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1.
This work reports the results obtained by a new enzyme-linked immunosorbent assay (ELISA) test developed for the serological diagnosis of canine leishmaniasis.The new ELISA is based on a recombinant protein obtained by joining different antigens of Leishmania infantum.Test performances have been evaluated through the screening 227 sera of dogs, infected and uninfected by L. infantum. The new ELISA test has been compared to the indirect immunofluorescent-antibody test (IFAT) as a reference assay of canine leishmaniasis, and to a commercial ELISA.Excluding from the total number of IFAT positive sera the 27 sera with IFAT titre 1:40 (considered doubtful), the recombinant ELISA showed 97.0% specificity, 93.9% sensitivity and 95.5% agreement with IFAT. The commercial ELISA showed 78.2% specificity, 94.9% sensitivity and 86.5% agreement with IFAT.The results demonstrate a higher performance of the new recombinant ELISA test for the detection of negative samples, with a greater agreement with the reference test (IFAT).  相似文献   

2.
An indirect immunofluorescent antibody test (IFAT) and an enzyme-linked immunosorbent assay (ELISA) for specific anti-Neospora antibodies in bovine sera and foetal fluids were compared with histological examination results on aborted foetal material. The agreement between serological and histological examination results was poor, while the two serological tests showed a high degree of agreement. Serological testing of diagnostic serum samples and foetal fluids suggests that the prevalence of anti-Neospora antibodies in cattle which recently aborted is around 40%, in line with previous estimates of the number of abortions in dairy cattle caused by Neospora sp. A sero-epidemiological approach to the diagnosis of Neospora abortions in cattle may be suggested from these data.  相似文献   

3.
In this study, we developed two immunochromatographic tests (ICTs), which are nitrocellulose membrane-based immunoassays for the convenient and rapid serodiagnosis of bovine babesiosis caused by Babesia bovis (BoICT) and Babesia bigemina (BiICT). The efficacy of two ICTs was evaluated using 13 positive sera from experimentally infected cattle with B. bovis or B. bigemina. Clear results showed that the BoICT and ELISA detected antibodies in sera collected from 14 to 93 days post-infection, while BiICT and ELISA detected from 13 to 274 days post-infection. In additon, non-infected cattle, Neospora caninum, and Cryptosporidium parvum were negative in two ICTs. To evaluate the field utility of the ICTs, we tested 186 field bovine sera collected from cattle living in Yanbian (China) and Mato Grosso do Sul (Brazil). The results of ICTs were compared to those of classical serodiagnostic methods, enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence assay (IFAT). The overall concordances of BoICT were determined as 92.5 and 90.3% when the results of ELISA and IFAT were set as the reference standards, respectively. In contrast, those of BiICT showed 96.8 and 92.5% relative to the results of standard ELISA and IFAT, respectively. Conventional and rapid diagnosic devices for bovine babesiosis may provide a valuable tool in clinical and field applications.  相似文献   

4.
The aim of this study was to compare two serologic tests used to support a diagnosis of equine protozoal myeloencephalitis (EPM). Serum and cerebrospinal fluid (CSF) samples were analyzed for antibodies to Sarcocystis neurona and Neospora hughesi by indirect fluorescent antibody testing (IFAT) and surface antigens of S. neurona and N. hughesi by enzyme-linked immunosorbent assay (ELISA). The samples originated from neurologic horses with confirmed and suspected EPM (nine S. neurona, three N. hughesi), from neurologic horses with confirmed neurologic diseases other than EPM (16 horses) and from healthy horses (10). The IFAT on CSF and ELISA titer ratios showed equal sensitivity in diagnosing EPM caused by S. neurona. The ELISA titer ratios showed slightly greater specificity in diagnosing EPM than the IFAT on CSF. Overall agreement between the IFAT on CSF and ELISA titer ratio was 90.9%. The IFAT on CSF and ELISA serum/CSF ratio are indicated to help support a laboratory diagnosis of EPM.  相似文献   

5.
Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiver-operating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P(neg)=0.83), but a poor positive concordance (P(pos)=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninum was significantly associated with T. gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T. gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T. gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis.  相似文献   

6.
Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assay's negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.  相似文献   

