological parameters between the various age-groups demonstrated that a time-span of approximately 1 month of age did not play a significant role for the severity of CSF disease in young, weaned pigs. The detailed analysis of various haematological and cellular immunological parameters proved to provide a valuable set of objective reference values for healthy control pigs and for pigs with mild clinical CSF disease. Despite that only mild disease occurred in the infected pigs, modulations of haematological and immunological parameters were observed. Depletion of B cell and a number of T cell populations in peripheral blood was observed in both age-groups, however, the changes being most pronounced in the 6-week-old pigs. In the infected pigs, but not in any of the controls, a population of large granulocytes (LG) developed in peripheral blood. The LG, which were demonstrated to be identical to low-density granulocytes, appeared before the development of viraemia. Therefore, we suggest detection of LDG to be used as an additional tool in early CSF diagnosis. The observation that pigs with a unique, high sanitary status only developed mild disease after infection with CSFV strain Eystrup emphasizes the important role of the host in the CSFV virulence puzzle.
Veterinary Immunology and Immunopathology 12/2010; 138(3):159-73. · 2.08 Impact Factor
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ABSTRACT: Aleutian mink disease virus (AMDV) is a severe progressive disease causing multiple different clinical syndromes in mink. In Denmark, the disease is notifiable and under official control. The control programme, based on serological screening, has confined successfully AMDV to the northern part of Denmark. However, re-infections and new introductions of virus into farms require a confirmatory virological test to verify the positive test results of single animals and ultimately to investigate disease transmission. A one step PCR amplifying a 374-base fragment of the NS1 gene of AMDV was compared to the counter-current immune electrophoresis (CIE) routinely used in the serological screening programme. Mink organs (n=299) obtained from 55 recently infected farms and 8 non-infected farms from 2008 to 2010 were tested by PCR, and the results were found to have a high correlation with the serological status of the mink. The relative diagnostic sensitivity of the PCR was 94.7%, and the relative diagnostic specificity was 97.9% when read in parallel with the CIE. PCR positive samples were sequenced and phylogenetic analysis revealed high similarity within the analysed AMDV strains and to AMDV strains described previously.
Journal of virological methods 10/2010; 171(1):81-5. · 2.13 Impact Factor
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ABSTRACT: The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study. The assay can detect ASFV DNA in a range of clinical samples. Sensitivity was equivalent to the Office International des Epizooties (OIE) recommended TaqMan assay. In addition the assay was found to have a detection limit 10-fold more sensitive than the conventional PCR recommended by the OIE. Linear range was ten logs from 2x10(1) to 2x10(10). The assay is rapid with an amplification time just over 2h. The development of this assay provides a useful tool for the specific diagnosis of ASF in statutory or emergency testing programs or for the detection of ASFV DNA in research applications.
Journal of virological methods 09/2010; 168(1-2):141-6. · 2.13 Impact Factor
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ABSTRACT: The prophylactic use of vaccines against exotic viral infections in production animals is undertaken exclusively in regions where the disease concerned is endemic. In such areas, the infection pressure is very high and so, to assure optimal protection, the most efficient vaccines are used. However, in areas considered to be free from these diseases and in which there is the possibility of only limited outbreaks, the use of Differentiation of Infected from Vaccinated Animals (DIVA) or marker vaccines allows for vaccination while still retaining the possibility of serological surveillance for the presence of infection. This literature review describes the current knowledge on the use of DIVA diagnostic strategies for three important transboundary animal diseases: foot-and-mouth disease in cloven-hoofed animals, classical swine fever in pigs and avian influenza in poultry.
Expert Review of Vaccines 01/2010; 9(1):73-87. · 4.25 Impact Factor
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ABSTRACT: The applicability of an electronic monitoring system using microchip transponders for measurement of body temperatures was tested in 6-week-old conventional Danish weaners infected with classical swine fever virus (CSFV). Subcutaneous tissue temperatures obtained by the implantable transponders were compared with rectal temperatures, recorded by a conventional digital thermometer.
