Antigen Enzyme-linked Immunosorbent Assay Research Articles

Development of an indirect competitive ELISA for the detection of doxycycline residue in animal edible tissues

A synthetic hapten doxycycline (DOX) with a spacer-arm (para-aminobenzoic acid (PABA)) was attached to bovine serum albumin (BSA) or ovalbumin (OVA) by the diazonium coupling reaction and mixed anhydride methods. Then, DOX–PABA–OVA conjugate was used as a coating antigen in enzyme-linked immunosorbent assay (ELISA), while DOX–PABA–BSA was used as an immunogen to produce polyclonal antibodies. A reliable and sensitive indirect competitive enzyme-linked immunosorbent assay was developed and applied to the quantitative determination of DOX residue in muscle and liver samples. After the optimisation of the main parameters, 50% inhibition was 8.74 µg/l and the limit of detection was 1.96 µg/l. A weak cross-reactivity (CR) was also observed with other structurally related compounds, such as oxytetracycline (10.71%), tetracycline (4.10%) and chlortetracycline (1.89%). The CRs with other antibiotics were all below 0.1%. With the ELISA method, the recoveries were demonstrated to be from 80.19 to 89.41% in liver samples and 83.98–94.75% in muscle samples. The mean of the coefficients of variation with the intra-assay test were 5.75 and 7.53% in liver and muscle samples, respectively. For the inter-assay test, the average of the coefficients of variation was 5.92% in liver and 7.21% in muscle samples. The ELISA method is accurate and reliable for the detection of DOX residue in edible foods of animal origins.

Impact of individual‐donation nucleic acid testing on risk of human immunodeficiency virus, hepatitis B virus, and hepatitis C virus transmission by blood transfusion in South Africa

In 2005, the South African National Blood Service introduced individual-donation (ID) nucleic acid test (NAT) screening for human immunodeficiency virus (HIV) RNA, hepatitis C virus (HCV) RNA, and hepatitis B virus (HBV) DNA. At the same time the use of ethnic origin to prioritize the transfusion of blood according to a hierarchy of residual risk was discontinued. ID-NAT (Ultrio on Procleix Tigris, Chiron) and serology (PRISM, Abbott) repeat test and confirmation testing algorithms were designed to enable differentiation between false-positive and true-NAT and -serology yields. After 1 year, the NAT and serology yield rates in first-time, lapsed, and repeat donors were analyzed and used to estimate the residual risk of HIV, HBV, and HCV infections by blood transfusion. The HIV, HBV, and HCV ID-NAT window phase yield rates in 732,250 blood donations were 1:45,765, 1:11,810, and 1:732,200, respectively. Seven of 16 HIV window phase donations with viral loads above 16,000 copies/mL were HIV p24 antigen enzyme-linked immunosorbent assay positive. PRISM detected anti-HIV and hepatitis B surface antigen (HBsAg) in 89.4 and 73.9% of early infections in repeat donors. The Procleix assay detected viremia in 99.7 and 95.5% of anti-HIV- and HBsAg-positive first-time donors. In these donors, the occult HBV DNA carrier rate was 1:5200. The residual transmission risk of ID-NAT HIV, HBV, and HCV window phase donations was estimated at 1:479,000, 1:61,500, and 1:21,000,000 respectively. One-year ID-NAT screening of 732,250 donations interdicted 16 HIV, 20 HBV, and 1 HCV window phase donations and 42 anti-hepatitis B core antigen-reactive infections during an early recovery or a later stage of occult HBV infection.

Detection of Autoantibodies to Annexin A11 in Different Types of Human Cancer

Abstract Introduction Annexin A11 was previously identified as an autoantigen in 4.1–10.1% of patients with various systemic autoimmune diseases. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to investigate the occurrence and features of anti-annexin A11 autoantibodies in sera from patients with different types of cancer. Methods The recombinant protein of GST fused to the N-terminal domain (1–175 residues) of human annexin A11 was expressed and used as antigen in ELISA. A total of 246 serum specimens were analyzed, which includes sera from healthy women (77), patients with ovarian cancer (72), breast cancer (18), colon cancer (19), pancreatic cancer (20), prostate cancer (20), and diabetes (20). Results The overall titer of anti-annexin A11 autoantibodies in ovarian cancer patients (or primary tumors only) was found much higher than that in healthy controls (P < 0.05). At the cut-off value designating positive reaction, anti-annexin A11 autoantibodies were detected in 12.5% (5/40) of primary ovarian cancer patients with a significant difference from 2.6% (2/77) of the healthy controls (P < 0.05), but only in 6.25% (2/32) of recurrent tumors. ROC curve demonstrated the potential diagnostic value of anti-annexin A11 autoantibodies in primary ovarian cancer patients with an AUC of 0.62 (0.52–0.73). Anti-annexin A11 autoantibodies were also detected in 5.26% (1/19) of colon cancer and 10% (2/20) of diabetes patients but without significant difference from the healthy controls. Conclusion A convenient assay to detect anti-annexin A11 autoantibodies in patients was developed, and the experimental data are promising but need to be expanded to address their biological/clinical relevance.

