Diagnosis Of Classical Swine Fever Research Articles

Comparison of methods for improved RNA extraction from blood for early detection of Classical swine fever virus by real-time reverse transcription polymerase chain reaction

Classical swine fever (CSF) is a highly contagious disease of pigs. Early detection of the Classical swine fever virus (CSFV) in infected animals and routine surveillance is important for a rapid response and control of an outbreak of CSF. The current study investigated whole blood as a clinical specimen for the detection of CSFV by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in experimentally infected pigs. The virus was detectable in pre-clinical animals in whole blood and in different fractions of blood, including white blood cells, red blood cells (RBC), and serum. Based on an in-vitro binding assay, CSFV is retained in the RBC fraction. Naturally occurring PCR inhibitors of whole blood were shown to inhibit detection, and several commercial RNA extraction kits failed to remove these inhibitors. The commercial blood RNA extraction protocols were modified to achieve optimized single tube and high-throughput 96-well plate RNA extraction that efficiently removed PCR inhibitors from whole blood and enhanced detection of CSFV in experimentally inoculated pigs. This enabled detection 1-2 days earlier than observed using unmodified RNA extraction protocols. The results show effective use of whole blood as a clinical specimen for diagnosis and surveillance of CSF in pre-clinical animals.

Constructing naive Bayesian classifiers for veterinary medicine: A case study in the clinical diagnosis of classical swine fever

For diseases of which the clinical diagnosis is uncertain, naive Bayesian classifiers can be of assistance to the veterinary practitioner. These simple probabilistic models have proven to be very powerful for solving classification problems in a variety of domains, but are not yet widely applied within the veterinary domain. In this paper, naive Bayesian classifiers and methods for their construction are reviewed. We demonstrate how to construct full and selective classifiers from a data set and how to build such classifiers from information in the literature. As a case study, naive Bayesian classifiers to discriminate between classical swine fever (CSF)-infected and non-infected pig herds were constructed from data collected during the 1997/1998 CSF epidemic in the Netherlands. The resulting classifiers were studied in terms of their accuracy and compared with the optimally efficient diagnostic rule that was reported earlier by Elbers et al. (2002). The classifiers were found to have accuracies within the range of 67–70% and performed comparable to or even better than the diagnostic rule on the available data. In contrast with the diagnostic rule, the classifiers had the advantage of taking both the presence and the absence of particular clinical signs into account, which resulted in more discriminative power. These results indicate that naive Bayesian classifiers are promising tools for solving diagnostic problems in the veterinary field.

Evaluation of a primer-probe energy transfer real-time PCR assay for detection of classical swine fever virus

This study describes evaluation of a real-time PCR assay based on primer-probe energy transfer (PriProET) technology for detection of classical swine fever virus (CSFV). The PriProET technology allows melting curve analysis following PCR amplification and thus provides a higher specificity. The assay was compared with a TaqMan assay by testing a total of 203 samples including 175 clinical specimens and 28 batches of Hog Cholera Lapinized Virus (HCLV) vaccine. The two assays gave the same results for 184 (91%) samples. Compared with the TaqMan assay, 19 additional samples were found to be positive for CSFV using the PriProET assay. In an RNA mixture of both wild type CSFV and C-strain vaccine, the melting curves displayed only one curve: either a wild type-like or a vaccine-like depending on the dominating RNA. The PriProET assay can be a routine molecular tool or a confirmative tool for diagnosis of classical swine fever (CSF), especially in the case of samples that yield an inconclusive result by the TaqMan assay.

Assessment of international inter-laboratory comparison tests for the diagnosis of classical swine fever from 1998 until 2007

The inter-laboratory comparison tests for classical swine fever (CSF) laboratory diagnosis organised by the European Community Reference Laboratory for CSF are regularly performed within European Union Member States. The objective of this study was to evaluate the results of the inter-laboratory comparison tests carried out over the last decade, from 1998 until 2007, by using a statistical approach. A set of five or six lyophilised sera was sent to participants. These included sera containing CSF antibodies, sera containing antibodies against ruminant pestiviruses, sera containing CSF virus and negative sera. This study focused on the results of the diagnostic reference methods for CSF: the neutralisation test for the detection of CSF antibodies (including its interpretation) and virus isolation for the detection of CSF virus. For the detection of CSF antibodies, results were closest to what was expected by the Community Reference Laboratory when only neutralisation tests were performed. The percentage of correct results decreased as soon as the results of CSF antibody enzyme-linked immunosorbent assay were included or when sera with antibodies to ruminant pestiviruses were added to the panel. The results for the detection of CSF antibodies are still valid today, as no additional method has been introduced recently. Regarding CSF virus detection, CSF virus isolation is well established but on the way to being superseded as the reference test by reverse-transcription polymerase chain reaction.

