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1.
West Nile virus (WNV) is an emerging zoonotic pathogen with rapid global expansion. The virus circulation is confirmed in many countries of Mediterranean Basin and Southern and Central Europe. In our study detection of specific WNV antibodies was performed in horses and cattle sera samples collected from October 2010 to April 2011. Serum samples were randomly taken from different parts of Croatia and tested by IgG and IgM ELISA. Positive serological results were confirmed by virus neutralization assay (VN-assay) and plaque reduction neutralization test (PRNT). Results showed that WNV antibodies were present in 72 out of 2098 horse sera (3.43%) and 3 of 2695 cattle sera (0.11%). The highest seroprevalence was found in Eastern Croatia in counties next to Hungarian, Serbian and Bosnia and Herzegovinian state borders. In Adriatic part of Croatia positive animals were found only in the westernmost county, near Slovenian and Italian borders. Geographic distribution and number of positive horses indicated that WNV is highly present in Croatia and spreading from East to West. However, positive horses in westernmost part of country indicate possible second origin of spreading. Location of serological positive cattle supports the hypothesis that seropositive cattle could be indicators of high WNV activity in the respective geographic regions.  相似文献   

2.
Chikungunya virus (CHIKV) is a mosquito‐borne pathogen that causes CHIKV fever. Definitive diagnosis is crucial for patients experiencing symptoms similar to other arboviral diseases because they can vary in clinical consequences. An increasing number of patients experience long‐term rheumatic effects of CHIKV infection, but these cases may not be optimally detected by molecular assays and anti‐CHIKV IgM ELISAs (M‐ELISAs) used for confirmation and screening, respectively. The subsequent confirmatory serological test, the plaque reduction neutralization test (PRNT), is laborious and time‐consuming. In this study, we evaluated a new diagnostic algorithm in which the M‐ELISA is conducted in parallel with an anti‐CHIKV IgG ELISA (G‐ELISA) and observed that the Euroimmun M‐ELISA combined with the Euroimmun G‐ELISA or the Abcam G‐ELISA exhibited excellent sensitivity and specificity for CHIKV. The combinations demonstrated perfect and near perfect inter‐rater agreement with the PRNT, respectively, suggesting their potential to be used as alternatives to the confirmatory serological PRNT assay for CHIKV.  相似文献   

3.
OBJECTIVE: To compare neutralizing antibody response between horses vaccinated against West Nile virus (WNV) and horses that survived naturally occurring infection. DESIGN: Cross-sectional observational study. ANIMALS: 187 horses vaccinated with a killed WNV vaccine and 37 horses with confirmed clinical WNV infection. PROCEDURE: Serum was collected from vaccinated horses prior to and 4 to 6 weeks after completion of an initial vaccination series (2 doses) and 5 to 7 months later. Serum was collected from affected horses 4 to 6 weeks after laboratory diagnosis of infection and 5 to 7 months after the first sample was obtained. The IgM capture ELISA, plaque reduction neutralization test (PRNT), and microtiter virus neutralization test were used. RESULTS: All affected horses had PRNT titers > or = 1:100 at 4 to 6 weeks after onset of disease, and 90% (18/20) maintained this titer for 5 to 7 months. After the second vaccination, 67% of vaccinated horses had PRNT titers > or = 1:100 and 14% had titers < 1:10. Five to 7 months later, 33% (28/84) of vaccinated horses had PRNT titers > or = 1:100, whereas 29% (24/84) had titers < 1:10. Vaccinated and clinically affected horses' end point titers had decreased by 5 to 7 months after vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: A portion of horses vaccinated against WNV may respond poorly. Vaccination every 6 months may be indicated in certain horses and in areas of high vector activity. Other preventative methods such as mosquito control are warranted to prevent WNV infection in horses.  相似文献   

