Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.     

Influence of locoregional irradiation on local control and survival in breast cancer

A profiling ELISA was developed to detect antibody to the non-structural (NS) proteins Lb, 2C, 3A, 3D, and the polyprotein 3ABC, of foot-and-mouth disease virus (FMDV). The assay was used to examine panels of sera from naive cattle, and from experimentally infected or vaccinated animals. All sera from cattle experimentally infected with any of the seven serotypes of FMDV were positive for antibody to 2C, 3A, 3D and 3ABC, and the majority were positive for Lb. The three categories of sera could be differentiated on the basis of the presence or absence of antibody to the structural and/or NS proteins of FMDV. The assay is simple, rapid and reproducible and can be used to identify previous infection in animals which are seropositive for antibody to the structural proteins of the virus. Validating the assay with field sera demonstrated that antibody to 3ABC, and usually one or more of the other non-structural proteins, was detected only in animals reported to have shown clinical signs of FMD. Vaccinated cattle which had received less than five vaccinations, were frequently positive for antibody to 3D but were negative for antibody to 3ABC. Occasional animals which had received more than ten vaccinations had NS protein antibody profiles which were similar to those seen following infection.     

Isotype-specific antibody responses to foot-and-mouth disease virus in sera and secretions of "carrier' and "non-carrier' cattle

Isotype-specific antibody responses to foot-and-mouth disease virus (FMDV) were measured in the sera and upper respiratory tract secretions of vaccinated and susceptible cattle challenged with FMDV by direct contact or by intranasal inoculation. A comparison was made between cattle that eliminated FMDV and those that developed and maintained a persistent infection. Serological and mucosal antibody responses were detected in all animals after challenge. IgA and IgM were detected before the development of IgG1 and IgG2 responses. IgM was not detected in vaccinated cattle. Challenge with FMDV elicited a prolonged biphasic secretory antibody response in FMDV "carrier' animals only. The response was detected as FMDV-specific IgA in both mucosal secretions and serum samples, which gained statistical significance (P < 0.05) by 5 weeks after challenge. This observation could represent the basis of a test to differentiate vaccinated and/or recovered convalescent cattle from FMDV "carriers'.     

Large-scale use of liquid-phase blocking sandwich ELISA for the evaluation of protective immunity against aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina

Specific serum activity levels against four reference strains of foot-and-mouth disease virus (FMDV) were evaluated from 1634 animals vaccinated with commercial quadrivalent oil vaccines and from 746 unvaccinated, naive animals, using the liquid-phase blocking sandwich ELISA (lpELISA) test. Cows from the FMDV-free area of Argentina were tested for the absence of specific FMDV antibodies (sp FMDV Abs) and those showing lpELISA titres < 1.0 were grouped in lots of 16 animals. They were vaccinated and challenged at 90 days postvaccination (DPV) with one of four virus strains used for vaccine production and control (prototype strains). Serum samples from vaccinated and control cattle were collected 60 and 90 DPV and the level of sp FMDV Abs was determined by lpELISA. Animals were examined for clinical signs of disease. Results show that serum lpELISA titre levels directly correlate with the percentage of protected animals. It was seen that 100, 98, 93 and 87% of the vaccinated cattle with antibody titre levels > or = 2.1 were protected against challenge with serotypes C85, A87,01 Cas and A79, respectively. Evidence is also presented of seroconversion in a sample of 3-5-month-old calves vaccinated in the field, showing lpELISA titres compatible with protection against the four vaccine viruses as long as 150 DPV. Results reported in this paper strongly support the use of the lpELISA test for a rapid and reliable evaluation of the efficacy of FMDV commercial vaccines as well as for the assessment of the immunological status of cattle in FMDV-free and enzootic regions of South America.     

