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.     

The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle

A diagnostic assay to differentiate antibodies induced by foot-and-mouth disease virus (FMDV) infection from those induced by vaccination was developed. The test is an indirect-trapping ELISA which uses a monoclonal antibody to trap the non-structural 3ABC-FMDV polypeptide expressed in E. coli. Experimental and field sera from naive, vaccinated and infected cattle were examined. Using the established threshold of 0.20 optical density units, the sensitivity of the assay was 100%, as all the experimental post-infection sera (n degree = 137) gave values greater than this threshold, irrespective of the FMDV serotype used for the infection. In contrast, more than 99% of sera from vaccinated animals were negative (225 out of 228 primo-vaccinates and 159 out of 159 multi-vaccinates). A high degree of specificity was also confirmed by the finding that 99.5% (442 out of 444) of sera from naive animals gave negative results. Serum conversion against 3ABC was first detected 8 days post-infection and demonstrable levels of 3ABC specific antibodies were detectable at least 1 year post-infection. The described 3ABC-ELISA is safe, cheap and also easy to perform in large scale serological surveys. The high specificity and sensitivity makes this test an ideal tool for FMD eradication campaigns and control programs.     

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'.     

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.     

Localizations of NS3 and E proteins in mouse brain infected with mutant strain of Japanese encephalitis virus

Infection with a mutant Japanese encephalitis virus (JEV) strain RP-2ms showed reduced neurovirulence than wild type or RP-9 strains after inoculation in BALB/c mice. However, higher intracellular viral titer was detected in Rp-2ms infected cultured cells. Localizations of non-structural 3 (NS3) and envelope (E) proteins were demonstrated by immunocytochemistry. NS3 protein was primarily found in the pyramidal neurons in cerebrum, in the molecular and granular layers of cerebellum. Neither E nor NS3 protein was detected in Purkinje cells of the cerebellum. Immunoelectron microscopic observations showed that E and NS3 proteins were positive in JEV-induced membranous systems, mainly hypertrophic rough endoplasmic reticulum (rER) and membrane vesicle structure (MVS) but not smooth membrane structure. Virus particles were seen in the Golgi apparatus, rER, nuclear envelope, MVS and cytoplasmic vacuoles. Different mechanisms of intracellular trapping in vivo provide a possible basis for attenuation of RP-2ms strains of JEV.     

The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle

Antigens that were specific to Fasciola gigantica were obtained from the whole worm homogenate of the parasite by immunoaffinity chromatography in cyanogen bromide-activated sepharose 4B columns and used for the production of monoclonal antibodies. The F. gigantica-specific monoclonal antibody was labelled with horseradish peroxidase and used for the detection of circulating antigen by the direct ELISA method in the sera of cattle experimentally infected with the parasite. Circulating antigens were detectable in the sera of the animals as from the third week after infection while negative absorbance values were obtained 2 weeks after the termination of the infection by chemotherapy with oxyclozanide. This immunodiagnostic method offers an attractive alternative as a supplement to the conventional coprological diagnosis of fasciolosis.     

Hemagglutination with crane herpesvirus

A crane herpesvirus (CrHV) grown in chicken embryo fibroblast (CEF) cells was tested for hemagglutination (HA) with erythrocytes from a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with erythrocytes from mouse, ddY and BALB/c strains, but not with those from cattle, sheep and chicken. Mice, ddY strain, showed an individual variation in agglutinability of their erythrocytes and erythrocytes from BALB/c gave a higher HA titer. The HA activity was inhibited by the sera obtained from naturally infected cranes, experimentally infected duck and immunized rabbit with CrHV. HI antibody titers of these sera showed a closely positive correlation with their neutralizing antibody titers.     

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.     