In a preliminary study, transponders were inserted subcutaneously at 6 different positions of the body of 5 pigs. The transponders positioned by the ear base provided the best correlation to rectal temperature. To test the stability of the monitoring system in a larger group of pigs, transponders were therefore inserted by the left ear base in a subsequent infection experiment with 30 pigs.
Generally, the microchip transponders measured a subcutaneous tissue temperature, which was about 1 degrees C lower than the rectal temperature. However, a simple linear relationship between the measures of the two methods was found.
Our study showed that the tested body monitoring system may represent a promising tool to obtain an approximate correlate of body temperatures in groups of pigs. In contrast, however, the tested system did not constitute a suitable tool to measure body temperatures of individual animals in the present pig infection experiment.
Acta Veterinaria Scandinavica 01/2010; 52:29. · 1.00 Impact Factor
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Adám Bálint,
Miklós Tenk,
Zoltán Deim,
Thomas Bruun Rasmussen, Ase Uttenthal,
Attila Cságola,
Tamás Tuboly,
Attila Farsang,
Caroline Fossum,
Sirje Timmusk,
Mikael Berg,
Sándor Belák
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ABSTRACT: A real-time PCR assay, based on Primer-Probe Energy Transfer (PriProET), was developed to improve the detection and quantification of porcine circovirus type 2 (PVC2). PCV2 is recognised as the essential infectious agent in post-weaning multisystemic wasting syndrome (PMWS) and has been associated with other disease syndromes such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC). Since circoviruses commonly occur in the pig populations and there is a correlation between the severity of the disease and the viral load in the organs and blood, it is important not only to detect PCV2 but also to determine the quantitative aspects of viral load. The PriProET real-time PCR assay described in this study was tested on various virus strains and clinical forms of PMWS in order to investigate any correlation between the clinical signs and viral loads in different organs. The data obtained in this study correlate with those described earlier; namely, the viral load in 1 ml plasma and in 500 ng tissue DNA exceeds 10(7) copies in the case of PMWS. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of PCV2.
Acta Veterinaria Hungarica 10/2009; 57(3):441-52. · 0.67 Impact Factor
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ABSTRACT: Complete genome amplification of viral RNA provides a new tool for the generation of modified viruses. We have recently reported a full-genome amplification strategy for recovery of pestiviruses (Rasmussen et al., 2008). A full-length cDNA amplicon corresponding to the Border disease virus-Gifhorn genome was generated by long RT-PCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived.
Veterinary Microbiology 09/2009; 142(1-2):13-7. · 3.33 Impact Factor
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ABSTRACT: Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.
PLoS ONE 02/2009; 4(8):e6662. · 4.09 Impact Factor
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ABSTRACT: Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance.
Journal of Virological Methods 11/2008; 155(1):87-90. · 2.01 Impact Factor
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ABSTRACT: This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products could be a valuable instrument for virus rescue, conservation and further characterization.
Journal of Virological Methods 06/2008; 149(2):330-3. · 2.01 Impact Factor
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ABSTRACT: A chimeric pestivirus of border disease virus Gifhorn and bovine viral diarrhea virus CP7 (Meyers et al., 1996) was constructed. Virulence, immunogenicity and vaccine properties of the chimeric virus were studied in a vaccination-challenge experiment in pigs. The chimeric virus proved to be avirulent and neither chimeric virus nor viral RNA was detected in serum after vaccination. The safety of the vaccine was tested by horizontal transmission to sentinel pigs, which remained uninfected. The vaccine efficacy was examined by challenge infection with classical swine fever virus (CSFV) Eystrup. In 'challenge controls', the viral load of CSFV coincided with the development of pronounced clinical symptoms. In contrast, the vaccinated pigs showed transient and weak clinical signs. Analysis of the viral load in these pigs showed 1000-fold lower viral RNA levels compared to 'challenge controls' and horizontal transmission of challenge virus to sentinel pigs was not observed.