A histopathological comparison of the uvula between snorers and non-snorers

To study the specificity of serum antibodies against filamentous hemagglutinin (FHA) and pertactin for infection with Bordetella pertussis, we followed the acquisition of IgG serum antibodies against these 2 surface proteins of the organism in children who had been vaccinated with a monocomponent pertussis toxoid vaccine and who had experienced no symptoms of pertussis. Antibodies were estimated with enzyme-linked immunosorbent assay. In Part 1 of our study 5 consecutive samples obtained between 3 and 36 months of age from 71 children were available. Most had maternally derived antibodies to FHA (70 of 71) and pertactin (51 of 71) in the 3-month sera which declined in the subsequent sera. From about 1 year of age there were small but significant increases in antibodies against both antigens. At 3 years of age 71 of 71 had antibodies to FHA and 58 of 71 had antibodies to pertactin. In Part 2 of our study sera from 109 three-year old children were available. The 12 children with a history of family exposure to pertussis had significantly higher geometric mean titers of FHA antibodies than the 97 children with no history of family exposure. The geometric mean titers of pertactin antibodies did not differ. We suggest 3 explanations for the acquisition of FHA and pertactin antibodies in children with no history of pertussis: (1) asymptomatic B. pertussis infection in vaccinated children; (2) infection with Bordetella parapertussis; (3) infection with cross-reacting antigens from other organisms, e.g., nonencapsulated Haemophilus influenzae.     

An enzyme-linked immunosorbent assay (ELISA) for monitoring guineapigs and rabbits for Bordetella bronchiseptica antibodies

An enzyme-linked immunosorbent assay (ELISA) for monitoring antibodies specific to Bordetella bronchiseptica in guineapigs and rabbits was developed. In conventional and SPF colonies of guineapigs and rabbits, the ELISA was equally successful in detecting infected animals when compared to selective cultivation from the respiratory tract. The ELISA showed a sensitivity of 100% and a specificity of 90% in guineapigs. In rabbits the sensitivity and specificity of the ELISA were 97% and 91% respectively. In rabbit sera from infected colonies, ELISA activity showed a statistically significant correlation with titres obtained in the micro-agglutination test. Since serologically unrelated strains of the bacterium exist, the monitoring of animals for B. bronchiseptica infection by ELISA should be performed with various antigens.     

Detection of virus infection-associated antigen and 3D antibodies in cattle vaccinated against foot and mouth disease

The analysis of sera obtained from animals vaccinated or revaccinated with inactivated vaccines against foot and mouth disease (FMD) virus showed that these vaccines induced antibodies against the virus infection-associated (VIA) antigen, detectable by agar gel immunodiffusion (AGID). The present study evaluates the antibody response to protein 3D and the VIA antigen (VIAA) of FMD virus induced by different vaccines in a group of 51 calves. This response was detected using AGID and a liquid-phase blocking sandwich enzyme-linked immunosorbent assay (ELISA) for anti-3D antibodies (ELISA-3D). No anti-VIAA or anti-3D antibodies were detected after the initial vaccination. Following revaccination, animals giving positive results were detected by both methods. This immune response disappeared 60-120 days post-revaccination (dprv) according to the AGID method, and 90-180 dprv when ELISA-3D was used. Samples of oesophageal-pharyngeal fluid obtained from animals that remained positive for anti-VIAA antibodies at 90-120 dprv gave negative results for viral isolation, indicating that the transitional antibody response induced by the vaccine was due to the presence of non-structural antigens in the vaccine and not to viral infection. These results indicate that the ELISA-3D method could be used as a complementary method for sero-epidemiological studies as an indirect indicator of viral activity, as long as the age and vaccination status of the animals being sampled are taken into consideration.     

Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of na?ve cattle sera (n = 130) gave titres below log10 = 1.2, and none had a titre above log10 = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's rho, rs) between SNT and LPBE were highly significant (rs > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.     

