VN Test Research Articles

Abstract 13963: Redefining the Assessment of “Therapeutic” Platelet Response to Acetylsalicylic Acid

Background: Despite extensive investigations, the optimal assessment of the “therapeutic response” to acetylsalicylic acid (ASA) remains unclear. Limited information is available on the relation between serum thromboxane (Tx)B 2 ; arachidonic acid (AA)-induced platelet aggregation (PA) by light transmittance aggregometry (LTA); aspirin reaction units (ARU) by VerifyNow aspirin test (VN), and pharmacokinetics (PK). Methods: Serial PD and PK analyses were performed immediately after a single 162 or 650 mg dose of chewed and swallowed ASA in ten healthy volunteers. ASA response was defined as >95% inhibition of serum TxB 2, <550 ARU by VN test and ≤20% AA-induced PA. Correlation analyses between PK and PD measurements and receiver operating characteristic (ROC) curve analyses were performed. Results: ASA Response measured by VN, and AA-induced PA was achieved within 30 minutes of ASA administration. A good correlation was observed between VN ARU and 1 mM AA-induced maximum PA (r=0.80, p<0.001), serum TxB 2 (r=0.76, p<0.001), and inhibition of serum TxB 2 (r=0.87, p<0.001). The relation between platelet aggregation and TxB 2 inhibition appears to be dichotomous, with only modest inhibition of TxB 2 (~50% inhibition) associated with marked inhibition of platelet aggregation. ROC curve analyses indicated that ≤558 ARU and ≤7% AA-induced PA were associated with >95% inhibition of TxB 2 . A plasma ASA 686 ng/mL cut-off was associated with >95% inhibition of serum TxB 2 , ≤7% 1 mM AA-induced PA by LTA, and ≤585 ARU. Conclusion: Our study demonstrated an important relationship between PD and PK parameters measured immediately following oral ASA and cutoff values for VN assay and LTA that is associated with >95% inhibition of serum TxB 2 . We demonstrated that ~50% inhibition of TxB 2 is required to reach high levels of platelet function inhibition.

Development and Evaluation of a Blocking Lateral Flow Assay Strip for Detection of Newcastle Disease Virus Antibodies.

Newcastle disease (ND) is an acute septicemic infectious disease caused by Newcastle disease virus (NDV). Considering that vaccination is currently the main modality for the prevention of ND, it is essential to assess the effectiveness of clinical immunization. In this study, we have developed a blocking lateral flow assay (bLFA) strip for the rapid detection of NDV antibodies using the monoclonal antibody 9C1 against haemagglutinin-neuraminidase (HN), which allows for the determination of an NDV-specific antibody titer within 10 min at room temperature. In addition, the bLFA strip has no cross-reactivity with the positive serum of other avian pathogens including avian influenza subtypes H5, H7, and H9, MD, IBD, IB, EDS, and avian adenovirus. The ability of the bLFA strip for detecting a neutralizing antibody was also estimated. The results showed that the chicken NDV hyperimmunized serum had a complete blocking (100%) titer of 11 log 2, and half-blocking titer of 13 log 2, which are 4 times less than and the same as that of the HI test (13 log 2), and 8 and 2 times less than that of the VN test (14 log 2), respectively. A total of 510 clinical samples were tested for NDV antibodies. The coincidence rate between the results of the bLFA strip and HI test was 97.65%. Therefore, it is an ideal alternative method for assessing the clinical immunity of ND vaccines in the field in real-time.