A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89TCID50. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.

Virulence of Classical Swine Fever virus isolates from Europe and other areas during 1996 until 2007

Classical Swine Fever (CSF) has caused several outbreaks in EU Member States with grave economic consequences. Several times the diagnosis of CSF was made too late partially due to non-specific clinical signs which did not raise suspicion for CSF. Virulence of CSF virus isolates (CSFV) still remains a subject of discussion and speculation as sufficient knowledge is still not available. Six uncharacterised CSFV isolates from 1996 to 2007 were assessed in animal experiments for their clinical virulence in order to broaden the knowledge about circulating CSFV and thereby assist disease eradication. A clinical (CS) and pathological score was applied and further extended by additional parameters to a modified CS (mCS) including case fatality, antibody production and leukocyte count. The unknown CSFV isolates could be classified as moderately or highly virulent. The inclusion of additional parameters, especially case fatality, into the mCS gave a more reliable classification of virulence, proving that there are clinical signs and laboratory parameters of blood which can be recognised. Therefore a subclinical course of infection is unlikely, especially in weaner pigs.

Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus

Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF.

Meat juice as diagnostic sample for virological and serological diagnosis of classical swine fever.

The objective of this paper was to assess if meat juice is a suitable substrate for virological and serological diagnosis of classical swine fever (CSF). Fifty-six domestic pigs and 21 wild boars experimentally vaccinated and/or infected as well as 129 field samples from wild boars were involved in this study. Meat juice from diaphragm, forequarter and hindquarter was used for investigations. CSFV and viral RNA were detected in meat juice between days 5 and 21 post infection (pi). Animals which had survived the infection were diagnosed virologically negative and antibody-positive in muscle fluid. After vaccination or vaccination and subsequent infection of animals (n = 42), meat juice samples scored serologically positive. The antibody titres of these samples were significantly lower than in serum. Serological investigations of field samples derived from wild boars (n = 75) shot in Mecklenburg-Western Pomerania showed a clear correlation between the antibody-positive samples in serum and in meat juice, whereas the serological results of meat juice samples (n = 54) from wild boars collected in Lower Saxony were slightly different. The reasons for these differences are discussed. Nevertheless, meat juice seems to be a suitable substrate for CSF diagnosis, especially for wild boars.

Value of Skin Punch Biopsies for the Diagnosis of Acute Classical Swine Fever

The objective of this study was to determine if skin punch biopsies are appropriate for the diagnosis of classical swine fever. For this purpose, 6 wild boars and 2 domestic pigs were experimentally infected with the highly virulent classical swine fever virus (CSFV) Koslov and 5 domestic pigs with a CSFV field isolate (genotype 2.3 Uelzen) derived from wild boar. Skin biopsy specimens were tested using virus isolation, real-time reverse transcription polymerase chain reaction (rtRT-PCR), and fluorescent antibody test (FAT) on cryosections. Whereas CSFV Koslov was first detected at 4 days postinfection (DPI) by rtRT-PCR and virus isolation, FAT failed to detect CSFV antigen until 9 DPI. In domestic pigs infected with CSFV 2.3 Uelzen, viral RNA and CSFV were detected at 7 DPI. FAT was negative until 11 DPI. CSFV antigen was detected in endothelial cells of the vascular plexus in the upper dermis as shown by confocal laser-scanning microscopy and double labeling with von Willebrand factor. At 18 DPI, CSFV antigen was present diffusely in capillaries and spindle shaped cells of the dermis, multifocally within keratinocytes of the epidermis and in numerous cells of the inner and outer root sheath epithelium, hair bulb, and intravascular leukocytes. The rtRT-PCR proved to be the test with the highest sensitivity followed by virus isolation and FAT. Taken together, this study demonstrates that skin is easy to sample antemortem and is also suitable as postmortem tissue, and suggests that rtRT-PCR of skin should be included for CSF diagnosis in the acute period of disease.