Detection of Border Disease Virus in Fetuses, Stillbirths, and Newborn Lambs from Natural and Experimental Infections

The purpose of the present study was to evaluate the use of enzyme-linked immunosorbent assay (ELISA) antigen detection in blood or fetal fluids and reverse transcription polymerase chain reaction (RT-PCR) amplification in tissues for routine laboratory diagnosis of Border disease virus (BDV) infection. Samples from 67 fetuses, 6 stillbirths, and 11 lambs from 25 commercial flocks with suspicion of BDV abortion and 3 fetuses, 7 stillbirths, and 15 lambs obtained from an experimental infection with a local isolate (BDV genotype 4) were investigated. Presence of BDV was detected by RT-PCR in 7.9% of fetuses, 50% of stillbirths, and 50% of lambs from the commercial flocks analyzed, corresponding to 8 of the 25 farms (32%). A similar percentage of the lambs and stillbirths from the experimental infection were positive by RT-PCR of tissue samples (54.5%), and the highest positivity was detected in lymph node, thyroid gland, and kidney. The current study revealed that RT-PCR analysis of stillbirths and lambs with clinical symptoms is more suitable than the analysis of fetuses to confirm the presence of BDV in a flock. Pestiviral antigen was detected by antigen ELISA in a high proportion of fetuses (24/58) and stillbirths (3/4) from commercial flocks, but in lambs, the presence of colostral antibodies masked the detection of the antigen by ELISA. Nevertheless, in lambs from the experimental infection that were not fed colostrum, antigen ELISA was less efficient than RT-PCR in detecting viral presence in stillbirths and lambs. Antigen ELISA is therefore recommended for fetuses with advanced autolysis that can adversely affect RNA integrity.

Production of Polyclonal Antibodies to Potato virus X Using Recombinant Coat Protein

Polyclonal antibodies to recombinant Potato virus X (PVX) coat protein (PVX-CP) were developed and their effectiveness determined in different ELISA protocols. The PVX-CP gene was amplified by reverse transcription-polymerase chain reaction, cloned and expressed in Escherichia coli. For immunization, the CP fractions from bacterial lysate were purified either by simple fractionation or by excision from sodium dodecyl sulphate gels. The PVX-CP was injected into rabbits for antibody production. The PVX-CP antibodies reacted in an indirect plate trapped antigen enzyme-linked immunosorbent assay and immunoblot assay and were useful for the detection of a broad spectrum of isolates of PVX.

Cloning and expression of a truncated pigeon circovirus capsid protein suitable for antibody detection in infected pigeons

Infections with pigeon circovirus (PiCV) (also termed columbid circovirus) occur in meat and racing pigeons (Columba livia) of all ages and have been reported worldwide. A PiCV infection is associated with immunosuppression and the development of young pigeon disease syndrome. An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of virus-specific serum antibody was developed for research purposes. In the absence of a method to propagate PiCV in cell culture, the assay was based on a recombinant truncated capsid protein (rCapPiCV) produced by overexpression in Escherichia coli. A 6×His-Tag was fused to the N-terminus of the protein to facilitate purification by metal affinity chromatography and detection by anti-His antibody. PiCV-negative and PiCV-positive control sera were generated by inoculation of pigeons with tissue homogenate containing PiCV, followed by five weekly blood sample collections. Western blotting of the immune serum revealed a specific protein band of approximately 32 kDa, which was absent in the pre-immune sera. Using rCapPiCV as antigen in an indirect ELISA, PiCV-specific antibody was detected in sera of the experimentally PiCV-infected pigeons collected at 1 to 5 weeks post infection. By testing 118 field sera collected in the years 1989, 1991, 1994 and 2008 in the rCapPiCV ELISA, virus-specific antibody was detected in 89 (75%) of the sera. The results obtained demonstrate that the rCapPiCV-based indirect ELISA is able to detect PiCV-specific antibodies in pigeon sera and may be a useful tool for PiCV serodiagnosis.

