Seroepidemiological study of Rift Valley fever (RVF) in animals in Saudi Arabia

Serological prevalence of IgG antibodies against Rift Valley fever (RVFV) virus was investigated in 22 major localities in five different regions of Saudi Arabia where vaccination against RVF virus (RVFV) is not practiced. The study excludes the southwestern region where a major outbreak of RVF occurred in 2000 and where annual vaccination of ruminants is practiced. Sheep and goat IgG-sandwich ELISA were used to test serum samples from sheep and goats, and bovine IgG-sandwich ELISA was used to test cattle sera. A nonspecies-specific, nonantibody isotype-specific ELISA was used to test camel sera. A total of 3,480 sheep, goats, cattle and camels with no previous history of vaccination against RVFV were randomly tested. All tested animals were negative for IgG class antibodies against the virus except four out of 1,508 sheep and three out of 913 goats, which tested positive. All animals were clinically normal and no evidence was found of virus activity in the studied areas. It is, therefore, most likely that those rare positive cases, which constituted 0.002% of the total animals tested, were either false positives or vaccinates smuggled from the outbreak zone. The need for regular monitoring of animals both within the outbreak zone of 2000 and other parts of the kingdom is strongly emphasized.     

Competitive ELISA for the detection of antibodies to Rift Valley fever virus in goats and cattle

Rift Valley fever virus (RVFV) is one of the important emerging viral diseases of serious impact in public health and animal hygiene both in human and animal industries. In this study, we developed a monoclonal antibody-based competitive ELISA for the detection of antibodies to RVFV in goats and cattle. The recombinant N protein of RVFV was expressed in E. coli with a six-histidine tag, and the purified N protein was used for detecting antigen with a competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goats and cattle with a sensitivity of 94.7% (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, the C-ELISA did not show any cross-reactivity with positive sera against arboviruses such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever virus, which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results of the present study indicate that the C-ELISA is a simple, rapid and convenient serodiagnostic method for RVFV in goats and cattle.     

Recombinant nucleocapsid-based ELISA for detection of IgG antibody to Rift Valley fever virus in African buffalo

Wild ruminants are thought to serve as natural hosts for Rift Valley fever virus (RVFV) but the role of these animals as reservoirs for RVFV during inter-epidemic periods and as amplifiers during epidemics is not well understood. An indirect enzyme-linked immunoassay (I-ELISA) based on the recombinant nucleocapsid protein (rNp) of RVFV was validated for the detection of specific IgG antibodies in African buffalo. Data sets derived from testing buffalo sera from Kenya (n=405) and South Africa (n=618) were dichotomised according to the results of a virus neutralisation test. The assay characteristic performance was analysed using threshold values optimised by the two-graph receiver operating characteristics (TG-ROC) analysis, and by mean plus two, as well as by mean plus three standard deviations derived from I-ELISA PP values in uninfected animals. Among 1023 buffalo sera tested, 77 (7.5%) had detectable virus neutralising antibodies. The assay had high intra- and inter-plate repeatability in routine runs. At a cut-off optimised by the TG-ROC at 95% accuracy level, the diagnostic sensitivity of the I-ELISA was 98.7% and diagnostic specificity 99.36% while estimates for the Youden's index (J) and efficiency (Ef) were 0.98 and 99.31%. When cut-off values determined by traditional statistical approaches were used, the diagnostic sensitivity was 100% but estimates of J, Ef and other combined measures of diagnostic accuracy were lower compared to those based on cut-off value derived from the TG-ROC. Results of the study indicate that the I-ELISA based on the rNp would be useful for seroepidemiological studies of RVFV infections in African buffalo.     

Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera

An indirect enzyme-linked immunosorbent assay (I-ELISA) for the detection of specific IgG immunoglobulins against Rift Valley fever virus (RVFV) was validated in-house. A total of 3055 sera from sheep (n = 1159), goats (n = 636), cattle (n = 203), African buffalo (n = 928), and other wild ruminants (n = 129), including eland, kudu, and black wildebeest, was used. Sera from domestic ruminants were collected in West (n = 10), South (n = 1654) and East Africa (n = 334), and sera from wild ruminants (n = 1064) were collected in South Africa. In addition, 136 sera from eight experimentally RVFV-infected sheep, taken during a period of 28 days post infection (dpi), were used to study the kinetics of RVFV antibody production. Field sera were tested by the serum neutralization (VN) test and experimental sera by VN and haemagglutination-inhibition (HI) test. Based on VN test results, negative sera were regarded as reference controls from RVFV-free, and positive sera were regarded as reference controls from RVFV-infected subpopulations of animals. ELISA data were expressed as the percentage positivity (PP) of an internal high positive control. The two-graph receiver operating characteristics approach was used for the selection and optimization of I-ELISA cut-offs including the misclassification costs term and Youden index (J). In addition, cut-off values were determined as the mean plus two-fold standard deviation of the result observed with the RVFV-free subpopulations. Established optimal cut-offs were different for each of the data sets analyzed, and ranged from 1.65 PP (buffalo) to 9.1 PP (goats). At the cut-off giving the highest estimate of combined measure of diagnostic accuracy (highest J value), the I-ELISA test parameters were determined as follows: (1) Diagnostic sensitivity (%): cattle--84.31, buffalo--94.44, sheep--98.91, goats--99.18. (2) Diagnostic specificity (%): cattle--99.34, buffalo--98.28, sheep--99.16, goats--99.23 and other game ruminants--99.26. In the group of RVFV-experimentally infected sheep, seroconversion In all individuals was detected by VN on 4-6 dpi, by HI on 5-7 dpi, and by I-ELISA on 6-7 dpi. All tests showed the same kinetic pattern of immunological response. Antibody levels were low for a very short period before increasing to high titres, after which it was easily detectable by all tests. Compared to traditional tests, the lower sensitivity of I-ELISA in the detection of the earliest stage of immunological response may be practically insignificant, particularily when this assay is used in population-based, disease-surveillance programmes. The high sensitivity and specificity of I-ELISA established in this study, especially for the statistically more representative subpopulations of animals tested, seem to support this prediction. Test parameters determined in this study should, however, be regarded as in-house diagnostic decision limits, for which further updating is recommended, particularly for specimens from other countries, and preferably by applying a standardized method for sampling of new subpopulations of animals to be targeted by the assay.     

Clinical, virological and serological response of the West African dwarf sheep to experimental infection with different strains of Rift Valley fever virus.

West African dwarf sheep were inoculated with three different strains of Rift Valley fever virus (RVFV). Using infective mouse serum as the source of virus classical RVFV disease characterised by sudden onset, a sharp but transient febrile response, viraemia, abortions and the development of specific RVFV antibodies in surviving animals was observed. The severity of clinical response was, however, dependent on the strain of virus used, with animals inoculated with Smithburn's neuroadapted strain showing a milder response than those inoculated with either the Nigerian or Lunyo strain. The inoculation of sheep with RVFV infective mouse brain material of the three different strains resulted in a mild febrile response with low level viraemia. Immune sera from sheep inoculated with both the Nigerian and Smithburn's neurotropic strains did not neutralise the Lunyo virus strain in a mouse intracerebral neutralisation test; the reverse, however, was not the case. The findings indicate that the West African dwarf sheep is highly susceptible to RVFV infection and that previous reports of only a mild clinical response following inoculation with the Nigerian strain were due to infective mouse brain rather than infective mouse serum.     

Application of a recombinant protein for the serological diagnosis of canine leishmaniasis

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).     

Determination of IgM and IgG antibodies to Toxoplasma using the IFA test, ELISA, and Dot-ELISA procedures

The dot enzyme-linked immunosorbent assay (Dot-ELISA) and the enzyme-linked immunosorbent assay (ELISA) were compared with the immunofluorescent antibody test (IFA) for detection of IgM- and IgG-specific antibodies to human toxoplasmosis. Reciprocal titers were determined in all three assays using sera from 56 patients with suspected toxoplasmosis or with symptoms and diseases requiring exclusion of toxoplasmosis and control sera from 56 healthy persons. Using the Dot-ELISA, six patient sera (10.7%) were positive at titers of greater than equal to 1024 for IgM antibodies (titer range 1024-16 384) and 47 sera (84%) were positive for IgG antibodies (titer range 16-262 144) at a titer of greater than or equal to 16. One control serum was reactive for IgM (titer 1024) and 10 control sera (18%) were positive for IgG in the Dot-ELISA. In the ELISA, at titers of greater than or equal to 128, five sera (9%) were reactive for IgM (titer range 128-512) and 52 sera (92.8%) were reactive for IgG (titer range 32-8192) at a titer of greater than or equal to 32; no control sera gave positive reactions for IgM while 10 sera (18%) were positive for IgG in the ELISA. Using the IFA test at reciprocal titers of greater than or equal to 16, four sera (7.1%) were positive for IgM (titer range 32-512), and 51 sera (91%) were positive for IgG (titer range 16-8192). None was reactive for IgM, and eight sera (14%) were positive for IgG (titer range 32-128) in the IFA test. The Dot-ELISA correlated well with the IFA test (correlation coefficient = 0.895) and the ELISA correlated slightly higher with the IFA test (correlation coefficient = 0.910) for detection of IgG antibodies to Toxoplasma gondii.     