4.
In serum, tracheal wash fluid, and bile from chickens that were inoculated with live or inactivated Newcastle disease virus (NDV), the kinetics and immunoglobulin (Ig) class distribution of an antibody response were demonstrated. The Ig classes (IgM, IgG, and IgA) were captured using monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (Ig-capture ELISA). The antibody specificity of the captured Ig was confirmed by binding of NDV. After inoculation with live virus, antibodies of the IgG and IgM classes were mainly found in serum. IgM was produced early from day 4 postexposure (PE) onward, IgG was detected later from day 7 PE onward, and in the tracheal wash fluid and bile, all three Ig classes were demonstrated. After inoculation of inactivated virus, a delayed response of all three classes was observed in serum, and only IgM and IgG were recognized in the tracheal fluid and bile. The type of vaccine and the mute of antigen entrance may have determined the immunoglobulin class produced. The Ig-capture ELISA assay developed in this study can be useful for evaluating various strategies to improve the efficacy of Newcastle disease vaccines and to study the evoked immune mechanisms.  相似文献   

5.
One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein-based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0-48.0), although the rNSI MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine (P < 0.0001) or naturally infected with WNV (P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.  相似文献   

6.
Enzyme-linked immunosorbent assays (ELISAs) for the detection of porcine IgM, IgA, IgG1 and IgG2 antibodies directed against Aujeszky's disease virus (ADV) are described. ADV-specific IgA and IgM were detected in an antibody capture assay, and ADV-specific IgG1 and IgG2 were detected in an indirect double antibody sandwich assay. A selected set of samples was tested in the four ELISAs and in a 24 h virus neutralization assay. Comparison of the results showed that the ELISAs were isotype-specific, sensitive, and reproducible. Samples with ADV antibody of one isotype showed that ADV-specific IgG1, IgG2 and IgM were able to neutralize the virus in vitro. In vitro neutralization of virus can be enhanced by complement. ADV-specific IgA neutralized virus only weakly. ADV-infected cells activated complement in the absence of antibody. Specific IgG2 and IgM enhanced complement activation. Analysis of the time course of antibody responses after infection or vaccination revealed that the isotype-specific ELISAs are suitable to study the humoral antibody response of pigs to the virus in mucosal secretions. Wild-type virus (strain NIA-3) and an attenuated vaccine strain (Bartha) administered intranasally induced mucosal IgM and IgA responses to the virus. In contrast, a killed vaccine (Nobivac) administered intramuscularly induced only weak mucosal IgM responses. The attenuated vaccine strain primed for a mucosal IgA memory response evoked upon challenge infection with wild-type virus.  相似文献   

7.
The use of the four-layer enzyme immunoassay (EIA) for the detection of IgG, IgM and IgA antibodies against Aujeszky's disease virus in blood and oropharyngeal swabs of infected and vaccinated pigs is described. Mean antibody titres obtained using the four-layer EIA were 6.1 and 3829 times higher compared with the indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test, respectively. The VN test detected mainly IgG antibodies, while the IgM antibodies did not react. Using the EIA, the first antiviral antibodies in sera were demonstrated on Days 5-7 after infection or vaccination. Up to the 7th day, demonstrable antibodies were almost exclusively of the IgM class. In infected pigs high titres of IgM antibodies were still detected on Day 18, while in vaccinated animals they were absent by this time. Antibodies of the IgG class appeared in infected pigs sooner (Day 7) than in vaccinated pigs (Day 10) and reached higher mean titres. Antibodies of the IgA class were demonstrable from Day 10 only in samples from infected pigs. Similar antibody dynamics and distribution were detected in oropharyngeal swabs, except that the IgG and IgM titres were roughly 100 times lower than in sera. However, titres of IgA antibodies in oropharyngeal swabs were two times higher than in sera. The greatest differences between both groups of animals were recorded on Day 18; in the infected pigs, IgG, IgM and IgA antibodies were present in sera and oropharyngeal swabs at that time, while in vaccinated pigs only IgG antibodies were demonstrable. The effect of infection and vaccination on the pattern of the immune response as well as the importance of the detection of individual immunoglobulin classes for the specificity of the enzyme immunoassay are discussed.  相似文献   