Evaluation of real-time RT-PCR assay for the routine intra vitam diagnosis of classical swine fever

The aim of the study was to evaluate the diagnostic quality of the real-time RT-PCR assay described by Hoffmann et al. [Hoffmann, B., Beer, M., Schelp, C., Schirrmeier, H., Depner, K., 2005. Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever. J. Virol. Methods 130, 36–44] for the routine intra vitam diagnosis of classical swine fever (CSF). We compared the assay with conventional diagnostic methods by using defined diagnostic material from an animal experiment with pigs showing different clinical forms of CSF. Compared to virus isolation and antigen ELISA an enhanced sensitivity of the real-time RT-PCR could be shown. We were able to detect all infected pigs regardless of the clinical course of CSF. CSF infection was detected already during the incubation period, during the entire clinical phase as well as at the beginning of convalescence when the first antibodies were detected and no virus could be isolated any more. In most cases, positive PCR results were obtained 2 days earlier than with virus isolation and 2–4 days earlier than with the antigen ELISA.

野外豚からの牛ウイルス性下痢ウイルス抗体の検出

Pig sera collected from breeding sows in one farm in Hokkaido prefecture were diagnosed as antibody-positive with a commercial ELISA of classical swine fever (CSF) in 2002 and 2003. However, an on-site inspection and virological examination concluded that an outbreak of CSF in this pig farm was negative. As a result of further ongoing inspections, antibody-positive sera with this ELISA were confirmed again from the sows, which had no history of vaccination against CSF. To specify the causative virus that had infected these pigs, the neutralization titers of the sera against classical swine fever virus (CSFV) were compared with those against bovine viral diarrhea viruses (BVDVs) and border disease virus (BDV), since these viruses are classified in the samepestivirusgenus. As a result, neutralization titers against BVDV NOSE strain were markedly higher than those against CSFV, BDV and other BVDV strains. From the data and from epidemiological investigations revealing indirect contact between a pig and a cow, it is concluded that the pigs on this farm were infected with BVDV. This report concludes that infections of pigs with BVDV and BDV should be considered important cases for the laboratory diagnosis of CSF.

Procedimiento de diagnóstico de la peste porcina clásica en el jabalí una vez concluida la inmunización por vía oral

The purpose of this paper is to define diagnostic procedures for wild boar after the completion of oral immunisation against classical swine fever (CSF). Epidemiological analysis of CSF in wild boar in Germany demonstrated that it is vital to carry out virological investigations on all animals found dead, sick or involved in traffic accidents. In principle, this should ensure an effective and prompt diagnosis of CSF. In addition, a defined number of wild boar, especially young animals < or = 6 months old, should also be tested for CSF virus to guarantee a high confidence level in the virological monitoring. Which animals should be examined serologically depends on the age class investigated, the season in which vaccination was stopped and the period of time since completion of vaccination. Therefore, different serological procedures have been defined for different situations during the first three years after completion of oral immunisation.

Diagnostic methods for detection of Classical swine fever virus—Status quo and new developments

Classical swine fever (CSF) is a highly contagious disease causing major losses in pig populations almost worldwide. The disease occurs in many regions of Asia, Central and South America and parts of Europe and Africa. Some countries have eradicated the disease (Australia, USA, Canada, within the EU), yet it keeps recurring sporadically (South Africa, Germany, Netherlands, England). The causative virus is a member of the genus Pestivirus, family Flaviviridae. The first diagnosis of CSF is based on the recognition of clinical signs by the veterinarian in the field and by post mortem findings. Many signs are not exclusively associated with CSF and they may vary with the strain of virus, age and health status of the pigs. Since clinical signs may be confused with other pig diseases, laboratory diagnosis of CSF is indispensable. Both the Office International des Epizooties (OIE) and the European Union, have approved diagnostic manuals establishing sampling methods and diagnostic procedures for the confirmation of the disease. In this review, experiences with current tests will be analyzed and complemented with new developments, with emphasis on the polymerase chain reaction after reverse transcription of the RNA genome (RT-PCR).