Bentracimab demonstrates reversal of antiplatelet effects of ticagrelor: impact of hematocrit and generic versions of ticagrelor in vitro

Abstract Recent clinical studies demonstrated bentracimab provides immediate and sustained reversal of ticagrelor's antiplatelet effects in patients undergoing surgical procedures or with bleeding. Ticagrelor reversal was assessed by VerifyNow PRUTest (VN) and other platelet function testing assays in Phase I-III studies. As decreases in hematocrit (hct) may impact the VN result, it remains to be demonstrated whether this assay can detect platelet function restoration by bentracimab in bleeding patients with low hct. Additionally, bentracimab utility against generic ticagrelors is unknown. The purpose of this study was to determine the impact of decreased hct and generic forms of ticagrelor on platelet function and subsequent reversal by bentracimab, as measured by light transmission aggregometry (LTA) and VN. The hct level in blood samples collected from healthy volunteers (n=10) was sequentially lowered to 32, 30, 25, 22.5, 20 or 15% by addition of autologous platelet-rich plasma to whole blood with matching platelet counts. Prepared samples were treated with nothing, vehicle, or ticagrelor (3.0 μg/mL) followed by treatment with bentracimab (1.2 mg/mL). In a separate study, blood collected from healthy volunteers (n=10) was treated with nothing, vehicle, ticagrelor or generic ticagrelors (3.0 and 5.6 μg/mL; mean and peak plasma levels in the Phase 1 trial) prior to addition of bentracimab (0, 0.6, 1.2, 1.8 and 2.4 mg/mL; below the reported minimum to peak plasma level) to demonstrate a dose response. Reversal was assessed by either ADP-mediated LTA or VN or both assays. Results show a decrease in hct to <30% significantly increased the PRU in untreated samples (p<0.05). Ticagrelor treatment significantly decreased the PRU in all hct groups (p<0.0001). When normalized to each donor's baseline PRU values, bentracimab reversed the ticagrelor effects within 10-min, restoring the PRU value to 54–73% for all hct groups. PRU values were further restored to 85–93% of baseline PRU after 1-hr treatment. The PRU inhibition for both 10-min and 1-hr treatments were not significantly different across the hct groups, except for the 10-min treatment in the 15% hct group vs. unadjusted hct group (p<0.05), suggesting equivalent reversal by bentracimab across the hct range. In the second study, treatment with generic ticagrelor demonstrated significant platelet inhibition and was comparable to standard ticagrelor. Bentracimab (>0.6 mg/mL) immediately restored platelet function after ticagrelor treatment to 80–100% of baseline using the VN test. LTA data demonstrated similar results to VN, but as expected, reversal was less robust with 5.6 μg/mL ticagrelor + 0.6 mg/mL bentracimab combination (mean 55% of baseline values). Collectively, these data suggest the VN test can effectively measure the antiplatelet effects of ticagrelor and subsequent reversal, even at low hct, and bentracimab is also capable of reversing the antiplatelet effects of generic forms of ticagrelor. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): PhaseBio Pharmaceuticals, Inc

Investigation of Cross Neutralization of Egyptian Variant 2 among both Classic and Variant Vaccinal Infectious Bronchitis Viruses

Infectious Bronchitis Virus (IBV) is considered an important virus which may cause major losses in the poultry industry. One of the most important and effective tool that control the infection spread is the vaccination strategy. The present study was undertaken to determine the antigenic relationship between the currently used IBVs, classic and variant vaccinal strains, and the dominating Egyptian variant-2, IBV which could be relatively reflecting the quality of such vaccine. The recommended doses of the monovalent live IBV vaccines of Ma5, H120, 4/91 & CR88, and bivalent vaccine of H120-D274 were administrated via the eye-drop route in groups of 2-week-old SPF-chickens. Three weeks after vaccination, immunized and control chickens were bled, and serum samples were collected. Sera were tested individually for measuring the IBV-Haemagglutination Inhibiting (HI) antibodies against each of IBV-haemagglutinating antigen prepared from the reference classic M41 strain and the Egyptian variant.2 strain (Egy/12/2b spike protein), and IBV- neutralizing antibodies against the Egyptian variant-2 strain. The chicken groups vaccinated with the commercial live vaccines of strains Ma5, H120, 4/91, CR88 and H120-D274 of IBV respectively developed mean serum HI-antibody titers of 6.8, 6.5, 4.9, 5.0 and 5.5 log 2 with the reference classic strain M41-HA antigen, and 3.0, 3.0, 4.4, 4.0, and 4.6 log 2 with the Egyptian variant.2 strain-HA antigen. However, the obtained results revealed utility of VN test more than HI test for assessment of the antigenic relatedness between the vaccinal and challenge strains of IBV as well as testing quality of different commercial live IBV vaccines using the sera of vaccinated chickens against the field strain(s) matched with the homologous strain(s); the antigenic relatedness (r 1 ) between the dominating Egyptian variant-2 strain, and the vaccinal strains, CR88, D274-H120 and H120 of IBV were 33%, 55% and 17% respectively.