7.
A commercially available serum antibody detection enzyme-linked immunosorbent assay for Neospora caninum in cattle was evaluated against an immunofluorescent antibody test (IFAT) by applying it to 397 sera from normal adult cattle, 352 sera from cattle which had recently aborted, and 422 sera from two herds which had a history of N caninum-associated abortions. It was evaluated in two laboratories and showed high reproducibility, repeatability and almost perfect or substantial agreement with the IFAT.  相似文献   

8.
The dot enzyme-linked immunosorbent assay (Dot-ELISA) was used for the detection of IgG antibodies to Sarcocystis muris and compared with the enzyme-linked immunosorbent assay (ELISA) and the indirect fluorescent antibody test (IFAT). In experimentally infected mice, first positive reactions occurred in the Dot-ELISA between 18 and 32 days after infection (dpi), in the ELISA between 18 and 49 dpi, and in the IFAT between 11 and 25 dpi. Maximum titers were 1:40 960 in the Dot-ELISA, 1:1280 in the ELISA and 1:2560 in the IFAT. High titers persisted until the end of the examination period (182 dpi) in all 3 tests. In immunized mice, all 3 tests detected antibodies 7 days after the first injection of protein antigen. The highest titers of 1:5120 and 1:10 240 were recorded in the Dot-ELISA after 35 days; titers of 1:1280 and 1:2560 were observed in the ELISA, and titers of 1:160 and 1:320 in the IFAT after 42 days. No false-positive reactions occurred in the Dot-ELISA and in the IFAT when 177 sera from non-infected mice were examined, but 1% (2/177) of the sera reacted positively in the ELISA. Sixty-three percent (94/150) of sera from mice infected experimentally with Toxoplasma gondii showed slight positive reactions up to 1:40 in the Dot-ELISA; 9% (13/150) of the sera reacted positively up to 1:40 in the IFAT and 4% (6/150) up to 1:20 in the ELISA. The Dot-ELISA appears to be a good alternative to the ELISA and the IFAT in the serodiagnosis of sarcosporidiosis and should be further evaluated for the serodiagnosis of other parasitic diseases.  相似文献   

9.
Neospora caninum has recently been shown to be a cause of abortions of sheep in New Zealand. A commercially available enzyme-linked immunosorbent assay (ELISA) was validated for use in sheep with sera from experimentally infected sheep. A cut-off threshold was established that demonstrated sero-conversion between 7 and 14 days post-infection. Higher inocula led to earlier sero-conversion. This ELISA was applied to 640 sera collected from rams across New Zealand and 0.625% (+/-0.61%) (4/640) were shown to be serologically positive. The four positive sera were also demonstrated to be positive by indirect fluorescent antibody test (IFAT). The ELISA evaluated here lends itself more readily to large-scale investigations than IFAT. The low background of N. caninum infection in the New Zealand sheep population suggests that N. caninum abortions could be more easily diagnosed by serological means than in populations with higher background sero-prevalence.  相似文献   

10.
A total of 384 sheep serum samples collected from two organised sheep farms was tested by dot immunobinding assay (DIA) and indirect enzyme-linked immunosorbent assay (I-ELISA) for the presence of bluetongue virus (BTV) antibodies. The results of both these assays were compared to find a sensitive, specific, rapid, easily performed and economical test for the diagnosis of bluetongue disease. DIA detected BTV antibodies in 210 samples (54.94%) and I-ELISA detected 157 positive samples (40.88%). Competitive ELISA (C-ELISA) was performed to check the discrepancies in I-ELISA and DIA. On the basis of these tests the overall agreement, relative specificity and sensitivity between ELISA and DIA were 75%, 87.6% and 100%, respectively. DIA was found to be a rapid, sensitive, easily performed and economical test as compared to ELISA.  相似文献   

11.
Human visceral leishmaniasis is endemic in the northeast of Brazil, where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. In this study, we evaluated the clinical signs of canine visceral leishmaniasis (CVL), serum protein profile and the antileishmanial IgG antibody production in 86 dogs living in northeast endemic areas of leishmaniasis. Thirty dogs from a leishmaniasis-free area were used as a control group. The major clinical signs of CVL seen were emaciation and skin ulcers (80%), followed by onychogryphosis and conjunctivitis (73%). Depilation was observed in 60% of animals while lymphadenomegaly, splenomegaly, liver enlargement or kidney involvement was less frequent (< or =20%). VL seropositive dogs presented with serum hyperproteinemia, hypoalbuminemia, hypergammaglobulinemia and decreased albumin/globulin ratio. A lower sensitivity and higher specificity was observed for promastigote indirect fluorescent antibody test (IFAT) (83 and 100%, respectively) compared with enzyme-linked immunosorbent assay (ELISA) (94 and 90%), which uses a crude extract of Leishmania. There was a positive correlation between IFAT and ELISA titers of antileishmanial IgG antibodies (Spearman test, P < 0.05), which was augmented in CVL dogs. This study found that the determination of serum protein, A/G ratio and the use of two different leishmanial serological tests like IFAT and ELISA are essential in CVL screening.  相似文献   