Prevalence of cryoglobulinemia in chronic hepatitis C virus infection and response to treatment with interferon-alpha

A recombinant Schistosoma mansoni protein has been identified as a useful antigen for the detection of S. mansoni and Schistosoma haematobium antibodies. The purified recombinant protein, Sm22.3, was assayed using an enzyme-linked immunosorbent assay format against a battery of 491 well defined sera, including S. mansoni, S. haematobium, and Schistosoma japonicum infection sera, normal human sera, sera from 9 other parasitic infections, and sera from 2 additional infections. The sensitivity for detecting S. mansoni and S. haematobium infections with this single recombinant protein is 80.1%. The specificity is 94.8%. However, 15 of the 16 cross-reactive sera are malaria infection sera, and we have data suggesting that these malaria sera are actually recognizing an epitope on the vector-derived 6Xhistidine tag of recombinant Sm22.3. If this is the case, then, the actual specificity of the assay is 99.6%.     

Isotype responses to candidate vaccine antigens in protective sera obtained from mice vaccinated with irradiated cercariae of Schistosoma mansoni

In experimental schistosomiasis, sera of mice multiply vaccinated with radiation-attenuated cercariae of Schistosoma mansoni passively transfer resistance against cercarial challenge to naive mice. To further characterize these sera, we tested their protective capacities in two mouse strains (C57BL/6J and CBA/J) and compared the antigen-specific isotype compositions of the different protective sera by means of the enzyme-linked immunosorbent assay. By using an array of purified schistosomal antigens, the patterns of antibody titers and isotypes differed for each experimental group and antigen. In the most-protective C57BL/6J sera, high levels of immunoglobulin G1 (IgG1), IgG2a, and IgG2b bound to heat shock protein 70 and the integral membrane protein Sm23, whereas recognition of these antigens by less-protective CBA/J sera was lower. Glutathione S-transferase (GST) was recognized predominantly by IgM antibodies of all vaccinated groups, and a significant portion of this response was directed against carbohydrate epitopes. Antibodies specific for triosephosphate isomerase, paramyosin, and Sm32 (hemoglobinase) were present in less-protective sera and thus seem less relevant for passive transfer of resistance. The results of this study suggest a contribution of IgG antibodies specific for heat shock protein 70 and Sm23, and possibly a contribution of GST-specific IgM antibodies, to the protective effect of sera from C57BL/6J mice vaccinated with irradiated cercariae.     

Mechanomyography and oxygen consumption during incremental cycle ergometry

Brucella abortus strain RB51 was recently approved as an official brucellosis calfhood vaccine for cattle by the Animal and Plant Health Inspection Service branch of the United States Department of Agriculture. Currently available serologic surveillance tests for B. abortus do not detect seroconversion following SRB51 vaccination. The purpose of this study was to evaluate a dot-blot assay using gamma-irradiated strain RB51 bacteria for its specificity and sensitivity to detect antibody responses of cattle vaccinated with strain RB51. Dot-blot titers of sera at a recommended dosage (10(10) colony-forming units) were similar to those of sera from cattle vaccinated with similar numbers of B. abortus strain 19 and greater (P < 0.05) than titers of nonvaccinated cattle. In the first 12 weeks after vaccination with 10(10) colony-forming units of strain RB51, the RB51 dot-blot assay had 100% specificity for titers of 80 or less and a 53% sensitivity for titers of 160 or greater. Sensitivity of the RB51 dot-blot assay peaked at 4 weeks after vaccination with 10(10) colony-forming units of strain RB51. Dot-blot responses of sera from cattle vaccinated with a reduced dosage of strain RB51 (10(9) colony-forming units) did not differ (P > 0.05) from titers of sera from nonvaccinated cattle. Following intraconjunctival challenge with B. abortus strain 2308, titers on the RB51 dot-blot assay did not differ (P > 0.05) between nonvaccinated cattle and cattle vaccinated at calfhood with strain 19 or strain RB51.     