SEROSURVEILLANCE OF EQUINE INFECTIOUS ANAEMIA IN A REGION OF VOJVODINA

Equine infectious anemia is a consequence of a persistant infection of the horses with Lentivirus. Pathogenesis of the disease is very variable, what can bee seen through a wide range of clinical forms of the disease – from inaparrent infection to death. Diagnostics of EIA is based on clinical symptoms, detection of antibodies and virus. Antibodies can be identified with Hi, VN, CFIT, cELISA, SA-ELISA and AGID test. RT-PCR technique enables the detection of and/or quantifi cation of viral RNA level in blood of infected animal. First reliable serological test for EIA was AGID test. Modified AGID test is considered today as aknowledged, international standard for the detection of antibodies against EIA virus and it enables detection of more then 95% of ll positive animals. Horses with positive fi ndings with this test are considered infected and should be euthanized or placed in strict isolation. Further measures to control the spread of this disease are insectvector control and disinfection of surgical and other equipment in use on successive animals. Th e results of a study during a twenty year period, in the region of AP Vojvodina show that from the total of 11.972 horses blood samples, with te use of AGID test, positive results were found in 21 or 0,17% of horses.

Package of NDV-pseudotyped HIV-Luc virus and its application in the neutralization assay for NDV infection.

Newcastle disease virus (NDV) is a member of the Paramyxovirinae subfamily and can infect most species of birds. It has been a great threat for the poultry industry all around the world. In this report, we successfully produced infectious pseudotyped pNL4-3-Luc-R−E− (HIV-Luc) viruses with the HN and F envelope proteins of NDV. Further investigation revealed the cytoplasmic domains of HN and F, especially HN, plays a significant role in the infection efficiency of these pseudotyped HIV-Luc viruses. Replacement of, or direct fusion to the cytoplasmic domain of the HN protein by that of vesicular stomatitis virus G (VSV-G) could greatly enhance or destroy the infective potential of HN and F-pseudotyped (NDV-pseudotyped) HIV-Luc virus. We further established a novel neutralization assay to evaluate neutralizing antibodies against NDV with the NDV-pseudotyped HIV-Luc viruses. Comparative neutralization data indicate that the results determined by using the NDV-pseudotyped HIV-Luc viruses are as reliable as those by the conventional virus-neutralization assay (VN test) with native NDV. Moreover, the results show that the novel neutralization assay is more sensitive than the VN test.

Dispositivos poliméricos cardiovasculares: comportamento termomecânico e viabilidade celular

Nas últimas décadas têm sido desenvolvidos novos materiais sintéticos que possuam biofuncionalidade e biocompatibilidade para que se tornem um biomaterial. Polímeros bioestáveis têm uso generalizado no campo biomédico, sendo que muitos avanços em biomateriais poliméricos têm ocorrido na busca de melhorias aos implantes cardiovasculares. Atualmente, os materiais sintéticos mais utilizados para a fabricação de próteses vasculares são o PET e o PTFE devido a sua estabilidade química após o implante. Neste trabalho é apresentado um estudo das propriedades térmicas e mecânicas de dispositivos comerciais à base de PET e PTFE, bem como sua citotoxicidade em células de fibroblastos de camundongos, 3T3-NIH, através de testes para a avaliação da viabilidade celular (teste VN e MTT). Estes materiais apresentaram grande estabilidade térmica (acima de 300 ºC), mesmo após 270 dias de degradação in vitro e um comportamento elástico (deformação máxima de 186±22% para o PET e de 65±19% para o PTFE). A viabilidade celular por VN e MTT do dispositivo de PTFE foi superior a 80%, permitindo classificar o mesmo como não citotóxico. No teste de VN, o dispositivo de PET não apresentou efeito citotóxico, contudo os resultados por MTT indicaram que o mesmo causa alteração da funcionalidade mitocondrial, independente da dose e tempo avaliados.