12.
Babesia divergens antigen obtained from bovine and gerbil erythrocytes was compared for the diagnosis of bovine babesiosis by the indirect immunofluorescence antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA). Using a range of sera from the field and from experimental cattle, it was found that antigen from the two sources did not differ significantly in sensitivity or specificity. Antigen from gerbils may, therefore, be used for serological surveys of bovine babesiosis caused by B. divergens. The IFAT and ELISA were also compared and it was concluded that they are equally effective in the detection of positive samples. For small-scale surveys, the IFAT using gerbil-derived antigen is adequate, but for large surveys the ELISA is preferable.  相似文献   

13.
Specific antibody to Eperythrozoon ovis was detected by an enzyme-linked immunosorbent assay (ELISA) in the sera of infected sheep. In the presence of parasite antigen, positive control serum showed a reaction approximately eight times that of negative serum. When compared to an immunofluorescent antibody test (IFAT), the ELISA was eight times more sensitive. Positive control sera gave a titre of 1:3200 by IFAT and 1:25,600 by ELISA. Through the use of a reference titration curve ELISA could be used as a semi-quantitative system to determine antibody levels in test sera.  相似文献   

14.
An outbreak of Neospora caninum-associated abortion occurred in a South Carolina dairy wherein greater than 10% of the herd aborted over a 4-month period. Of the total number of cows at mid-late gestation, nearly 40% (28/71) aborted while the remaining 60% (43/71) gave birth to normal calves. Immunohistochemical examination of brain tissue from a subset of aborted fetuses confirmed N. caninum as the causative agent of abortion in these animals. A variety of serological assays, including indirect fluorescence antibody test (IFAT), recombinant enzyme-linked immunosorbent assay (rELISA), ISCOM-ELISA, avidity ELISA, and Neospora agglutination test (NAT), were used to evaluate sera collected during the outbreak from 240 cows for antibodies to N. caninum. IFAT and ISCOM-ELISA testing showed that nearly 80% of the dairy cows had antibodies to N. caninum. NAT and rELISA had similar levels of seropositivity relative to IFAT and ISCOM-ELISA, but the percentage of positive sera was dependent on the cut-off value chosen. As indicated by kappa coefficient statistical analysis, ISCOM-ELISA and IFAT exhibited the highest level of agreement in identifying N. caninum-positive and -negative cows. A decrease in the percentage of seropositive cows as determined by ISCOM-ELISA and IFAT with increasing cow age was noted. However, no significant difference was observed between cow age and abortion status. In addition to these tests, an avidity ELISA was performed on all sera with high (> or =0.4) ISCOM-ELISA readings. Avidity index (AI) increased with time post-abortion suggesting that most abortions were due to recent N. caninum infection. Of the cows at risk for abortion, the mean serological AI of aborting cows was significantly lower (P<0.05) than mean serological AI of non-aborting cows.  相似文献   

15.
Seroagglutination (SAT), enzyme-linked immunosorbent assay (ELISA), and indirect fluorescent antibody staining tests (IFAT) were compared for reliability in the detection of pilus antigens K99, K88, and 987P of Escherichia coli. Test sensitivities were compared using mixtures of piliated bacteria of several strains diluted to a constant optical density with a nonpiliated strain. Relative sensitivities and specificities of the 3 tests were also compared using 55 E. coli strains that had previously been serotyped and characterized for pilus genes by DNA probe. Although specificity was not a serious problem with any of the tests, the SAT was relatively nonsensitive. The IFAT showed the greatest sensitivity of the 3 tests in detecting K88, K99, and 987P E. coli.  相似文献   

