Evaluation of a real-time RT-PCR assay using minor groove binding probe for specific detection of Chinese wild-type classical swine fever virus

A one-step real-time RT-PCR assay using a minor groove binding probe was developed for the specific detection of Chinese wild-type classical swine fever virus (CSFV). The assay detected wild-type CSFV strains representing different genotypes, but did not amplify viral RNA from the Hog Cholera Lipinized Virus (HCLV) vaccine-strain and other porcine viruses. The assay had a detection limit of 10 copies/reaction or 3.0 median tissue culture infective dose/reaction. In comparison to the sequencing nested RT-PCR assay, the sensitivity and specificity of the assay were 98.3% and 94.3%, respectively, when testing 515 veterinary samples. Wild-type CSFV RNA was detected in nasal swabs 2-4 days before detection in serum samples from pigs exposed to infection by contact, and 2-4 days prior to the onset of clinical disease. HCLV RNA remained undetectable in nasal swabs and serum samples from vaccinated pigs. In conclusion, the novel assay described in this study provides a rapid and sensitive method for differentiating between wild-type and the HCLV-strain of CSFV. It could be used for monitoring in CSF outbreak areas or as a screening method for CSFV eradication strategies.     

Oral immunization of pigs against classical swine fever. Course of the disease and virus transmission after simultaneous vaccination and infection

The efficacy of simultaneous vaccination of pigs against classical swine fever (CSF) and challenge was evaluated. In this study, domestic weanling pigs were vaccinated orally with a conventional live virus vaccine based on CSF virus (CSFV) C strain and were challenged simultaneously with CSFV of different virulence. All the animals vaccinated and challenged with a high dose of highly virulent Koslov strain died while three of five animals challenged with a low dose of highly virulent Alfort 187 strain survived, shed the virus in nasal secretions, developed antibodies, and four of them showed a transient viremia. All the animals vaccinated and challenged with the low virulent field isolate MV 140/Riems survived, showed a short viremia and developed antibodies. No CSFV or CSFV RNA could be detected in the animals surviving the infection. This study demonstrates that oral vaccination of wild boars in an infected area bears no risk for the development of a persistent CSF infection.     

Monitoring of infectious diseases among wild boars

Sixty-seven serum samples and 43 pathological material samples from wild boars, taken in 5 regions of Russia and in the Kharkov Region of the Ukraine in 2002 to 2005 were studied. Wild boars in some regions of Russia were shown to be carriers of Aujeszky's disease virus, porcine parvovirus, porcine circovirus type 2, lymphotropic herpesvirus-1, porcine cytomegalovirus, Mycoplasma hyopneumoniae and Pasteurella multocida. The classical swine fever (CSF) virus genome was detected by polymerase chain reaction in the samples from 10 wild boars from 2 Russian regions (the Tver and Moscow Regions). Sequencing of the E2 gene 5'-terminal region of detected CSF virus isolates showed that they were closely related to two field virus isolates early found in domestic pigs in the Moscow and Vladimir Regions, which suggests that there is an epizootic relation between the SCF outbreaks among wild boars and domestic pigs in these regions. Tests for porcine reproductive and respiratory syndrome, transmissible porcine gastroenteritis, porcine influenza, enteroviruses, and actinobacillus-induced pleuropneumonia were negative in all the regions under study.     

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(?) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas.     

Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.

BACKGROUND: Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis. OBJECTIVE: To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens. STUDY DESIGN: After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay. RESULTS: Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80). CONCLUSIONS: The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.     

免疫去势和未去势猪睾丸上生长激素释放肽的定位及其基因表达

目的:探讨生长激素释放肽在猪睾丸中的定位及其基因表达,以及免疫去势对猪睾丸中生长激素释放肽表达的影响.方法:14头9周龄公猪随机分为免疫去势组和对照组(未去势组),25周龄时屠宰,取血清和睾丸组织,放射免疫法检测血清睾酮的水平,免疫组织化学PV-9000两步法确定生长激素释放肽在睾丸中的细胞定位,实时荧光定量PCR法分析睾丸中生长激素释放肽mRNA的变化.结果:免疫去势组猪血清睾酮水平显著地低于未去势组;免疫组织化学显示表达生长激素释放肽的细胞为精原细胞、初级精母细胞、精子细胞、支持细胞和间质细胞,且免疫去势猪阳性细胞数显著多于未去势猪.另外,猪睾丸中有生长激素释放肽mRNA,但免疫去势组与未去势组间差异无统计学意义.结论:猪睾丸中广泛存在生长激素释放肽及其mRNA,免疫去势导致生长激素释放肽的表达增多.     

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.     

Comparison of commercial kits for detection of cryptococcal antigen.