Comparação dos testes de virusneutralização contra os genótipos 1 e 2 do vírus da diarreia viral bovina (BVDV-1 e BVDV-2) em bovinos de rebanhos naturalmente infectados

Os resultados dos testes de virusneutralização (VN) contra os genótipos do vírus da diarreia viral bovina (BVDV-1 e BVDV-2), bem como os respectivos títulos de anticorpos, foram comparados em 1.925 amostras de soro sanguíneo obtidas de rebanhos bovinos naturalmente infectados e não vacinados contra o BVDV, provenientes dos Estados de São Paulo e Minas Gerais. A proporção de amostras reagentes entre os genótipos foi analisada pelo Teste de McNemar, e os títulos de anticorpos das amostras reagentes ao BVDV-1 e ao BVDV-2 foram comparados pelo Teste de Wilcoxon. Não foi verificada discordância na proporção de bovinos reagentes ao BVDV-1 e ao BVDV-2 (P>0,05). No entanto, houve discordância entre os títulos de anticorpos detectados nos testes de VN contra os genótipos 1 e 2 do BVDV (P<0,0001). Embora a proporção de animais reagentes contra ambos os genótipos do BVDV tenha sido semelhante, resultados falso-negativos seriam obtidos se 67 amostras (3,5%) tivessem sido submetidas apenas ao teste de VN, para o BVDV-1, e em 51 amostras (2,65%), apenas para o BVDV-2. Alguns rebanhos apresentaram títulos de anticorpos superiores para o BVDV-1, enquanto outros para BVDV-2, demonstrando assim a ocorrência da infecção pelos diferentes genótipos do vírus entre os rebanhos analisados. Portanto, tais resultados demonstraram a necessidade da inclusão de ambos os genótipos do BVDV nos testes de VN.

Identification of Different Serotypes of Infectious Bronchitis Viruses in Allantoic Fluid Samples with Single and Multiplex RT-PCR

Background and Aims: Infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory and kidney disease of chickens which results in significant economic losses in commercial broilers, layers and breeders. Rapid identification of IBV serotypes involved in respiratory complex is a problem in the differential diagnosis. Materials and Methods: In this study, a single and multiplex RT-PCR was used to detect group III, Massachusetts and 793/B serotypes of IBV that have been circulating in Iranian poultry industry since 1999. Results: Only seven out of 49 allantoic fluid samples showed positive results in single RTPCR using group specific primers. Two of these samples belonged to IBV positive group in VN test. Whereas; four samples belonged to H9N2 positive group and one sample belonged to IBV/AIV negative groups. The high sensitivity of single RT-PCR led to detect some of the missed IBV infections as compared with other virological tests. Three out of 7, IBV isolates identified with single RT-PCR, classified as 793/B serotype and four of them categorized as Massachusetts serotype in multiplex RT-PCR. Conclusion: The results indicated that multiplex RT-PCR method, can be a valuable test for rapid identification of Massachusetts and 793/B serotypes in complex respiratory complex infection of chickens

Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) is a causative agent of severe diarrhea which leads to death in piglets. Because of the high mortality which is up to 100% in suckling piglets, PED is an important porcine disease in Korea. In this study, we developed a prophylactic candidate using single-chain Fvs to prevent the PEDV infection. ScFvs of mouse monoclonal antibody which was verified to neutralize PEDV was expressed in Escherichia coli expression system. After the confirmation of PEDV neutralizing activity of purified recombinant scFvs by VN test, scFvs were expressed on the surface of E. coli cells. The signal sequence and autotransporter β domain of protease IgA (IgAP) of Neisseria gonorrhoeae were introduced to endow scFvs with the direction to the cell surface and the support as a transmembrane domain. 5 × 10 6 CFU of E. coli expressing scFvs against PEDV showed promising result of 94% foci reduction compared to wild type E. coli. This result demonstrated that E. coli expressing scFvs on the cell surface retained functional potency of parent antibody and therefore blocked PEDV infection into target cells in vitro. This in vitro assay result proposes the perspective of recombinant E. coli cells expressing scFvs as a novel prophylactic against PEDV infection.

Prevalence of Feline Coronavirus Antibodies in Cats in Bursa (Turkey) by An Enzyme-Linked Immunosorbent Assay

Since feline coronavirus (FCoVs) are ubiquitous pathogens, the seroprevalence was studied to monitor infection in Turkey. One hundred sera, collected from cats belonging to catteries or community shelters and to households, were tested. The VN test was performed and compared with ELISA. Each sample was heat-inactivated and serial two-fold dilutions starting from 1/2 were mixed with 100TCID50/50μl of FCoV type II strain 25/92. Subsequently, freshly trypsinized CRFK cells were added and the plates were incubated at 37°C for 96h.

Técnica rápida de neutralização viral para a detecção de anticorpos contra o vírus da Diarréia Viral Bovina (BVDV) no leite

A identificação de rebanhos positivos para o vírus da Diarréia Viral Bovina (BVDV) através de detecção de anticorpos no leite pode viabilizar programas de controle em larga escala. Com esse objetivo, a técnica de soro-neutralização (SN) foi adaptada para a pesquisa de anticorpos em amostras de leite. A adaptação consistiu na redução do tempo de incubação do teste, seguida da detecção de antígenos virais por imunofluorescência. A redução do tempo de incubação minimizou os efeitos tóxicos do leite sobre as células de cultivo, além de permitir a obtenção dos resultados em 24 horas. A técnica rápida (SNR) foi inicialmente testada em 1.335 amostras de soro bovino, apresentando sensibilidade de 93,7% e concordância de 91,1% em relação à SN tradicional. A SNR foi também utilizada para testar 423 amostras de soro bovino que apresentaram toxicidade para as células na SN tradicional, detectando 316 (74,7%) amostras positivas. O teste de amostras de soro e leite de 520 vacas em lactação demonstrou que a SNR pode detectar anticorpos no leite de vacas com títulos séricos a partir de 10. Atividade neutralizante anti-BVDV no leite foi detectada em 97,4% (191/196) de vacas com títulos séricos ³ 320; em 92,9% (79/85) de vacas com títulos de 160; em 88% (59/67) de vacas títulos de 80. A freqüência de animais positivos na SNR foi de 76,9% (40/52) para animais com títulos séricos de 40; 61,3% (19/31) com títulos de 20 e de 33,3% (10/30) para vacas com títulos de 10. Esses resultados demonstram que a técnica de SNR é adequada para a pesquisa de anticorpos anti-BVDV no leite, principalmente em animais com títulos moderados e altos de anticorpos. Essa técnica pode ser utilizada para testar amostras coletivas de leite e identificar rebanhos com atividade viral. A utilização dessa técnica pode viabilizar programas regionais de combate à infecção, pois permite testar um grande número de amostras e identificar rebanhos positivos através do leite enviado rotineiramente para contagem de células somáticas (CCS), reduzindo significativamente os custos com a coleta individual, transporte e teste de amostras.

Development and validation of a monoclonal antibody blocking elisa for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4)

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement ( κ = 0.75, confidence limits = 0.63–0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres ( r = 0.76, p ⪡ 0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.