An evaluation of urine-CCA strip test and fingerprick blood SEA-ELISA for detection of urinary schistosomiasis in schoolchildren in Zanzibar

To develop better monitoring protocols for detection of urinary schistosomiasis during ongoing control interventions, two commercially available diagnostic tests – the urine-circulating cathodic antigen (CCA) strip and the soluble egg antigen enzyme-linked immunosorbent assay (SEA-ELISA) – were evaluated for detection of Schistosoma haematobium infections in 150 schoolchildren from Zanzibar. The children originated from five primary schools representative of different levels of disease endemicity across the island; using standard urine filtration assessment with microscopy, mean prevalence of S. haematobium was 30.7% (95% confidence interval (CI) = 23.4–38.7%) and a total of 35.3% (95% CI = 27.7–43.5%) and 8.0% (95% CI = 4.2–13.6%) children presented with micro- and macro-haematuria, respectively. Diagnostic scores of the urine-CCA strip were not satisfactory, a very poor sensitivity of 9% (95% CI = 2–21%) was observed, precluding any further consideration. By contrast, the performance of the SEA-ELISA using sera from fingerprick blood was good; a sensitivity of 89% (95% CI = 76–96%), a specificity of 70% (95% CI = 60–79%), a positive predictive value of 57% (95% CI = 45–69%) and a negative predictive value of 90% (95% CI = 86–98%) were found. At the unit of the school, a positive linear association between prevalence inferred from parasitological examination and SEA-ELISA methods was found. The SEA-ELISA holds promise as a complementary field-based method for monitoring infection dynamics in schoolchildren over and above standard parasitological methods.

Construction of a Complementary DNA Library of Parelaphostrongylus tenuis and Identification of a Potentially Sero-Diagnostic Recombinant Antigen

Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms, and ligation with the vector lambda-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned and expressed using the pET expression vector to produce a glutathione S transferase (GST)-His-S.Tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An enzyme-linked immunosorbent assay utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, 10 out of 10) that had been inoculated with 6 - 150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.

Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine to human or light chains

An alphaCD30xalphaCD16 bispecific monoclonal antibody (MAb) was previously shown to induce remission of Hodgkin's disease refractory to chemo- and radiotherapy through specific activation of natural killer (NK) cells, but the appearance of a human anti-mouse antibody (HAMA) response prevented its use for prolonged therapy. Here, we describe an effort to humanize the Fab arm directed against FcgammaRIII (CD16), which-in context with the previously humanized CD30 Fab fragment-provides the necessary component for the design of a clinically useful bispecific antibody. Thus, the CDRs of the anti-CD16 mouse IgG1/lambda MAb A9 were grafted onto human Ig sequences. In a first attempt, the murine V(lambda) domain was converted to a humanized lambda chain, which led, however, to complete loss of antigen-binding activity and extremely poor folding efficiency upon periplasmic expression in Escherichia coli. Hence, its CDRs were transplanted onto a human kappa light chain in a second attempt, which resulted in a functional recombinant Fab fragment, yet with 100-fold decreased antigen affinity. In the next step, an in vitro affinity maturation was performed, wherein random mutations were introduced into the humanized V(H) and V(kappa) domains through error-prone PCR, followed by a filter sandwich colony screening assay for increased binding activity towards the bacterially produced extracellular CD16 fragment. Finally, an optimized Fab fragment was obtained, which carries nine additional amino acid exchanges and exhibits an affinity that is within a factor of 2 identical to that of the original murine A9 Fab fragment. The resulting humanized Fab fragment was fully functional with respect to binding of the recombinant CD16 antigen in enzyme-linked immunosorbent assay and in cytofluorimetry with CD16-positive granulocytes, thus providing a promising starting point for the preparation of a fully human bispecific antibody that permits the therapeutic recruitment of NK cells.

Testing of Cattle Ear Notch Samples Using a Bovine Viral Diarrhea Virus NS3 Antigen ELISA Kit for Detection of Persistently Infected Animals

Identification and elimination of persistently infected (PI) cattle is important to controlling bovine viral diarrhea virus (BVDV) in herds. Compared to many diagnostic laboratory techniques, ELISA (enzyme linked immunosorbent assay) is convenient due to the ability to test large number of samples rapidly and consistently. A virus can be detected by probing for a specific nonstructural antigen like the NS3 (p80 in former nomenclature) or a structural antigen like the glycoprotein Erns (E0 or gp48 in former nomenclatures). Ear notch samples can be obtained easily and quickly from cattle. Another important advantage of ear notch skin biopsy samples is that they contain a high concentration of BVDV antigen. Few publications can be found describing the detection of NS3 in ear notches from cattle. The objective of this study was to compare the performance of a current commercial ELISA which specifically detects the NS3 antigen on ear notches from cattle to other tests utilized in diagnostic laboratories.