Diagnostic Evaluation of a Real‐Time RT‐PCR assay for Routine Diagnosis of Classical Swine Fever in Wild Boar

The aim of the study was to evaluate a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for routine diagnosis of classical swine fever (CSF) in wild boar by using 1000 spleen homogenates that were tested previously negative by virus isolation and 26 homogenates from which CSF virus could be isolated. All 26 positive samples in the virus isolation assay also were found to be positive in real-time RT-PCR. Additionally further 10 samples were detected by real-time RT-PCR out of the 1000 negative samples in the virus isolation. With a commercial CSF antigen-ELISA only 14 out of the 36 real-time RT-PCR positive samples could be detected. The sequence analysis of all ten samples that tested positive by real-time RT-PCR and negative by virus isolation revealed CSF virus-specific sequences. Based on the assumption that all samples with a CSF virus-specific sequence or positive in the virus isolation test originated from truly CSF virus-infected wild boar, the following sensitivity values were calculated as antigen-ELISA, 39%; virus isolation, 72% and real-time RT-PCR, 100%. The use of real-time RT-PCR instead of antigen-ELISA and virus isolation as a routine tool for control and eradication of CSF in wild boar populations is recommended.

Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever

A fully validated, ready-to-use, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, multiplexed for simultaneous detection of an internal control, for the simple and rapid diagnosis of classical swine fever (CSF) was developed. Primers and FAM-labeled TaqMan-probes specific for classical swine fever virus (CSFV) were selected from the consensus sequence of the 5′ non-translated region (5′ NTR) of 78 different CSFV strains. For determining analytical sensitivity, an in vitro transcript (T7-PC3alf) of the 5′ NTR was constructed and tested. In addition, the T7-PC3alf transcript was further used as a positive control and a standard for quantitation of CSFV genome copies. A second heterologous in vitro transcript based on a specific primer–probe HEX-system was designed as an internal positive control for the RNA isolation step and RT-PCR. By using limited primer concentrations for the internal control, no adverse effects on the sensitivity of the CSF-system could be observed, and the newly designed duplex real-time RT-PCR proved to have a sensitivity of approximately eight copies. The primer–probe combination selected was strictly CSFV-specific and no amplification was observed in all non-CSFV pestiviruses tested.

Nictitating Membrane as a Potentially Useful Postmortem Diagnostic Specimen for Classical Swine Fever

The gold standard for diagnosis of classical swine fever (CSF) is cell culture virus isolation combined with reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescent antibody test (FAT) in cryosections of tonsils, spleen, various lymph nodes, ileum, and kidney. Autolytic and heterolytic samples render correct FAT evaluation difficult and can even yield false-negative or ambiguously positive results. To extend the spectrum of CSF diagnostic specimens, the authors tested whether the nictitating membrane (NM) might be a useful adjunct diagnostic specimen in wild boars and domestic pigs. To accomplish this, results of virus isolation, FAT, and RT-PCR were compared on NM samples and lymphoid tissues, which are the routine specimens of choice for CSF diagnosis. Wild boars (n = 30) and domestic pigs (n = 8) were experimentally challenged with various CSF virus (CSFV) strains or isolates of different virulence. The FAT revealed CSFV antigen in surface and tubular adenoid epithelium as well as in lymphatic follicles of the NM. In wild boars and domestic pigs with CSF, a strong agreement was found between results of FAT, virus isolation, and RT-PCR on NM and lymphoid tissues. These results suggest that NM is a useful additional specimen that can provide valuable data for postmortem diagnosis of CSF. The NM is relatively easy to sample at necropsy, and postmortem autolysis and heterolysis of this tissue is minimal compared with internal organs.

When can a veterinarian be expected to detect classical swine fever virus among breeding sows in a herd during an outbreak?

The herd sensitivity (HSe) and herd specificity (Hsp) of clinical diagnosis of an infection with classical swine fever (CSF) virus during veterinary inspection of breeding sows in a herd was evaluated. Data gathered from visits to herds during the CSF outbreak in 1997–1998 in The Netherlands were used for the analysis. Herds were visited one or more times by the same or by different veterinarians. On the basis of the veterinarians’ reports, each visit was coded as 0 (negative clinical diagnosis) or 1 (positive clinical diagnosis). The HSe for clinical diagnosis of CSF was modelled as a function of days elapsed since introduction of the virus. The moment of introduction of the CSF virus in the CSF-positive herds was unknown, so for each herd, a probability distribution for the unknown number of days since introduction was derived from serum samples collected at depopulation. The information from the reports of the veterinarians and from the test results of the serum samples at depopulation was combined in a Bayesian analysis. Data from CSF-negative herds were analysed to estimate HSp of clinical diagnosis of CSF. The HSe of clinical diagnosis was 0.5 at 37 days after virus introduction (95% CI: 31, 45) and reached 0.9 at 47 days after virus introduction (95% CI: 41, 54). The estimated herd specificity was 0.72 (95% CI: 0.64, 0.79). Dependence of HSe and HSp on characteristics of the veterinarians and the herds also was studied. Specialisation of the veterinarian significantly, although not markedly, affected the HSe.