Protective Immunity against Newcastle Disease: The Role of Cell-Mediated Immunity

The role of cell-mediated immunity (CMI) in protection of birds from Newcastle disease was investigated by two different strategies in which only Newcastle disease virus (NDV)-specific CMI was conveyed without neutralizing antibodies. In the first strategy, selected 3-wk-old specific-pathogen-free (SPF) birds were vaccinated with either live NDV (LNDV), ultraviolet-inactivated NDV (UVNDV), sodium dodecyl sulfate-treated NDV (SDSNDV), or phosphate-buffered saline (PBS) (negative control) by the subcutaneous route. Birds were booster vaccinated 2 wk later and challenged with the velogenic Texas GB strain of NDV 1 wk after booster. All vaccinated birds had specific CMI responses to NDV as measured by a blastogenesis microassay. NDV neutralizing (VN) and hemagglutination inhibition (HI) antibody responses were detected in birds vaccinated with LNDV and UVNDV. However, birds vaccinated with SDSNDV developed antibodies that were detected by western blot analysis but not by the VN or HI test. Protection from challenge was observed only in those birds that had VN or HI antibody response. That is, birds with demonstrable CMI and VN or HI antibody response were protected, whereas birds with demonstrable CMI but no VN or HI antibody response were not protected. In the second strategy, birds from SPF embryos were treated in ovo with cyclophosphamide (CY) to deplete immune cells. The birds were monitored and, at 2 wk of age, were selected for the presence of T-cell activity and the absence of B-cell activity. Birds that had a significant T-cell response, but not a B-cell response, were vaccinated with either LNDV, UVNDV, or PBS at 3 wk of age along with the corresponding CY-untreated control birds. The birds were booster vaccinated at 5 wk of age and were challenged with Texas GB strain of NDV at 6 wk of age. All birds vaccinated with LNDV or UVNDV had a specific CMI response to NDV, VN or HI NDV antibodies were detected in all CY-nontreated vaccinated birds and some of the CY-treated vaccinated birds that were found to have regenerated their B-cell function at 1 wk postbooster. The challenge results clearly revealed that CY-treated birds that had NDV-specific CMI and VN or HI antibody responses to LNDV or UVNDV were protected, as were the CY-nontreated vaccinated birds. However, birds that had NDV-specific CMI response but did not have VN or HI antibodies were not protected from challenge. The results from both strategies indicate that specific CMI to NDV by itself is not protective against virulent NDV challenge. The presence of VN or HI antibodies is necessary in providing protection from Newcastle disease.

Antibody response of calves to immunoaffinity-purified bovine respiratory syncytial virus VP70 after vaccination and challenge exposure

Summary Immunoaffinity-purified bovine respiratory syncytial virus (brsv) fusion (F) protein elicited anti-brsv-specific antibody responses in brsv-seronegative calves. After primary vaccination, all calves seroconverted to brsv as determined by the virus neutralization (vn) test and developed anti-F protein antibodies detectable by protein immunoblot analyses. Subsequent vaccinations induced > twofold increase in vn titer in 3 of 9 (33%) calves, and 1 calf became vn-negative, but still had nonneutralizing antibody detectable by protein immunoblot analysis. This calf remained seronegative after challenge exposure. Two groups of calves were vaccinated im with immunoaffinity-purified brsv F protein. Each dose was 2 ml containing 20 μg of purified F protein. Freund's adjuvants were used for all vaccinations, with Freund's complete adjuvant used for the primary vaccination and Freund's incomplete adjuvant for subsequent vaccinations. The vaccine was administered to both groups at weeks 0 and 3; the first group received a third vaccination at week 21. Group-1 and -2 vaccinated calves and non-vaccinated contact controls were intranasally aerosol challenge-exposed with low cell culture-passage brsv on weeks 22 and 9, respectively. Eight of 9 vaccinated calves did not develop a humoral anamnestic response following challenge exposure, as demonstrated by vn test and protein immunoblot analyses. Calf 14 from group 1 which had a 1:2 vn antibody titer prior to vaccination, was the only calf that developed an anamnestic response. This suggests that vaccine-induced antibodies interfered with the immune response or that the challenge virus (and the virus that calf 14 was infected with before challenge exposure) contained different F protein epitopes, compared with the purified F protein immunogen.