The Studies on the Aetiology of Diarrhoea in Neonatal Calves and Determination of Virulence Gene Markers ofEscherichia coliStrains by Multiplex PCR

SummaryThe purpose of this study was to determine aetiological agents of diarrhoea in neonatal calves and to investigate virulence gene markers of Escherichia coli strains isolated from calves by multiplex polymerase chain reaction (PCR). Eighty‐two diarrhoeic calves and 18 healthy calves were used as subjects. Faeces were taken from the rectums of all the calves and were subjected to bacterial culture. Antigen enzyme‐linked immunosorbent assay (ELISA) was performed to detect rotavirus, coronavirus and E. coli K99 in faeces of all the calves. A multiplex PCR was used to characterize E. coli strains in all the calves. Escherichia coli was isolated from 37 faeces samples, Enterococcus ssp. was isolated from 22 faeces samples and Salmonella was isolated from one faeces sample in diarrhoeic calves. Furthermore, only E. coli was isolated from all 18 faeces samples of healthy calves. Of the 37 E. coli isolated from diarrhoeic calves, K99 (18.9%), F41 (18.9%), heat‐stable enterotoxin a (STa) (18.9%), Shiga toxin 1 (Stx1; 13.5%) and Shiga toxin 2 (Stx2; 5.4%) and intimin (8.1%) genes were identified by multiplex PCR. Of the 18 E. coli isolated from healthy calves, K99 (16.6%) and intimin (55.5%) genes were identified by PCR. A total of 15 rotavirus, 11 coronavirus and 11 E. coli K99 were detected in diarrhoeic calves by the antigen ELISA. As a result, this study shows that rotavirus, coronavirus, E. coli and Enterococcus ssp. were determined to play a role in the aetiology of diarrhoea in the neonatal calves. K99, F41, STa, Stx1 and Stx2 were found as the most common virulence gene markers of E. coli strains isolated from calves with diarrhoea. Multiplex PCR may be useful for characterization of E. coli isolated from calves.

Analysis of Commercial Entamoeba histolytica ELISA Kits for the Detection of Entamoeba invadens in Reptiles

Entamoeba invadens is pathogenic in multiple reptile species and has caused severe outbreaks in zoos and other facilities worldwide. Infections can be difficult to diagnose and to differentiate from other reptilian Entamoeba species. The goal of this study was to determine if kits developed to identify the human pathogen Entamoeba histolytica could be used to detect E. invadens in reptile species at the Maryland Zoo. The E. histolytica II antigen enzyme-linked immunosorbent assay and the ProSpecT E. histolytica microplate assay did not react with cultured E. invadens controls or with fecal samples from multiple reptiles, demonstrating the need for a sensitive and specific test for E. invadens.

BACTERIOLOGICAL AND SEROLOGICAL STUDIES ON PASTEURELLA MULTOCIDA INFECTION IN RABBITS

Pastruella multocida is a mucosal pathogen that colonizes the upper respiratory tract of rabbits. Respiratory infections can result, but also the bacteria can invade the circulatory system producing abscesses or septicemia. Pasteurella mulocida (P.multocida) produce extracelluler sialidase that encode enzymes which is believed to augment colonization of the respiratory tract and the production of lesions in the active infection. The recombinant antigen enzyme-linked immunosorbent assay (ELISA) based on the sialidas enzyme of P.multocida and rabbits that were experimentally colonized with P.multocida produce detectable immunoglobulin (IgM) and (IgG) in serum, although they demonstrated no clinical signs of pasteurellosis. In addition clinically ill pet rabbits infected with P.multocida possessed IgM and/orIgG antibody against sialides. The sialides ELISA may be useful for the diagnosis of P.mulocida infection in sick rabbits as well as for screening for carriers rabbits.