Dot enzyme-linked immunosorbent assay (Dot-ELISA): comparison with standard ELISA and complement fixation assays for the diagnosis of human visceral leishmaniasis

The dot enzyme-linked immunosorbent assay (Dot-ELISA), standard ELISA and the complement fixation (CF) tests were compared in the serodiagnosis of African visceral leishmaniasis (kala-azar). Assay sensitivity was determined using sera from 44 patients with parasitologically confirmed kala-azar. Using the Dot-ELISA, 42 of 44 patients (95%) were positive at a reciprocal titer of greater than or equal to 32 (titer range 512-524 288). In the standard ELISA technique, 43 of 44 patients (98%) were positive (titer range 32-32 768). At a reciprocal titer of greater than or equal to 8 in the CF test, 35 patients (80%) were positive, 1 (2%) was negative and 8 patients (18%) showed anticomplementary (AC) activity (titer range 8-2048). Specificity, determined using 33 sera from healthy individuals not living in endemic areas, was 97% in both the Dot-ELISA and the standard ELISA (32 of 33 sera); in he CF test, all sera were negative except 1 (3%) which showed AC activity. Sera from patients with Chagas' disease cross-reacted in the dot-ELISA up to a titer of 512. In the standard ELISA, cross-reactions occurred mainly using sera from patients with Chagas' disease, malaria and syphilis, and to a lesser extent with sera from amebiasis, schistosomiasis and trichinosis patients. Overall titer agreement in replicate experiments was highest in the Dot-ELISA (89%), followed by the standard ELISA (80%) and the CF test (72%).     

Could bats act as reservoir hosts for Rift Valley fever virus?

The inter-epizootic reservoir host of Rift Valley fever virus (RVFV) remains unknown, although the namaqua rock rat, Aethomys namaquensis, as well as bats have been implicated. Bats can be asymptomatically infected with rabies, as well as several arboviruses; the possibility that they can act as host for RVFV therefore exists. To examine this possibility, 350 different samples (brain, liver, salivary glands and brown fat) obtained from 150 bats (comprising seven species) were tested for RVFV antigen using an enzyme linked immunosorbent assay (ELISA). None of the samples tested positive, but the ELISA proved to have limited sensitivity (> or = 10(3) TCID50/ml). In order to determine whether bats could be infected with RVFV, one Miniopterus schreibersii and two Eptesicus capensis bats were inoculated by the oral or intramuscular route with 100 ml and 30 ml, respectively, of a RVFV suspension with a titre of 10(6) TCID50/ml. None of the bats developed any clinical signs. A low concentration of RVFV antigen was found in the liver and urine of M. schreibersii, but not in brain tissue. A third E. capensis bat was inoculated by the intramuscular route and sacrificed on day 18. A low level of antigen was detected in the brown fat. These results demonstrate that bats can be infected with RVFV, and that further studies should be done to determine the potential of different bat species to act as reservoir hosts for RVFV during inter-epizootic periods.     

Evaluation of an enzyme-linked immunosorbent assay kit to detect Babesia bovis antibodies in cattle

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) to detect antibodies; to Babesia bovis was evaluated in 1000 sera from Holstein heifers. Five hundred of them were from cattle naturally or experimentally infected with B. bovis and 500 from uninfected heifers born and raised in a region free of the vector of cattle babesiosis. Additionally, the ELISA was evaluated and compared with an indirect immunofluorescent antibody (IFA) test in 374 heifers inoculated with different kinds of B. bovis antigens in four trials. The cross-reaction was also evaluated in 50 heifers infected with Babesia bigemina and 50 heifers infected with Anaplasma marginale. The mean percentage positivity of negative sera in relation to the ELISA strong positive sera was 8%. The seropositive/seronegative cutoff point was set as twice the mean percentage positivity of negative cattle sera ( = 16%). The sensitivity of the ELISA was 98% with a 95% confidence interval (CI) of 96–99%. The specificity was 95% (CI 93–97%). The agreement was 97% and the kappa value was 0.93. The predictive values of positive and negative results were 95% and 98% respectively. ELISA showed a similar sensitivity to that of the IFA test to detect antibodies to different B. bovis antigens. Its sensitivity ranged from 97.1% to 100% (CI 89–100%), while the sensitivity of the IFA test ranged from 92.8% to 100% (CI 83–100%). ELISA cross-reacted in 8% and 6% of the sera carrying B. bigemina and A. marginale antibodies, respectively, while the IFA showed 4% cross-reaction in each situation. The ELISA evaluated has the advantages of a proper sensitivity, objectivity and capacity to be adapted to test large number of samples in a short period of time. The results indicate that the ELISA is a suitable replacement for the IFA test to detect B. bovis antibodies in cattle sera, especially in epidemiological studies.     