An immunohistochemical method of detecting Mycoplasma species antigens by use of monoclonal antibodies on paraffin sections of pneumonic bovine and caprine lungs

Lung samples from pneumonic lesions in cattle and goats, naturally or experimentally infected with strains of the Mycoplasma mycoides cluster, were fixed in formalin and embedded in paraffin. An immunohistochemical technique using monoclonal or polyclonal antibodies was performed on tissue sections in order to detect Mycoplasma antigens. Four monoclonal antibodies (MAbs), one (2A3) raised against M. mycoides ssp. mycoides small colony (SC) and large colony (LC), two (1D3 and 5E5) against M. mycoides ssp. capri, and one (5A10) against M. bovis, were used. A range of polyclonal antibodies, raised to the individual subspecies of the M. mycoides cluster, and one to Pasteurella haemolytica, was also used. The MAb 2A3 showed positive immunostaining in lung sections from cattle and goats naturally and experimentally infected with M. mycoides ssp. mycoides SC and LC, but not with pneumonic lesions of cattle and goats due to other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 1D3 showed immunostaining in lung sections from goats naturally and experimentally infected with M. mycoides ssp. capri, but again not with pneumonic lesions caused by other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 5E5 immunoreacted in sections from pneumonic lesions from all animals infected with one of the three M. mycoides cluster subspecies used in the study, but not with M. bovis or Pasteurella infected tissue. Immunoreaction was mainly found in the cell debris around necrotic areas, as well as in macrophages, neutrophils and epithelial cells. The localization of antigens of the M. mycoides cluster using polyclonal antisera followed basically the same pattern as that obtained with the monoclonals. However, a wide cross reactivity was found between different antisera and relatively high background immunostaining was also seen, especially in necrotic areas. The results suggest that immunohistochemical methods using monoclonal antibodies are useful tools for the diagnosis and study of the pathogenesis of pneumonia caused by the Mycoplasmas of the M. mycoides cluster.     

Piroplasms of domestic animals in the Macedonia region of Greece. 1. Serological cross-reactions

During a serological survey on haemoparasites in Macedonia, serum samples were collected from cattle, sheep and goats. All sera were tested by the indirect immunofluorescence test (IFAT); the cattle sera against Theileria orientalis, T. annulata, Babesia bigemina, B. bovis, B. divergens and B. major antigens; the sheep and goat sera against T. ovis, B. ovis, B. motasi and B. crassa antigens. Parallel tests of negative and positive control sera against all the antigens showed the existence of cross-reactions of different degrees between species of the same genus. In cattle, the most important cross-reactions were obtained against B. bigemina antigen, especially with the anti-B. bovis serum, in small ruminants against B. motasi with the anti-B. crassa serum. In the field sera, there was a high correlation between the antibody titres of B. bigemina and B. bovis, and also between the titres of these two Babesia spp. and B. divergens. A high correlation was also found between B. motasi and B. crassa, and lower ones between these two and B. ovis. The correlations of the sera titres were due to mixed infections or to cross-reactions. Therefore, the use of the IFAT is not always satisfactory for diagnosing infections in regions where animals are infected with different piroplasms.     

Somatic antigens of adult Dicrocoelium dendriticum recognised by bile antibodies of naturally infected cattle

Bile samples, from slaughtered cattle harbouring between 120 and 280 adult lancet flukes, were used to investigate the range of somatic proteins inducing local antibody responses in naturally infected animals. Lancet fluke infections induced local (bile) antibody responses against Tris-buffered saline (TBS) soluble, sodium dodecyl sulphate (SDS) soluble and 2-mercaptoethanol (2-Me) soluble somatic proteins of adult Dicrocoelium dendriticum. IgA antibody isotypes predominated in the response against buffer-soluble somatic antigens, whereas SDS-soluble and 2-Me-soluble proteins induced similar level of both IgA and IgG1 antibodies. Analysis of the antigens recognised by particular isotype-specific bile antibodies suggests that different antigens preferentially induce isotype restricted antibody responses. The bile antibody response was highly species specific, only one antigen from somatic protein extracts of Fasciola hepatica being precipitated by bile samples showing the highest reactivity against D. dendriticum.     