8.
Serum samples from 786 red foxes shot between January 1995 and August 1996 in the southern half of Northrhine‐Westphalia, located in western Germany, were tested for the presence of antibodies against tick‐borne encephalitis (TBE) virus using the Immunozym FSME IgG All Species‐ELISA® (Immuno, Heidelberg, Germany) as a screening test: 759 sera were negative, 23 (2.9 %) were borderline, and four (0.5 %) were positive. Nine of the 27 ELISA reactive sera were confirmed by the TBE Western‐Blot (Immuno, Heidelberg, Germany). Furthermore these 27 sera were tested for neutralizing antibodies by means of a plaque reduction neutralization test (PRNT) against TBE and West Nile viruses. Only one single serum was found to have a neutralization titre (+ 1 : 800 PRNT80) against TBE virus. All other 26 sera were negative for neutralizing antibodies against TBE or West Nile virus. Since the titre of the single serum is low, it can be interpreted that if TBE virus is present, its prevalence is extremely low. Northrhine‐Westphalia is not classified as a TBE‐endemic area. Further calculated serological testing of game and virological investigation of collected ticks in the affected area seem to be meaningful and necessary.  相似文献   

9.
West Nile virus (WNV) is a single-stranded, enveloped RNA virus capable of causing encephalitic disease in horses. Unvaccinated horses are at risk for developing WNV disease in endemic geographic regions. Effective vaccination reduces disease frequency and diminishes disease severity in vaccinated individuals that become infected with WNV. Recent data indicate CD4+ lymphocytes are required for effective protection against disease; in particular, cross talk between CD4+ and CD8+ lymphocytes must be functional. The objective of this project was to investigate immune responses in horses throughout a series of three vaccinations using a commercial inactivated vaccine under natural conditions. Immune responses to vaccination were determined by neutralizing antibody titers with plaque reduction neutralization test (PRNT), IgM titer (capture ELISA), WNV specific antibody Ig subclass responses, WNV lymphocyte proliferative responses and intracellular cytokine expression. Horses were vaccinated with a series of three vaccines at 3-week intervals using an inactivated product. An initial measure of immune activation following vaccination was determined by evaluating changes in lymphocyte cytokine expression. Interferon (IFN) gamma and interleukin (IL)-4 expressing CD4+ lymphocytes significantly increased 14 days following initial vaccination compared to unvaccinated horses (P<0.05). IFN-gamma expressing CD8+ lymphocytes also increased and remained elevated for 110 days. Antigen specific lymphocyte proliferative responses were significantly increased up to 90 days following the third vaccination (P<0.05). As expected, vaccinated horses produced increased neutralizing antibody based on PRNT data and WNV antigen-specific Ig subclass responses compared with unvaccinated horses (P<0.05). Our data indicate that WNV vaccination with an inactivated product effectively induced an antigen-specific antibody responses, as well as CD4+ and CD8+ lymphocyte activation.  相似文献   

10.
A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.  相似文献   

11.
West Nile virus (WNV) is a zoonotic pathogen of global importance. In horses with neurological signs, detection of WNV-specific immunoglobulin M (IgM) in serum is widely used to identify clinical cases of WNV encephalitis. Here, we describe the development of two monoclonal antibodies (mAbs) to equine IgM which were used in a WNV IgM-specific enzyme-linked immunosorbent assay (ELISA). Their performance was compared to an established assay based on polyclonal anti-IgM. Check test serum samples from the National Veterinary Service Laboratory (NVSL) were used to evaluate the performance of the three anti-IgM antibodies. The anti-IgM 1-22 mAb correctly identified all NVSL samples. Both the polyclonal antibody and monoclonal anti-IgM 2B-63 identified eight out of ten samples correctly. The three assays were then compared using serum samples from clinically healthy animals (n=33) and horses with neurological signs (n=21). High Spearman rank correlations (0.76-0.86) were found among the ELISA results. Inter-test agreements (weighted kappa) for assay interpretation resulted in strong agreement (0.95) of the results obtained by the mAbs and moderate agreements when monoclonal and polyclonal anti-IgM-based assays were compared. To determine the analytical sensitivities of anti-WNV IgM detection, serial dilutions of WNV IgM-positive serum samples were analyzed. The highest sensitivity was obtained by using the anti-IgM 1-22 mAb to capture IgM from equine serum. In conclusion, the use of monoclonal anti-IgM antibodies can improve the sensitivity of IgM detection in the acute phase of WN disease.  相似文献   