Although kits to detect cryptococcal antigen are used widely to diagnose cryptococcal infection, the comparative performance of commercially available assays has not been evaluated in the past decade. Therefore, we compared the sensitives and specificities of five commercially available kits for detecting cryptococcal antigen (four latex agglutination test kits--Calas [Meridian Diagnostics])--Crypto-LA [International Biological Labs], Myco-Immune [MicroScan], and Immy [Immunomycologics]--and an enzyme immunoassay kit, Premier [Meridian Diagnostics]) with culture for the diagnosis of cryptococcal meningitis and fungemia. Of 182 cerebrospinal fluid (CSF) and 90 serum samples submitted for cryptococcal antigen and fungal culture, 49 (19 and 30 samples, respectively) from 20 patients had a culture positive for Cryptococcus neoformans. For CSF specimens, the sensitivities and specificities of all kits were comparable (sensitivity, 93 to 100%; specificity, 93 to 98%). There was a significant difference in sensitivities of the kits when serum samples were tested with the International Biological Labs and MicroScan kits, which do not pretreat serum with pronase. These kits were less sensitive (sensitivity, 83%) than the Immy and Meridian latex kits (sensitivity, 97%), which do pretreat with pronase. The sensitivity of the Meridian enzyme immunoassay kit was comparable to that of the pronase-containing latex kits. These kits were of equivalent specificities (93 to 100%) when testing serum. Some of the currently available kits have limitations that need to be recognized for proper interpretation of results. Specifically, the use of pronase on serum samples reduces the number of false-positive results, and a titer of < or = 1:4 can be a false-positive result when CSF samples are being tested.     

Technical and clinical validation of three commercial real-time PCR kits for the diagnosis of neuroborreliosis in cerebrospinal fluid on three different real-time PCR platforms

This study reports the evaluation of the technical and clinical validation of the O-DiaBorburg kit (DIA), Borrelia burgdorferi PCR kit, ISEX (GENE), and Borrelia burgdorferi sensu lato Real-TM (SAC) for the diagnosis of neuroborreliosis in cerebrospinal fluid based on both Borrelia DNA and CSF samples from patients with clinical suspicion of neuroborreliosis. This validation study was done by analysing the kits on the Rotorgene Q (RGQ), CFX96, and LightCycler480 (LC480). For all kits, the linear range was larger on RGQ than on CFX96 and LC480. A good reproducibility was obtained for all assays on all instruments. Storage at ?20 °C resulted in a decreased reproducibility for SAC. Results of the limit of detection (LOD₉₅) experiments indicated a better sensitivity than described in the kit insert for all kits on all PCR platforms. No cross-reactivity was found for genetically related organisms nor for other pathogens which may be present in CSF. All species of the Borrelia burgdorferi sensu lato complex were detected with the GENE and SAC kits. The DIA kit failed to detect B. lusitaniae. The results seemed to indicate a better overall performance for the GENE kit on RGQ. However, its diagnostic value could not be confirmed in the clinical validation study, wherein none of the 103 CSF samples from clinical neuroborreliosis cases showed a positive real-time PCR result with the GENE kit analysed on RGQ.     

A generic real-time TaqMan assay for specific detection of lapinized Chinese vaccines against classical swine fever

Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N^pro gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.     

Differentiation of Classical Swine Fever Virus Infection from CP7_E2alf Marker Vaccination by a Multiplex Microsphere Immunoassay

Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV E^rns, and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV E^rns antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV E^rns ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV E^rns antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals.     

Comparative evaluation of commercial rubella virus antibody kits.

Comparative evaluation of six commercial rubella virus antibody kits is described. A set of serum samples skewed toward nonreactive and low-positive rubella antibody levels was used because the goal of an immunity screening test is the detection of susceptible individuals. The M. A. Bioproducts Rubastat and the Hynson, Westcott and Dunning Rubascan were superior in achieving high sensitivity, specificity, and reproducibility. They also were the easiest and fastest tests to perform, and they require equipment available in most laboratories.     

Pathogenicity and kinetics of virus propagation in swine infected with the cytopathogenic classical swine fever virus containing defective interfering particles

Summary.  To analyze the pathogenicity and in vivo kinetics of the cytopathogenic (cp) classical swine fever virus (CSFV) WB82 strain, which is composed of cp defective interfering (DI) particles and noncytopathogenic (noncp) helper virus (WB82/E⁺ strain), WB82 and WB82/E⁺ strains were administered separately to domestic pigs. After inoculation with either strain, all pigs showed typical symptoms of classical swine fever (CSF), such as leucopenia and high fever. There were few differences in clinical signs and survival times between each group. However, the appearance of some symptoms of CSF had a tendency to be delayed following infection with the WB82 strain, when compared with the WB82/E⁺ strain. Virus isolation and detection of subgenomic (sg) and full-length viral (flv) RNA by RT-PCR was carried out using sera, 10% homogenized organs and oral, nasal and rectal swabs. Both noncytopathogenic helper virus and cp DI particles were detected in samples from pigs infected with the WB82 strain, but only noncp phenotype virus was isolated from pigs infected with the WB82/E⁺ strain. Interestingly, the cp DI particles appeared six to seven days later than helper virus in sera from pigs infected with the WB82 strain. Although active cp DI particles could not be isolated from swabs, sg RNA as well as flv RNA was detected in swabs from animals infected with the WB82 strain. These results suggest that progeny cp DI particles are replicated from parent DI particles after noncp virus replication, and subsequently discharged from infected animals. Furthermore, propagation of DI particles or replication of sg RNA, following propagation of helper virus, appears to inhibit the appearance of CSF symptoms induced by virulent helper CSFV. Received January 14, 2002; accepted August 19, 2002