Journal of General Virology 03/2007; 88(Pt 2):481-6. · 3.36 Impact Factor
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ABSTRACT: Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous detection and differentiation of three Office International des Epizooties (OIE) classified vesicular viruses: foot-and-mouth disease virus, vesicular stomatitis virus and swine vesicular disease, causing clinically indistinguishable vesicular diseases in swine. The multiplex assay consists of extraction of total RNA from clinical samples; reverse transcription to cDNA using random primers and one-tube real-time amplification of cDNA using multiplex PriProET with specific fluorescent-labelled primers and probes for detection of the three viruses from the vesicular disease complex. The probes are labelled with unique reporter fluorophores, which during amplification are excited by donor fluorophores incorporated in the 5' end of specific amplicons by primer extension. The sensitivity of the multiplex assay was approximately 100 TCID(50), which is 10-fold lower compared to the individual PriProET assays for the three vesicular viruses.
Journal of Virological Methods 07/2006; 134(1-2):176-82. · 2.01 Impact Factor
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ABSTRACT: Bovine virus diarrhoea virus (BVDV)-1f was isolated from a Lesser Malayan Mousedeer in Copenhagen Zoo during a routine screening. Analysis of animals related to the Copenhagen mousedeer revealed that its mother and all siblings were virus positive, a pattern also seen for persistently infected (PI) cattle. BVDV could be transmitted from the PI mousedeer to a calf after indirect contact. The host spectrum for BVDV seems to be even wider than expected; the implications for BVDV control are discussed.
Preventive Veterinary Medicine 12/2005; 72(1-2):87-91; discussion 215-9. · 2.05 Impact Factor
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ABSTRACT: In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating nucleotide analogues in the primers, both serotypes were amplified with similar efficiencies. The generation of specific amplicons resulted in fluorescent signals for either of the two serotypes, and the specificities of the reactions were confirmed from the melting temperature profiles of the fluorescent probes. The limits of detection were found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid identification of VSV.
Journal of Clinical Microbiology 02/2005; 43(1):356-62. · 4.15 Impact Factor
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ABSTRACT: We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID(50) produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak.
Veterinary Microbiology 05/2003; 92(3):197-212. · 3.33 Impact Factor
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ABSTRACT: The classical swine fever (CSF) epidemic in the Netherlands in 1997-1998 lasted 14 months, during which 429 infected and 1300 at risk herds were culled, at an estimated economical cost of 2 billion US dollars. Despite the overwhelming scale of the epizootic, the CSF virus (CSFV) strain causing the outbreak has remained largely uncharacterized. The Dutch epizootic is epidemiologically linked to a small CSF outbreak in 1997, in Paderborn in Germany. E2 and partial 5' NTR sequencing has shown that the index Paderborn isolate, and several Dutch isolates taken during the 1997-1998 epizootic, are virtually identical, confirming that the Paderborn isolate triggered the Dutch outbreak, and furthermore showing that this single isolate was stable throughout the whole Dutch outbreak (the above reviewed in [C. Terpstra, A. J. de Smit, Veterinary Microbiol. 77 (2000) 3-15]). We determined the nucleotide sequence of the 5' NTR (by 5' RACE) and the complete open reading frame of the Paderborn isolate (GenBank AY072924). Our sequence was identical to previously published partial 5'NTR and E2 sequences for the index Paderborn 1997 and Dutch 1997 (Venhorst) isolates, confirming the identity of the virus we sequenced. Phylogenetic analysis based on the complete open reading frame showed that Paderborn is genetically very different from common European laboratory reference strains. Neutralization studies showed that Paderborn is also antigenically very different from common laboratory strains such as Alfort 187. Paderborn is the only recent European CSFV field isolate for which a complete sequence is available, and given Paderborns genetic and antigenic uniqueness, the Paderborn sequence may have practical use for diagnostic and vaccine antigen development.
Veterinary Microbiology 05/2003; 92(4):311-25. · 3.33 Impact Factor