Renal Expression of Adhesion Molecules in Anca-Associated Disease

Anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated disease among other manifestations can underlie rapidly progressive glomerulonephritis (RPGN), with crescentic and necrotizing GN. Differences in pathogenic immune mechanisms in RPGN may provide differences in the renal expression of adhesion molecules mediating these lesions. Renal intercellular adhesion molecule 1 (ICAM-1; CD54) and vascular cell adhesion molecule 1 (VCAM-1; CD106) were assessed in 40 patients with type I RPGN (anti-glomerular basement membrane antibodies, n = 4), type II (immune complexes, n = 17), and type III (ANCA, n = 19). Enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin G antibodies against the Goodpasture's antigen and indirect immunofluorescence and ELISA for myeloperoxidase (MPO) and proteinase 3 (PR3) were performed for ANCA testing. Ten normal renal tissues were used as controls. Relationships between ICAM-1 and VCAM-1, histopathologic features, and CD18, CD14, and CD3 cells were analyzed. Abnormal ICAM-1 and VCAM-1 in tubule was seen in >80% of biopsies with RPGN. Abnormal VCAM-1 in glomerular tuft was seen in >60% of biopsies with RPGN. Glomerular ICAM-1 was associated with less glomerulosclerosis (chi (2) = 6.719, p = 0.01), less interstitial fibrosis (chi (2) = 4.322, p < 0.05), and less tubular atrophy (chi (2) = 8.547, p < 0.005). Glomerular VCAM-1 was associated with glomerular leukocyte infiltration (chi (2) = 4.698, p < 0.05). Glomerular tuft stains of ++/+++ for VCAM-1 was observed in 10% from MPO-ANCA-GN patients but in 60% from PR3-ANCA-GN (Fi = 8.538, p = 0.03). The following conclusions can be made from this study. (1) The renal expression of ICAM-1 and VCAM-1 is upregulated in RPGN, and this is associated with the histological activity. (2) De novo expression of VCAM-1 on glomerular tuft suggests that endothelial cells play a role in RPGN. (3) De novo tubular expression of ICAM-1 and VCAM-1 suggests that epithelial cells may participate in adhesive interactions in RPGN. (4) De novo expression of VCAM-1 at the glomerular tuft in PR3-ANCA positive patients seems greater than in MPO-ANCA positive patients, which suggests that testing specific immune activation mechanisms may play a role in ANCA-associated GN.

Frequency of antibodies to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burdgorferi in cattle from the northeastern region of the state of Pará, Brazil

Babesiosis, anaplasmosis, and trypanosomosis are relevant diseases, potentially causing morbidity in cattle, leading to economic losses. Borreliosis is import as a potential zoonosis. The objective of this study was to determine, by indirect enzyme-linked immunosorbent assay (ELISA), the frequency of seropositive cattle to Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma vivax and Borrelia burgdorferi in cattle from the Northeastern region of Pará, Brazil. Sera samples from 246 female adult cattle from municipalities of Castanhal and São Miguel do Guamá were used. Crude antigens ELISAs were used to detect antibodies to all agents, except to A. marginale, to which an indirect ELISA with recombinant major surface 1a protein (MSP1a) antigen was used. Overall frequencies of seropositive animals were: B. bigemina--99.2%; B. bovis--98.8%; A. marginale--68.3%; T. vivax--93.1% and B. burgdorferi--54.9%. The frequencies of seropositive cattle to B. bovis and B. bigemina suggest a high rate of transmission of these organisms by tick in the studied region, which can be classified as enzootically stable to these hemoprotozoans. The low frequency of seropositive cattle to A. marginale may be attributed to a lower sensitivity of the recombinant antigen ELISA utilized or a distinct rate of inoculation of this rickettsia by ticks, as compared with Babesia sp. transmission. The high frequency of seropositive cattle to T. vivax indicates that this hemoprotozoan is prevalent in herds from the Northeastern region of Pará. The rate of animal that showed homologues antibodies to B. burgdorferi indicates the presence of the tickborne spirochaetal agent in the cattle population in the studied region.

