Characterization of specific antibodies and the establishment of sandwich ELISA and ELISPOT systems for swine IL-4

We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.     

Observations on mouse-infective stocks of Cowdria ruminantium: microscopical demonstration of the Kwanyanga stock in mouse tissue and the carrier-status of the Senegal stock in mice

The behaviour of two different stocks of Cowdria ruminantium was investigated in mice. The mouse-pathogenic Kwanyanga stock of C ruminantium was microscopically demonstrated in mice in capillary endothelial cells of the lung, spleen, kidney, liver and brain. Mice of the ninth passage of the Senegal stock, which is infective but not pathogenic to mice, were kept alive for a year. Their blood and homogenised spleens, inoculated intravenously, caused fatal heartwater in a goat. However, the Senegal stock could not be demonstrated microscopically in mice. These results indicate the possible role of rodents in the epidemiology of heartwater.     

Comparison between 3 antigens for the serodiagnosis of heartwater disease by indirect immunofluorescence

A cell line of bovine endothelial cells (E5), infected with 3 different stocks of Cowdria ruminantium, was used as antigen in an indirect fluorescent antibody test for the serodiagnosis of heartwater. These antigens were compared to peritoneal macrophages from mice infected with the Kümm stock and to caprine neutrophils in primary cultures from goats infected with 4 different stocks of Cowdria. The use of endothelial cell cultures proved to be superior in all respects. The antigens can be produced in large quantities at a low cost, contrary to the other types. The reaction is easily and quickly read, compared to the laborious reading of neutrophil or macrophage antigens which often contain few and small colonies of Cowdria. Moreover, not all stocks are suitable for the preparation of neutrophil antigens, while macrophage antigen can only be obtained with the Kümm stock. Endothelial cell antigens also distinguish serotypes in C. ruminantium, but these differences seem to be less pronounced than those found with neutrophil antigens. Finally, the specificity of endothelial cell antigens appears to be better than that of Kümm antigen and comparable to that of neutrophil antigens. The use of Kümm antigen may have been responsible to a large extent for past unexplained positive serological results on certain Caribbean islands where it has not been possible to isolate Cowdria and where no clinical evidence of the disease has been found.     

Development of a monoclonal blocking ELISA for the detection of antibodies against fowlpox virus

To provide a fast and easy method to detect antibodies against fowlpox virus (FWPV) particularly in high numbers of chicken sera we established a monoclonal blocking enzyme-linked immunosorbent assay (ELISA). We chose two different monoclonal antibodies (mAb), anti-FWPV 3D9/2B3 and anti-FWPV 8F3/2E11, which are both directed against the 39-kDa protein of FWPV strain HP-1. The blocking ELISA depends on the blocking of mAb binding to solid-phase antigen in the presence of positive serum. For an epidemiological study a total of 184 serum samples from Gambian chicken flocks were analysed against each of the mAbs. Four of the sera were shown to contain FWPV antibodies. These four sera showed a positive cut-off value of more than 50% inhibition exclusively in the test against the mAb anti-FWPV 8F3/2E11. This phenomenon can be explained by the binding of the mAbs to distinct epitopes on the same protein.     

Cowdria ruminantium: stability and preservation of the organism

Blood collected in either sodium heparin or disodium edetate vacutainers from febrile goats infected with 4 isolates of Cowdria ruminantium and cryopreserved with 10% dimethyl sulphoxide at -70 degrees C and -196 degrees C was an effective stabilate to initiate heartwater infections in goats. A homogenized pool of whole Amblyomma variegatum ticks in Snyder's buffer, maintained at -196 degrees C, was used to infect a goat with C. ruminantium. Liver and spleen collected from Swiss mice infected with the Kwanyanga isolate of C. ruminantium were homogenized in Snyder's buffer, maintained at -196 degrees C and were used to initiate infections in mice. Fresh blood collected from febrile goats and maintained at 4 degrees C for as long as 72 h was infectious to mice. Neutrophils separated from blood of C. ruminantium infected goats and maintained in modified RPMI medium at 37 degrees C for 68 h were infectious for a goat. Similarly neutrophils from a 2nd infected goat maintained for 96 h at 37 degrees C were infectious for mice.     