Differential effect of donor-specific blood transfusions after kidney, heart, pancreas, and skin transplantation in major histocompatibility complex-incompatible rats

The excretory-secretory (E-S) products of helminths are considered to comprise immunogenic molecules of high diagnostic value. In the present study, the serodiagnostic potential of the E-S products released in vitro by cultured female Onchocerca volvulus was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting using 190 serum samples from persons infected with O. volvulus and unexposed persons. The sensitivity of detection of anti-O. volvulus E-S antibodies was 94% for sera from patients with the generalized form of onchocerciasis and 100% for sera from patients with the chronic hyperreactive form (sowda). 95% of the sera from amicrofilaridermic persons, who subsequently became microfilaridermic within 2 years, reacted with O. volvulus E-S antigens and the donors were therefore regarded as having had a prepatent infection when first examined. These sera gave higher (P < 0.05) ELISA optical densities than sera from the same persons obtained when they had become patent, indicating a loss of antibody reactivity after emergence of microfilariae. The specificity of the E-S ELISA was 100% when sera of subjects infected with Wuchereria bancrofti were used, and at least 88% for Mansonella perstans sera. In Western blot analysis, the sera of persons with generalized onchocerciasis recognized 7 protein bands. Many E-S proteins were stained less intensely by the sera of subjects with generalized onchocerciasis than by the sera of sowda patients. Similar antigen bands were demonstrated using sera from the persons with prepatent infections.     

Immunity to porcine rubulavirus infection in adult swine

The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae. It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Detection of anti-Ro(SSA) antibodies in autoimmune diseases: comparison of five methods

Counterimmunoelectrophoresis (CIE), RNA precipitation, ELISA and immunoblotting against cytoplasmic HeLa cell extract (IB-HeLa) and erythrocyte extract (IB-RBC) were applied to detect anti-Ro(SSA) antibodies in 93 sera selected from patients with various autoimmune diseases [47 were anti-Ro(SSA) positive by CIE]. The RNA precipitation assay, which demonstrated the highest sensitivity was selected as the reference method. CIE was found to be reliable with a specificity of 100% and a sensitivity of 89%. ELISA showed a comparable specificity (95%) but somewhat lower sensitivity (72%). Antibodies to 52 or 60 kDa Ro(SSA) proteins by IB-HeLa demonstrated a high specificity (95 and 97% respectively) but a low overall sensitivity (36 and 17% respectively). Anti-Ro(SSA) antibodies to 52, 54 and 60 kDa erythrocyte proteins by IB-RBC, had a variable overall specificity (95, 97 and 57%) and sensitivity (51, 13 and 34%). The anti-52 kDa antibodies detected by IB-HeLa correlated to those found by IB-RBC (P < 0.001) and occurred predominantly in primary Sj?gren's syndrome (P < 0.001, sensitivity: 71 and 77%) as well as in sera with anti-Ro(SSA) and anti-La(SSB) antibodies (P < 0.001). These findings confirm that RNA precipitation assay has the highest sensitivity and specificity for anti-Ro(SSA) antibody detection. However, until a more sensitive ELISA is available, CIE because of its reliability appears to be the method of choice. Finally IB-RBC was found to be more sensitive than IB-HeLa for the detection of anti-Ro52 kDa antibodies.     

Relationship of impairment of schistosome 28-kilodalton glutathione S-transferase (GST) activity to expression of immunity to Schistosoma mattheei in calves vaccinated with recombinant Schistosoma bovis 28-kilodalton GST