The development and evaluation of an ELISA for the detection of antibodies to caprine arthritis-encephalitis virus in goat sera

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to caprine arthritis encephalitis virus (CAEV) in goat sera. The system was evaluated using some 1500 sera from flocks of known clinical history. From this data the interpretation limits of the system were determined. The ELISA system was compared with a gel precipitin test using 5800 sera. Of the positive sera, ELISA detected 97.3% and AGPT 61%. Further evaluation was made using 60 sera of known CAEV reactivity from the USA, and results agreed 100%. Indications are that antibody to the envelope glycoprotein gp135 is being detected. The ELISA system is more sensitive than the precipitin test and is presently being used in a CAEV flock accreditation scheme.     

Development of an antibody-detection ELISA for Fasciola hepatica and its evaluation against a commercially available test

An ELISA was developed for the detection of Fasciola hepatica antibody in serum of cattle. The assay was applied to sera from 258 naturally infected cattle, 256 non-infected cattle and six calves experimentally infected with F. hepatica. The diagnostic sensitivity and specificity of the ELISA test was 98% (95% confidence intervals, 96-100%) and 96% (95% confidence intervals, 93-98%) respectively at a cut-off value of 15% positivity. The results using sera from the experimentally infected calves showed that antibodies were first detected 2-4 weeks after infection. The ELISA test was also compared to the commercially available Bio-X bovine F. hepatica ELISA kit. A subset of 39 positive sera and 47 negative sera were selected from the samples used to evaluate the in-house test. The results indicated that the agreement between the two tests was almost perfect (k statistic=0.82).     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

Bovine leukemia virus infection in Taiwan: evaluation of the enzyme-linked immunosorbent assay and agar gel immunodiffusion test.

I evaluated an enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test simultaneously for the detection of bovine leukemia virus (BLV) antibodies. Total 1,293 serum samples were tested for ELISA and AGID test and the results were compared. The results of ELISA and AGID agreed by 1,156 out of 1,293 (89.4%). All of AGID-positive 356 sera were positive by ELISA. However, of 451 ELISA-positive sera, 95 sera were either negative or equivocal by AGID test. Eleven animals which showed ELISA-positive but AGID-negative or equivocal became AGID-positive in a year. It may be inferred that ELISA detects infected cattle earlier and with greater sensitivity than AGID.     

Evaluation of enzyme-linked immunosorbent assay in comparison with complement fixation test for the diagnosis of subclinical paratuberculosis in cattle.

An enzyme-linked immunosorbent assay (ELISA) was evaluated and compared in parallel with the standard complement fixation test (CFT) for the diagnosis of bovine subclinical paratuberculosis. Bovine sera preabsorbed with the mixture of Mycobacterium phlei and kaolin suspension were assayed for antibody activities to the crude protoplasmic antigen of Mycobacterium paratuberculosis in the ELISA. ELISA antibody titer was expressed as ELISA antibody index (EAI) value: EAI = (At-An)/(Ap-An), where At, Ap and An are the absorbance values of a 1:200 dilution of unknown test sera, a 1:400 dilution of positive control serum, and a 1:200 dilution of negative control serum. An EAI of 0.6 or greater was established as a reasonable cutoff point for a positive antibody titer by ELISA. Of the 156 sera from cattle with subclinical M. paratuberculosis-infection, 106 (67.9%) were positive by ELISA and 41 (26.3%) by CFT. Of the 3,880 sera from cattle in the herds which had no history or evidence of paratuberculosis, 3,875 (99.9%) were negative by ELISA, and 3,787 (97.6%) by CFT. Positive ELISA titers were detectable 1 to 5 months earlier than positive CFT titers in experimentally infected cattle, and 7 to 10 months earlier in naturally infected cattle. These results indicate that the ELISA should replace the CFT as the routine test of choice for the diagnosis of bovine paratuberculosis.     

Comparison of serologic techniques for the detection of antibodies against feline coronaviruses.

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.     

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

Enzyme-linked immunosorbent assay for detection of bovine immunoglobulin G1 antibody to a protoplasmic antigen of Mycobacterium paratuberculosis

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to Mycobacterium paratuberculosis in the sera of cattle. This assay was designed to minimize the nonspecific ELISA reactions caused by immunoglobulin (Ig)M by measuring only IgG1 antibodies against a protoplasmic antigen from the organism. The ELISA detected IgG1 antibodies in the sera of 58% of cattle with positive fecal cultures for M paratuberculosis compared with detection of 45% of culture-positive animals with an immunodiffusion test. In addition to its sensitivity, the ELISA apparently is highly specific because only 4% of the sera from fecal culture-negative animals gave a false-positive result.