Serotypes in Cowdria ruminantium and their relationship with Ehrlichia phagocytophila determined by immunofluorescence

Two tick-borne rickettsial pathogens of ruminants, Cowdria ruminantium (causative agent of heartwater disease) and Ehrlichia phagocytophila (causative agent of tick-borne fever), were successfully cultivated in caprine or ovine neutrophilic granulocytes. Infected cultures were subsequently used as antigens in the indirect fluorescent antibody test. Low-level bilateral serological cross-reactions could be detected between Cowdria and Ehrlichia. In addition, comparison of five Cowdria stocks using immunofluorescence demonstrated the existence of distinct serotypes within the genus of Cowdria. It is concluded that the occurrence of these serotypes will considerably complicate the current serodiagnosis of heartwater.     

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.     

The application of the indirect fluorescent antibody test in research on heartwater

The preparation of the antigen, details of the reagents, the titration of the antispecies conjugates and the execution of the indirect fluorescent antibody test are described. The sensitivity and specificity of the test and its applicability to the detection of antibodies to Cowdria ruminantium are recorded. The test is both highly specific and sensitive and can be applied to a wide range of studies on heartwater, including epidemiology, determination of the C. ruminantium infection rate of Amblyomma ticks and the evaluation of immunization against heartwater. The test can also be used to detect antibodies to the heartwater agent in the sera of game.     

Heartwater serology: some problems with the interpretation of results

Antibodies in the sera of domestic ruminants that have been infected with Ehrlichia bovis and other ehrlichial agents cross-react with the Kümm strain of Cowdria ruminantium used in the indirect fluorescent antibody test as antigen. These cross-reactions are also shown by the Elisa test in which the Ball 3 strain of the heartwater agent is used as antigen.     

A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle

Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Preliminary observations on the use of the capillary flocculation test for the diagnosis of heartwater (Cowdria ruminantium infection).

A capillary flocculation test was developed to diagnose heartwater disease of ruminants. Antigen was prepared from the brains of cattle and goats highly infected with Cowdria ruminantium. Sera were obtained from experimentally infected ruminants which either recovered naturally or with the aid of oxytetracycline treatment. Antibodies were first detected one to two weeks after clinical recovery or after treatment, and persisted for periods varying between one and four weeks. Control sera collected from cattle (sheep) and goats in the Netherlands where heartwater does not occur, or from animals serologically positive for Anaplasma marginale or Eperythrozoon ovis infections, did not react to the test.     

Increased pathogenicity of an Ehrlichia-like agent after passage through Amblyomma hebraeum: a preliminary report

After being passaged through 3 generations of Amblyomma hebraeum, an Ehrlichia-like agent isolated from an adult Hyalomma truncatum female became more pathogenic and elicited a disease in sheep indistinguishable from heartwater. Cross-immunity between this agent and several stocks of Cowdria ruminantium and high levels of antibody elicited by the agent against 2 stocks of C. ruminantium in the indirect fluorescent antibody test, confirmed its close relationship to Cowdria.     

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.     

Comparing the detection of exposure to Ehrlichia ruminantium infection on a heartwater-endemic farm by the pCS20 polymerase chain reaction assay and an indirect MAP1-B enzyme linked immunosorbent assay

Detection of heartwater is not always easy especially because all the serological assays so far available either have poor sensitivity or specificity. The indirect MAP-1B ELISA has been reported to be the most specific test for heartwater, although it does also detect antibodies to some closely related ehrlichial agents. This study was undertaken to compare two methods for the detection of heartwater infection caused by the ehrlichial agent Ehrlichia (Cowdria) ruminantium. Fifteen cattle on a heartwater-endemic farm infested with high numbers of Amblyomma hebraeum ticks, and hence exposure to E. ruminantium infection were monitored over an 8-week period by pCS20 PCR and an indirect MAP-1B ELISA. Infection was detected by pCS20 PCR in most animals with the highest number of positives (60%) in week 6 of the study. Similarly, exposure to E. ruminantium was detected by indirect MAP-1B ELISA in some animals, with the highest number of seropositives (27%) at weeks 2-6 of the study. The data demonstrated a fluctuating rickettsaemia in cattle in a heartwater-endemic area. Comparison of the two tests indicated that the pCS20 PCR assay was more reliable because it detected more infections than the indirect MAP-1B ELISA and would therefore be the method of choice for detection of E. ruminantium infection.     