Sera from calves vaccinated with the recombinant Schistosoma bovis-derived 28-kDa glutathione S-transferase (28GST) and subsequently naturally or experimentally exposed to Schistosoma mattheei were studied for their content of specific immunoglobulin G (IgG) and IgA antibodies to recombinant S. bovis 28GST as well as for their capacity to inhibit the enzymatic activity of the antigen. The results were analyzed in regard to the presence (natural infection) or absence (experimental infection) of a protective effect(s) (reductions in worm burden, egg load, fecal egg counts, and excretion of viable eggs) toward S. mattheei challenge. Under such conditions, no differences in the IgG- and IgA-specific antibodies to recombinant S. bovis 28GST or in the ability to block the catalytic function of the antigen between the two groups were recorded. Nevertheless, correlation analysis between the specific antibody responses to recombinant S. bovis 28GST and the inhibition of GST activity suggested an association with IgG in experimentally infected vaccinated animals, while in naturally infected vaccinated calves, the inhibitory activity appeared to be linked to a greater degree with IgA. These results suggest that in contrast to schistosomiasis in humans, IgG antibodies in calves with schistosomiasis may exhibit inhibitory functions toward GST enzymatic activity or have a modulatory effect on IgA antibody properties. Furthermore, sera from animals immunized with recombinant S. bovis 28GST recognized the native S. mattheei 28GST and achieved comparable levels of inhibition of activity of recombinant S. bovis 28GST and S. matthei 28GST, indicating the presence of cross-reactive epitopes on these two molecules.     

Protection of swine by live and inactivated vaccines prepared from a leader proteinase-deficient serotype A12 foot-and-mouth disease virus

Previously, we demonstrated that a genetically engineered variant of foot-and-mouth disease virus (FMDV) serotype A12 lacking the leader proteinase-coding region (A12-LLV2) was attenuated and induced an immune response that partially protected cattle from FMD. In this study, A12-LLV2 was tested in swine as a live or chemically inactivated vaccine. Animals vaccinated with chemically inactivated A12-LLV2 or wild-type (WT) virus in oil adjuvant developed high levels of neutralizing antibodies and were protected from FMD upon challenge. Animals vaccinated with live A12-LLV2 did not exhibit signs of FMD, did not spread virus to other animals, developed a neutralizing antibody response and antibodies to nonstructural protein 3D, and were partially protected from FMD. Animals given a similar dose of chemically inactivated A12-LLV2 in the absence of adjuvant developed a poor immune response and were not protected from FMD, indicating that limited replication was responsible for the improved immune response found in animals vaccinated with live A12-LLV2. The results demonstrate the potential of A12-LLV2 as a live-attenuated vaccine as well as a safe source of antigen for chemically inactivated vaccines.     

Fee code creep among general practitioners and family physicians in Ontario: why does the ratio of intermediate to minor assessments keep climbing?

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939-1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen.     

Influence of orthodontic treatment in the primary dentition upon development of the dentition and craniofacial growth

The aim of the present study was to compare the sensitivity, specificity and usefulness of the DIG-ELISA, DOT-ELISA and Indirect ELISA tests for determining the seroprevalence of fasciolosis in cattle under tropical conditions in Mexico. To standardize the tests, positive and negative sera to F. hepatica from 88 Holstein Freisian adult cows located in an enzootic area of fascioliosis and 88 crossbred adult cattle from a fluke-free area were used. For the epidemiological study, 85 crossbred cattle between 1 to 7 years of age were used. Animals were bled every two months, from March 1995 to September 1996 and the sera obtained were stored at -70 degrees C, until used. Indirect ELISA showed a sensitivity of 96.5% and a specificity of 98.8%, DIG-ELISA 97.5% and 80.0% and DOT-ELISA 93.1% and 95.4%, respectively. During 1995, Indirect ELISA yielded the highest levels of IgG anti-F. hepatica antibodies. However, in 1996, after animal treatment with triclabendazole, DIG-ELISA tended to show higher percentages of antibody-positive animals, but it was not significantly different (p>0.05) from the other tests. Comparisons made in parallel to the faecal sedimentation test demonstrated that all serological tests detected higher percentages of positive animals. Only one serum out of ten (10%) of Paramphistomum spp. cross-reacted with the DOT-ELISA test, but no cross-reaction was observed with sera from animals with other parasites. All ELISA tests were highly sensitive and specific; they may be recommended for use in seroepidemiological surveys for F. hepatica.     

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus)

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.