Quality management in reference tests for the diagnosis of classical swine fever

Inter-laboratory comparison tests for the diagnosis of classical swine fever (CSF) have been established by the national swine fever laboratories of European Union (EU) Member States. They provide a method of measuring both the quality of the results of diagnostic tests performed by laboratories and the competence with which they were performed. The objective is that all laboratories obtain the same result when investigating the same sample. This study evaluates the results of serological and virological reference tests for CSF (neutralisation test and virus isolation) performed over a period of three years. The sensitivity of the serological diagnosis for the detection of CSF antibodies was very good and revealed a tolerance limit of the scored antibody titres of one dilution step. Results on the same sample in two consecutive years were similar. The variation of the scored antibody titres was larger when testing sera with a low CSF antibody titre. The interpretation of the antibody titres as 'CSF positive or negative' was only slightly altered by these variations. The backtitration of a neutralisation test (used as a control measure) is a more mathematical value which does not correlate directly with the biological system. Commercial CSF antibody enzyme-linked immunosorbent assays still display a lower sensitivity on individual samples compared to the reference neutralisation test. Classical swine fever virus isolation was well established in all participating laboratories and caused very few problems. Specificity of CSF diagnosis by investigating CSF antibody and CSF virus negative sera was not problematic either. In general, the reference tests for CSF diagnosis are well established in the EU. They are based on living systems, e.g. cells and virus, and consequently they have a different tolerance limit than pure mathematical values. What is important is that the interpretation of the test result is identical in all laboratories.

Diseño y desarrollo de un sistema de calidad en el Centro Nacional de Sanidad Agropecuaria de Cuba para el diagnóstico de enfermedades exóticas de los animales

A quality system for the diagnosis of exotic animal diseases was developed at the national centre for animal and plant health (CENSA), responsible for coordinating the clinical, epizootiological and laboratory diagnosis of causal agents of exotic animal diseases in Cuba. A model was designed on the basis of standard ISO 9001:2000 of the International Organization for Standardization (ISO), standard ISO/IEC 17025:1999 of ISO and the International Electrotechnical Commission, recommendations of the World Organisation for Animal Health (OIE) and other regulatory documents from international and national organisations that deal specifically with the treatment of emerging diseases. Twenty-nine standardised operating procedures were developed, plus 13 registers and a checklist to facilitate the evaluation of the system. The effectiveness of the quality system was confirmed in the differential diagnosis of classical swine fever at an animal virology laboratory in Cuba.

The effect of sample degradation and RNA stabilization on classical swine fever virus RT–PCR and ELISA methods

Classical swine fever (CSF), also known as hog cholera, is a highly contagious viral infection of swine caused by a member of the genus pestivirus of the family, Flaviviridae. The need for accurate laboratory diagnosis of CSF is particularly important as it is more reliable than clinical diagnosis. CSF is endemic in many tropical countries where the climate is characterized by high ambient temperature and humidity. This study details the effect of sample quality on CSF antigen-capture ELISA (AC–ELISA) and reverse transcriptase–polymerase chain reaction (RT–PCR) methods. RT–PCR assessment of AC–ELISA-positive spleen samples stored in a conventional glycerol/saline buffer demonstrated that the RT–PCR was detrimentally affected by poor sample quality. To provide a more accurate representation of this effect, a 14 days study was performed to determine the effect of tropical ambient conditions on CSF virus-positive spleen samples stored in two transport media; glycerol/saline and a proprietary RNA preservation solution (RNA later™). A protective effect was demonstrated in both assays with RNA later™ as samples were positive in both assays until day 14 post-exposure. Samples stored in glycerol/saline were negative at RT–PCR at day 3 post-exposure although AC–ELISA was still positive at day 14 post-exposure.