16.
A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.  相似文献   

17.
A serological study was conducted with calves experimentally infected with the protozoan parasite Neospora caninum. The animals were inoculated with either a low or high dose of N. caninum tachyzoites and temperature responses monitored daily for the first 2 weeks after inoculation. Blood samples were collected before inoculation, and at regular intervals thereafter for 1 year. Serological analysis was achieved using an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and an IgG avidity ELISA.Injection of Neospora produced a significant rise in rectal temperature in the high dose group. In addition, the lymph node draining the site of inoculation increased in size following injection in all animals, in both infected groups, before returning to normal by day 14 after injection. Both groups given N. caninum produced specific antibody that was detected by the IFAT and the ELISA, which remained elevated for the 12-month duration of the experiment. The specific Neospora antibodies produced did not cross-react in an IFAT for the detection of antibodies to Toxoplasma gondii. IgG avidity increased 2 weeks after inoculation, in both infected groups, until week 12 when infection was well established. There was a little difference between the two infected dose groups. This study demonstrates that the two different doses of N. caninum produced a similar antibody response, and that the higher dose also induced a febrile reaction. The IgG avidity ELISA was successful at distinguishing between recent and long-standing infection in this study. However, in both groups, there was fluctuation in the levels of specific antibody throughout the yearlong study, which accords with similar experiments in pregnant cattle, where it has been suggested that fluctuation may indicate periodic recrudescence of infection and a re-stimulation of antibody production by antigen.  相似文献   

18.
Neospora caninum infection provokes neurological disorders, recurrent abortion and death in dogs and cattle. Dogs are both intermediate and definitive host of N. caninum. Thus, the development of sensitive and specific immunoassays to diagnose canine neosporosis is essential to control this disease. This work investigated serum anti-neosporal IgG and IgE antibodies in 140 dogs represented by 30 healthy animals (group I), 11 dogs showing acute N. caninum infection (group II), 50 urban dogs with serological evidence of canine neosporosis in indirect fluorescent antibody test (IFAT) (group III) and 49 urban dogs without clinical and laboratory evidences of neosporosis (group IV). Enzyme-linked immunosorbent assay (ELISA) and western immunoblotting, both using a soluble N. caninum tachyzoite antigen (SNA), investigated these two isotypes of antibodies, while a Urea-ELISA measured the avidity of the IgG antibodies. Anti-Toxoplasma gondii IgG antibodies were also investigated in the animals. Anti-neosporal IgG was found in all animals from groups II and III, whereas 32.7% (16/49) of dogs from group IV were reactive. IgG antibodies of low avidity were demonstrated in dogs from group II (median 35.3%), while animals from groups III and IV had IgG antibodies of high avidity (medians of 61.5% and 61.7% respectively). IgE antibodies were found in four (13.3%) and five (16.6%) dogs from groups III and IV respectively. Dogs presenting acute infection (group II) or chronic infection (group III) had IgG antibodies to several neosporal antigens, mainly of 29-30 and 35 kDa, while 13 of 16 dogs from group IV recognized antigens from 14 to 170 kDa. Antibodies to T. gondii were detected in 36 of 50 (72%) sera from group III and 25 of 49 (51%) sera from group IV. We concluded that IgG-ELISA and Urea-ELISA with SNA may substitute for IFAT in both laboratory routine and epidemiological studies of canine neosporosis.  相似文献   

19.
The seroprevalence of feline coronavirus (FCoV) antibodies was studied in cats in southern Italy. One hundred twenty sera collected from cats belonging to catteries or community shelters and to households were tested for FCoV type I and II antibodies. The virus neutralization (VN) was performed and compared with indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Ninety-six sera tested positive for FCoV antibodies by VN and ELISA. Interestingly, ELISA revealed 2 more positive sera than did the VN test and 3 more positive sera than did the IFAT. All results were confirmed by Western blotting. ELISA proved to be more sensitive and detected a seroprevalence of about 82%. Considering the cross-reactivity of FCoV type I and type II, ELISA was able to detect antibodies against both serotypes, allowing the use of the assay as a reference test for sera screening. The high prevalence of antibodies observed indicates that FCoVs are common in southern Italian cat populations.  相似文献   

20.
A competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of antibodies against vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-IN) was compared with the serum neutralization test (SNT) using 1,106 serum samples obtained from dairy cattle on sentinel study farms in the Poás region of Costa Rica. Kappa coefficients between the C-ELISA and the SNT were 0.8871 (95% confidence interval [CI]: 0.8587-0.9155) and 0.6912 (95% CI: 0.6246-0.7577) for the VSV-NJ and VSV-IN tests, respectively. These results indicate good to excellent agreement between the 2 tests under these conditions.  相似文献   

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