Detection of the putative E2 protein of hepatitis C virus in human liver

The question was asked whether a predicted envelope protein, considered to be processed from the polyprotein precursor encoded by the putative E2/NS1 region of the hepatitis C virus (HCV) genome, may be observed in HCV-infected humans. Two polyclonal antibodies against recombinant E2/NS1 proteins were prepared and their reactivity tested against liver extracts from HCV-infected patients by immunoblotting analysis. A band corresponding to a size of 44 kDa was detected in liver extracts from patients who were positive for the HCV-specific antibody anti-C100-3 but not in liver extracts from patients who did not have anti-C100-3 antibody. Additionally, no band was detected using preimmune sera or antisera which had been preabsorbed with recombinant E2/NS1 proteins. Deglycosylation studies demonstrated that the 44 kDa protein was a glycosylated form of a 38 kDa protein which corresponds to the predicted molecular weight of the putative E2/NS1 protein. These results suggest that the 44 kDa protein is a product of the E2/NS1 region. Frequent observation of the 44 kDa band in cases of chronic active hepatitis C suggests a correlation between the expression of this protein and the progression of hepatitis.     

Serological indication for persistence of bovine respiratory syncytial virus in cattle and attempts to detect the virus

To identify putative persistent bovine respiratory syncytial virus (BRSV) infections in cattle, seven cattle that had experienced BRSV infections were treated with corticosteroids for two periods of 5 days. During the 5-day periods and the 3 weeks after treatment, attempts were made to isolate BRSV from lung lavage fluid and nasal swab specimens. Fluorescent antibody tests were used to detect BRSV antigen in lung lavage cells. A BRSV specific polymerase chain reaction (PCR) assay was developed, and was performed on lung lavage samples of all seven cattle as well as on various tissues of five of the cattle. In addition, nasal swabs of 74 over-one-year-old cattle, in a closed dairy herd were also assayed by PCR. The virus or its RNA was not detected in putative carriers, by any of the methods used, whereas all positive controls were positive. After corticosteroid treatment, three of the seven cattle showed a fourfold rise in antibody titre, suggesting induction of virus replication. BRSV-seronegative sentinel calves, that were housed together with each corticosteroid-treated animal, did not develop antibodies to BRSV indicating that BRSV was not shed by corticosteroid-treated cattle, or was shed at a very low level. In addition BRSV was not detected in seropositive cattle in a closed farm in summer. Although we consider the rises in antibody titres against BRSV an indication for persistence of BRSV in cattle, BRSV or its RNA was not detected in infected cattle.     

Serologic studies on the occurrence of bovine ehrlichiosis in the cantons Zürich, Schaffhausen, Thurgau, St. Gallen and Obwalden

The purpose of this study was to investigate the geographical distribution of bovine ehrlichiosis in the Cantons Zürich, Schaffhausen, Thurgau, St. Gallen and Obwalden. To this end, an indirect immunofluorescence assay was established. The diagnostic sensitivity and specificity was estimated to be close to 100% based on the results obtained with several reference sera from the US National Veterinary Services Laboratories and the seroconversion panels of 20 heifers and cows. Blood samples from 2557 healthy cattle were tested for antibody response against Ehrlichia phagocytophila. Ninety-three animals (3.6%) were seropositive with titers ranging from 1:20 to 1:320. Most of the positive animals were found in the area north of lake Zürich spanning to the german border, in the area between Walensee and Bad Ragaz, the Rhine valley and Klein Melchtal. All seropositive animals had grazed on pastures or alps with a favourable biotope for the tick Ixodes ricinus.     

Seroprevalence to bovine immunodeficiency virus and lack of association with leukocyte counts in Italian dairy cattle

We report herein on the first serological detection of antibodies to bovine immunodeficiency virus (BIV) in Italy. According to criteria of a stratified-random sampling of dairy cattle reared in the Parma area (a province in the Po Valley, Northern Italy), sera from 3166 cows belonging to 272 herds were collected. In addition, sera of 138 bulls from eight artificial-insemination (AI) centres were sampled. Seventy-eight cows (2.5%) from 16 herds (5.8%) and seven bulls (5.1%) from two AI centres were positive for BIV-R29 antibodies in the IFA-test. IFA-positive sera assayed by Western blot had reaction to different viral proteins: 81 out of 85 sera showed antibody to p26 (considered the BIV major internal core protein); four sera reacted to other viral proteins but not to p26. Peripheral blood leukocytes of 60 seropositive and 60 seronegative animals, belonging to eight BIV-infected herds, were enumerated to assess any effect of BIV infection on white-blood cells. No significant differences were detected between the two groups. These data indicate that BIV infection is present in Italian dairy cattle--but the role of BIV in inducing disease remains unclear.     