12.
The virus neutralization (VN) test and two enzyme-linked immunosorbent assays (blocking and indirect ELISAs) were used to detect antibodies to pseudorabies virus on serum samples of 1,000 pigs from the central part of Thailand. The results of these tests were compared to those of VN test. Using the VN test as standard, the blocking and indirect ELISAs showed respectively 95.12% and 99.37% relative sensitivity and 92.0% and 93.5% relative specificity. The two ELISAs were considered both as practical alternatives to the VN test. However, the indirect ELISA was the more suitable test for the routine screening for antibodies to pseudorabies virus in Thailand.  相似文献   

13.
Japanese encephalitis virus immunoglobulin M antibodies in porcine sera   总被引：1，自引：0，他引：1  
A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of porcine immunoglobulin (Ig)M antibodies to Japanese encephalitis virus (JEV). Antibodies in sera were captured onto the solid phase of Microtiter plates sensitized with mouse monoclonal antibodies to porcine mu heavy chain. Virus antigen binding to the lawn of IgM was quantitated by subsequent binding of peroxidase-labeled human hyperimmune anti-JEV IgG, which in the final step, catalyzed a substrate color change. In sucrose density-gradient fractionated sera from recently infected pigs, the peak of ELISA JEV IgM activity corresponded to the peak of 18-S, 2-mercaptoethanol-sensitive hemagglutination-inhibiting (HAI) antibody activity. Within 2 to 3 days, JEV-infected sentinel pigs developed high JEV IgM activity; this activity decreased within 2 weeks. Among specimens collected from 99 random swine at abattoirs in Thailand during a period of low JEV transmission, none of 25 JEV HAI-negative sera had JEV IgM activity, 7 of 74 JEV HAI-positive sera did have JEV IgM activity, and the remaining 67 sera had readily detectable JEV HAI antibodies, but lacked JEV IgM. The JEV IgM solid-phase ELISA was useful for rapidly diagnosing active or recent JEV infections in swine.  相似文献   

14.
The dominant immunoglobulin against eastern equine encephalomyelitis (EEE) virus and its duration and the longevity of the EEE virus haemagglutination inhibition (HI) antibodies were determined in sentinel and 125 immunised and hyperimmunised domestic chickens by enzyme-linked immunosorbent assay and the HI test respectively. The chickens ranged in age from 10 weeks to 18 months, were of varied pedigrees and from different countries. Results show that the HI antibody (IgG) is short-lived. It peaks and disappears within 30 days. The secondary response is dominated by the IgM immunoglobulin which is relatively long-lasting. These results are contrary to classical expectations and were observed in all the chickens studied. If these observations are found to be characteristic of birds generally, the present standard method of EEE virus seroepidemiological surveillance must be modified to be effective.  相似文献   

15.
The extreme decreased levels of IgG in dysgammaglobulinemic UM-B19 chickens are linked with decreased antibody activity. Antibody activity to T-dependent (although diminished) and T-independent antigens is present but is restricted to IgM and IgA antibodies. Complete Freund's adjuvant enhances the existing isotype pattern of serum immunoglobulins and antibodies. The antibody response to a "T-independent" antigen (B. abortus) is greatly increased by CFA in dysgammaglobulinemic chickens. Beside the appearance of high levels of IgG in dysgammaglobulinemic chickens during the first weeks of life and in transitory dysgammaglobulinemia, remarkable IgG synthesis can be temporarily induced by the mitogenic activity of LPS and even more by the regulatory function of Levamisole. Furthermore, LPS and Levamisole induce IgG antibody synthesis to concomitantly administered antigen, the IgG antibodies appearing within the normal time. Contrary to a missing feedback inhibition from total IgG to IgM serum immunoglobulins, a feedback inhibition from IgG to IgM antibodies is found. No correlation can be found between Levamisole-induced IgG immunoglobulin concentrations, and IgG antibodies. Germfree rearing for one week or longer prevents the dysgammaglobulinemic defect. The following conclusions are drawn: Early antigenic stimulation seems to be the inducing factor for dysgammaglobulinemia in UM-B19 chickens. A BG cell pool is still present in adult dysgammaglobulinemic chickens. This BG cell pool is probably diminished to a varying extent. T cell helper functions seem to be present (albeit they may be disturbed) and can be stimulated. IgG specific T cell suppression is probable. From these conclusions the etiology of the dysgammaglobulinemia in UM-B19 chickens is hypothesized to be primarily due to delayed bursal development: Immature BG cells are eliminated by environmental antigens during the neonatal period in a process similar to tolerance induction. This event, in turn, induces suppressor mechanism(s) or disturbance in helper mechanism(s).  相似文献   