Immunodominant antigens of zoospores from ovine isolates of Dermatophilus congolensis.

Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.     

A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice injected with tick homogenates

Amblyomma hebraeum ticks, collected in the field and individually homogenized, were injected into mice. Thirteen out of 240 ticks were shown to be infected with the heartwater agent. Antibodies against Cowdria ruminantium were detected in the sera of the mice by means of the indirect fluorescent antibody test. Giemsa-stained smears, prepared from the haemocytes of the ticks, revealed morphologically different forms of the heartwater agent. A strain of C. ruminantium, designated the Welgevonden strain, was isolated in mice from one of the infected ticks and passaged in mice for 8 generations. When inoculated intravenously, it was highly infective to mice, sheep and cattle. The murinotropism of the Welgevonden strain is compared with that of other strains previously described.     

Cowdria ruminantium is recognized by a monoclonal antibody directed against the major outer membrane protein of Chlamydia trachomatis.

The relationship between Cowdria ruminantium and Chlamydia trachomatis was studied by immunofluorescence. A monoclonal antibody directed against the major outer membrane protein of C. trachomatis recognized rickettsial colonies of C. ruminantium in infected goat brain. No specific fluorescence was observed in non-infected brain. Two commercial Chlamydia-specific monoclonal antibodies as well as polyvalent anti-Chlamydia rabbit serum recognized C. trachomatis, but did not recognize Cowdria. Moreover, polyvalent Cowdria antiserum failed to recognize C. trachomatis cultivated in HeLa cells. It is concluded that Cowdria and Chlamydia are to a certain extent related, confirming similarities in ultrastructure and developmental cycle.     

A mouse lethal dose assay for detection and titration of Cowdria ruminantium (Kwanyanga strain) in goats and ticks

A mouse lethal dose assay was used to detect a mouse pathogenic strain (Kwanyanga) of Cowdria ruminantium, the etiological agent of heartwater in goats and ticks. The titer of the rickettsial organisms in goat blood was directly related to the febrile response of the goat and the rickettsia were undetectable after the fever subsided. The maximum rickettsial titer in goat blood was 10(3) mouse LD50 ml-1. Cowdria-infected goat blood was shown to retain infectivity when held on ice for up to 2 h, but when held at room temperature infectivity declined by greater than 50% in 2 h. The mouse assay detected Cowdria in feeding female Amblyomma variegatum only on the eighth day of feeding and in feeding males on the second and eleventh days of feeding. Cowdria was shown to persist in the hemolymph of the soft tick Ornithodoros coriaceus for a period of at least 2 years.     

Seroprevalence of antibodies against Borrelia burgdorferi and Anaplasma phagocytophilum in cats

OBJECTIVE: To determine whether cats in the northeastern United States develop serum antibodies against antigens of Borrelia burgdorferi and Anaplasma phagocytophilum and whether coinfection with the 2 organisms occurs. SAMPLE POPULATION: Serum samples from 84 healthy cats and 9 cats with lameness, fever, anorexia, or fatigue. PROCEDURE: Serum antibodies against B. burgdorferi and A. phagocytophilum were measured with an ELISA incorporating a whole-cell preparation or purified recombinant antigens, by means of Western blot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: ELISA results indicated that 44 of 93 (47%) sera contained antibodies against > or = 3 B. burgdorferi antigens, whereas 43 (46%) were reactive to whole-cell B. burgdorferi. Serum reactivity to protein 35, VlsE, and outer surface proteins A and F was most common. Seropositivity to > or = 3 antigens occurred at the same rate (5/9) in the 9 ill cats as in the 84 healthy cats (46% [39/84]). Of 13 sera reactive to recombinant antigens, 9 were seropositive as measured by Western blot testing with whole-cell antigen. Seropositivity rates of 30% and 38% were detected for antibodies against A phagocytophilum via IFA and ELISA testing, respectively. Fifteen (16%) sera had antibodies against both pathogens. CONCLUSIONS AND CLINICAL RELEVANCE: Cats living in areas infested by Ixodes scapularis ticks are exposed to B. burgdorferi and A. phagocytophilum and, in some instances, may be coinfected. Most cats appeared healthy. An ELISA incorporating specific recombinant antigens may be used adjunctively with Western blot and other assays to confirm B. burgdorferi and A. phagocytophilum infection in cats.