Duck viral hepatitis type 1 vaccination: Monitoring of the immune response with a microneutralisation test in pekin duck embryo kidney cell cultures

A microneutralisation test in Pekin duck embryo kidney cell cultures with attenuated duck hepatitis virus (DHV) type 1 is described for monitoring and evaluating the immune response following vaccination of Pekin ducks against duck hepatitis type 1. Serum samples from 19 flocks of commercial fattening ducks 7 to 8 weeks of age had no detectable antibodies. Breeding ducks experienced exposure to DHV type 1 during the growing period as evidenced by some positive reactions in the VN test. Two consecutive subcutaneous vaccinations prior to the first breeding season resulted in high, and in most cases, uniform titres against DHV with small standard deviations and mostly narrow 95% confidence limits of the respective mean values. Additional vaccinations prior to further breeding seasons did not result in augmentation of the humoral immune response.

Immunofluorescence in the serological diagnosis of parainfluenza type 3 and respiratory syncytial virus infection in calves

The fluorescent antibody (FA) test is compared with the haemagglutination inhibition (HI) test for parainfluenza virus type 3 (PI-3) and virus neutralisation (VN) test for respiratory syncytial (RS) virus for detection and titration of virus-specific antibodies. In experimentally inoculated calves PI-3 and RS virus FA tests detected seroconversion at the same time as HI and VN tests, however, in serially diluted sera, the FA test was positive to higher dilution. In studies with paired samples from calves from four farms with respiratory problems, the FA test gave similar results to PI-3 HI and RS virus VN tests. Large increases in antibody titre to RS virus detected by FA and VN tests indicated this was the problem on two of the farms. Individual animals showed large rises to PI-3 by FA and HI test on three farms. It is concluded that the FA test provides a rapid and sensitive alternative to the more conventional serological tests for respiratory viruses.

A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus: II. Application

The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. alternative to the existing VN test for the quantification of antibodies against FMD virus.

Detection of stimulus-related (evoked response) activity in the electroencephalogram (EEG).

In clinical neurophysiology the visual evaluation of averaged evoked waveforms may be rather difficult, allowing no definite answer to the question of whether stimulus-related activity is present or not. This problem calls for appropriate statistical procedures testing for the presence of significant response activity. In the present work the commonly used averaging technique was supplemented by phase value measurements of Fourier components in the poststimulus sample functions. If the latter contain no evoked activity, the phase values may be assumed to be uniformly distributed in the interval 0°-360°(the null hypothesis assumption). Contrary to this, an aggregation of phase values and, hence, a nonuniform distribution will result if evoked activity is present. The statistical problem may thus be reduced to testing a null-hypothesis assumption of uniformity. In the present investigation a statistical procedure of the Kolmogorov-Smirnov type, Kuiper's VN test, appeared to be a valuable aid in the detection of stimulusrelated EEG activity.

Comparison of a newly developed enzyme-linked immunosorbent assay with complement fixation and neutralisation tests for serology of bovine respiratory syncytial virus infections

An indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and titration of serum antibodies to bovine respiratory syncytial virus (BRSV). The ELISA was compared with a complement fixation (CF) test and a test for virus neutralising antibody in serum (virus neutralisation [VN] test). Testing sera collected in dairy herds revealed the closest correlation between the results of the ELISA and the CF test with respect to BRSV antibody titres. The VN test detected BRSV antibodies in a higher percentage of acute phase sera compared to the other two tests in field samples and in early bleedings of experimentally infected calves. However, the VN test was less effective in making a diagnosis of BRSV infections on the basis of a significant titre increase in paired sera. For this purpose the ELISA was found to be the most sensitive test.