ISCOM vaccine against contagious bovine pleuropneumonia (CBPP). 1. Biochemical and immunological characterization

A better vaccine than the existing ones against contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) would improve the chances for eradication of CBPP. In such an effort, immunostimulating complexes (ISCOMS) have been prepared from the whole detergent-solubilized cells of MmmSC and characterized biochemically and immunologically. The most efficient detergent for solubilization of the mycoplasma was MEGA-10 which yielded a high recovery of proteins in the ISCOMS. The ISCOMS showed the typical cage-like structure by EM and sedimented as 19S by sucrose gradient centrifugation. The protein pattern of the ISCOMS, analyzed in SDS-PAGE, revealed a great number of bands distributed along the gel as high and low molecular weight polypeptides. The Western blot developed with a serum from a CBPP infected animal detected a reduced number of polypeptides. In samples from whole mycoplasma cells and in ISCOMS, lectin blots revealed more than 20 carbohydrate structures. The ISCOMS induced a strong primary antibody response in mice measured by ELISA and the boost resulted in a 6-fold increase of the serum antibody response. The IgG response was distributed into various IgG subclasses with high IgG1, IgG2a and IgG2b titres while the IgG3 response was low. In cattle the ISCOM vaccine induced strong primary and long lasting secondary antibody responses of similar magnitudes as those of naturally infected animals as recorded by ELISA which persisted more than a year. IgG response was equally distributed in IgG1 and IgG2 subclasses. Also a cell-mediated immune response measured by proliferation assay was induced by low dose of ISCOMS. In the growth inhibition test, sera from vaccinated cattle readily inhibited colony growth already after the first immunization.     

A radiomicroassay for cytotoxic antibody to human spermatozoa. Quantification by tritiated actinomycin d

A radiomicroassay for titration of spermocytotoxic antibody is described. The assay used [3H]AACTINOMYCIN D ([3H]Act D) to label damaged spermatozoa in a fashion analogous to penetration by vital dye. Optimal conditions for and some kinetics of the assay are presented. The assay is sensitive, reliable, simple to perform and uses only small amounts of serum and spermatozoa. Applied to sperm antibody positive human postvasectomy sera, the assay compared favourably in sensitivity eith vital dye microscopic observations and with parallel titration by the Isojima's immobilization tests.     

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.     

Experimental infection with Babesia divergens in cattle persistently infected with bovine virus diarrhoea virus

Nine Norwegian Red cattle, aged 7-14 months, persistently infected with bovine virus diarrhoea virus (BVDV) were inoculated with a Swedish strain of Babesia divergens. Six persistently infected cattle of the same age and breed were kept as controls. Blood and serum samples were collected regularly during the observation period. Rectal temperatures were recorded every morning for 25 days post infection, and the animals were examined clinically on a daily basis. Sera were examined for antibodies to B. divergens by indirect immunofluorescence antibody test (IFAT). Eight of the infected animals developed fever of 2-5 days duration. Babesia divergens organisms appeared in the erythrocytes of all infected animals on the day after inoculation. The parasitaemia lasted for 4-11 days. One animal had a transient haemoglobinuria. Compared with the control group, there was a 20% decrease in the haematocrit. There was a transient lymphopenia and thrombocytopenia during the period of fever. There were no differences in mean numbers of neutrophils between the two persistently infected groups. Compared with cattle free of BVDV, the persistently infected cattle had consistently lower total leucocyte count that was mainly due to decreased mean numbers of neutrophils and monocytes. All infected animals develop antibodies > or = 1:1280 between day 7 and 10 post infection. The magnitude of the antibody response was considerably lower than that of BVDV-free animals inoculated with the same strain and dosage of B.divergens.