Enzyme-Linked Immunosorbent Assays Using Novel Japanese Encephalitis Virus Antigen Improve the Accuracy of Clinical Diagnosis of Flavivirus Infections

The cross-reactive antibodies induced by flavivirus infections confound serodiagnosis and pathogenesis, especially in secondary infections caused by antigenically closely related yet distinct flaviviruses. The envelope (E) glycoprotein fusion peptide contains immunodominant cross-reactive determinants. Using a recombinant Japanese encephalitis virus (JEV) premembrane and E expression plasmid producing JEV virus-like particles (VLPs), dramatic reductions in cross-reactivity were produced by the G106K-L107D (KD) double-mutant VLP against a panel of flavivirus murine monoclonal antibodies. Human serum panels from patients with recent flavivirus infections were analyzed to compare the accuracy of JEV wild-type (WT) and KD VLPs as serodiagnostic antigens in enzyme-linked immunosorbent assays. Statistical analysis demonstrated significant differences in assay performances for accurate determination of current JEV infections between WT and KD antigens by detecting immunoglobulin M antibodies at a serum dilution of 1:4,000 (likelihood ratios = 2.74 [WT] and 22 [KD]). The application and continued development of cross-reactivity-reduced antigens should improve both flavivirus infection serodiagnosis and estimates of disease burden.

Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses

A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.

Detection of the Antibody to Lymphocytic Choriomeningitis Virus in Sera of Laboratory Rodents Infected with Viruses of Laboratory and Newly Isolated Strains by ELISA Using Purified Recombinant Nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.

Laboratory Diagnosis of Ebola Hemorrhagic Fever during an Outbreak in Yambio, Sudan, 2004

Between the months of April and June 2004, an Ebola hemorrhagic fever (EHF) outbreak was reported in Yambio county, southern Sudan. Blood samples were collected from a total of 36 patients with suspected EHF and were tested by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G and M antibodies, antigen ELISA, and reverse-transcription polymerase chain reaction (PCR) of a segment of the Ebolavirus (EBOV) polymerase gene. A total of 13 patients were confirmed to be infected with EBOV. In addition, 4 fatal cases were classified as probable cases, because no samples were collected. Another 12 patients were confirmed to have acute measles infection during the same period that EBOV was circulating. Genetic analysis of PCR-positive samples indicated that the virus was similar to but distinct from Sudan EBOV Maleo 1979. In response, case management, social mobilization, and follow-up of contacts were set up as means of surveillance. The outbreak was declared to be over on 7 August 2004.

First Report of Anthracnose Caused by Colletotrichum acutatum on Strawberry in the Czech Republic.

Colletotrichum acutatum J. H. Simmond is the causal agent of anthracnose rot and strawberry blackspot. This pathogen is listed by the EPPO as a regulated (formerly quarantined) organism for all European countries and is widely distributed throughout Europe (e.g., the United Kingdom, France, and Germany) (2). In the autumn of 2005, typical symptoms of anthracnose caused by C. acutatum (circular, dark, and sunken spots on fruit, dark, sunken lesions on petioles, and withering of the leaf, buds, and flowers) were repeatedly observed on field-grown strawberry plants in the Mělník Region of central Bohemia and Břeclav Region in southern Moravia, Czech Republic. Strawberry fruits and petioles showing typical symptoms were surface sterilized (30 s in 70% ethanol, 1 min in 10% NaOCl, and 15 s in 70% ethanol), rinsed in sterile water, dissected under aseptic conditions, and plated on 2% malt extract agar or placed in wet chambers and incubated at room temperature (18 to 20°C) for 10 days. All isolated strains were independently identified by morphological characteristics, plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) (1), and PCR with the C. acutatum-specific primers ITS4 and CaInt2 (3). Morphological studies of C. acutatum were carried out on potato dextrose agar (4). The colonies were white, cream, grayish, or rose-orange before sporulation and the colony reverse was cream to orange with brown spots. The mycelial growth rate was 7.5 mm per day at 25°C. The conidia were one-celled, hyaline, cylindrical, 11.3 to 19.7 × 3.6 to 5.5 μm, and the majority of conidia were pointed at either or both ends. The appresoria were brown, globose to ellipsoidal, 5.0 to 7.5 × 5.0 to 6.2 μm, and the sclerotia were absent. Ten strawberry plants with green fruits of each cultivar Elsanta and Kama were sprayed with 500 ml of suspension of C. acutatum conidia (104 conidia per ml). This test was carried out in the glasshouse under quarantine conditions at 20 to 25°C. C. acutatum caused withering of the flowers or dark brown spots on green fruits on five plants of cv. Elsanta and on four plants of cv. Kama after a 6-week incubation period. Isolation and identification of the pathogen from the diseased tissues were done as described above. C. acutatum was reisolated from three fruits, four leaf blades, and four petioles from five plants of cv. Elsanta and four fruits, four leaf blades, and two petioles from four plants of cv. Kama. The fungus was not reisolated from the control strawberry plants. In three cases, the pathogen was detected in the crown of plants of cv. Elsanta by PCR and ELISA.