16.
本试验以新城疫病毒（ＮＤＶ）感染的鸡胚尿囊液经差速离心，庶糖密度梯度离心提纯的ＮＤＶ为包被抗原，以纯化的鸡ＩgG、IgM及ＩgA免疫，制备辣根过氧化物酶标记的兔抗鸡ＩgG、IgM及ＩgA，分别建立了检测ＤＮＶ特异性ＩgG、IgM、ＩgA的间接ＥＬＩＳＡ方法，抗原最适包被量为１．３４μg/孔。酶标兔抗鸡ＩgG、IgM、ＩgA的最佳稀释度分别为１：４００，１：４００，１：１００，血清样品检测ＩgG时，最适工作浓度为１：４００，检测IgM时为１：５０。该方法具有特异性强、灵敏度快，快速简便等优点，适合于对各类新城疫特异性抗体的动态监测和大批检测，同时也为城疫免疫要理研究及流行病学调查提供了可靠手段。  相似文献   

17.
Fowl adenoviruses free of avian adenovirus-associated virus, representing 10 serotypes, were tested for cross-reactivity in an enzyme-linked immunosorbent assay (ELISA). All antigens and antisera prepared in chickens, along with uninfected control antigen and normal chicken serum, were reacted in a checkerboard pattern with ELISA. There was considerable cross-reactivity among all serotypes tested. Homologous reactions were generally, but not always, stronger than heterologous reactions. ELISA was about as sensitive as virus neutralization in detecting antibodies. The high sensitivity plus broad-spectrum reactivity should make ELISA a preferred test for the detection of adenovirus antibodies in poultry flocks.  相似文献   

18.
A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.  相似文献   

19.
West Nile virus (WNV) was recently detected in Culex pipiens mosquitoes in Morocco. The aim of this study was to evaluate the seroprevalence of WNV in humans and in domestic birds in two regions of Morocco by the detection of IgG antibodies. Blood samples were obtained from 91 human patients and 92 domestic birds from September to December 2019. All study samples were tested using competitive enzyme-linked immunosorbent assay (cELISA) and WNV neutralization tests (VNT) were performed on positive sera. Of all samples, 4 (4.39 %) humans and 4 (4.34 %) birds were found to be seropositive for flaviviruses by the cELISA test. The VNT revealed that three of the four human samples detected positive by cELISA contained neutralizing antibodies against WNV. Two bird samples were confirmed positive by VNT. These results show a significant seroprevalence of anti-WNV antibodies and therefore suggest the active circulation and exposure of human and bird populations in the northwest of Morocco.  相似文献   

20.
Enzyme-linked immunosorbent assay (ELISA) was used for detection of immunoglobulin (Ig) M and IgG antibodies against a serologically common antigen (original endotoxin protein), protease, and elastase of Pseudomonas aeruginosa. The P aeruginosa antibody in horse sera was measured, using ELISA. Horseradish peroxidase-labeled rabbit anti-horse IgM and IgG antibodies were used for enzyme-labeled antibody conjugate. 5-Aminosalicylic acid and H2O2 were used for substrate. Sera collected from a vaccinated horse, a newborn foal, and 72 healthy racehorses were investigated for antibodies against P aeruginosa by ELISA and passive hemagglutination procedure. Changes in IgM and IgG antibody titers with vaccination were clear by ELISA. In the newborn foal, significant amounts of IgM and IgG antibodies from colostrum were present on the 1st day after birth. It was shown by ELISA that the level of antibodies in the newborn decreased initially and then increased. Some antibodies against original endotoxin protein, protease, and elastase of P aeruginosa were detected in almost all the healthy racehorses investigated.  相似文献   

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