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.     

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.     

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.     

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.     

Accelerated viraemia in cats vaccinated with fixed autologous FIV-infected cells

We have vaccinated cats with fixed autologous FIV infected PBMC to determine whether autologous presentation of antigen is capable of inducing a protective immune response against homologous challenge. To this end autologous PBMC were infected with a FIV molecular clone (19k1). When infection was established, cells were inactivated by dialysis against paraformaldehyde. Upon vaccination, cats developed a virus specific immune response as measured by ELISA against the Gag protein of FIV. No antibodies against the envelope protein were detected with a peptide ELISA. Virus neutralizing antibodies however could be detected with a neutralization assay based on infection of CrFK cells, but not in an assay based on infection of primary T-cells. Although vaccination led to the induction of these virus-specific immune responses, vaccinated cats were not protected against homologous challenge but showed an accelerated viraemia upon infection. This was shown both by PCR and cell-associated viral load. The possible mechanisms underlying this observation are discussed in this paper.     

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.     

Relations in families with a mentally retarded child from the perspective of the siblings

Five monoclonal antibodies (MoAbs) against Indian reference/vaccine strain of foot-and-mouth disease (FMD) virus subtype A22 (IND17/77) and a guinea pig antibody against a synthetic peptide representing amino acids (aa) 136-151 of VP1 polypeptide of A22 virus were used in the study. All the antibodies either failed to react or showed a reduced reactivity with trypsin-treated (TT)-146 S virus particles in enzyme-linked immunosorbent assay (ELISA), and could neutralize the infectivity of the reference virus. The antibodies were hence identified as specific to a trypsin-sensitive neutralizable antigenic site of the virus. Using the antibodies we isolated mutants which showed either no or reduced reactivity with the homologous as well as heterologous antibodies in ELISA. The mutants could not be neutralized with the respective antibodies but were efficiently neutralized with the serum from vaccinated cattle (BVS). These results indicated that the antibodies elicited in cattle following vaccination protected them adequately against the mutants selected and that the trypsin-sensitive neutralizable antigenic site of FMD A22 virus as identified by the MoAbs may not be dominant in eliciting a neutralizing antibody response in vaccinated cattle.     

Genetic dissection of interaction between poliovirus 3D polymerase and viral protein 3AB

Poliovirus RNA-dependent RNA polymerase 3D and viral protein 3AB are both thought to be required for the initiation of RNA synthesis. These two proteins physically associate with each other and with viral RNA replication complexes found on virus-induced membranes in infected cells. An understanding of the interface between 3D and 3AB would provide a first step in visualizing the architecture of the multiprotein complex that is assembled during poliovirus infection to replicate and package the viral RNA genome. The identification of mutations in 3D that diminish 3D-3AB interactions without affecting other functions of 3D polymerase is needed to study the function of the 3D-3AB interaction in infected cells. We describe the use of the yeast two-hybrid system to isolate and characterize mutations in 3D polymerase that cause it to interact less efficiently with 3AB than wild-type polymerase. One mutation, a substitution of leucine for valine at position 391 (V391L), resulted in a 3AB-specific interaction defect in the two-hybrid system, causing a reduction in the interaction of 3D polymerase with 3AB but not with another viral protein or a host protein tested. In vitro, purified 3D-V391L polymerase bound to membrane-associated 3AB with reduced affinity. Poliovirus that contained the 3D-V391L mutation was temperature sensitive, displaying a pronounced conditional defect in RNA synthesis. We conclude that interaction between 3AB and 3D or 3D-containing polypeptides plays a role in RNA synthesis during poliovirus infection.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Detection of antibodies to the nonstructural 3C proteinase of hepatitis A virus

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and nonstructural proteins of the virus. However, vaccination with an inactivated vaccine produces antibodies exclusively to the structural proteins. Current diagnostic assays, such as the Abbott HAVAB test, used to determine exposure to HAV detect antibodies only to the structural proteins and as a result are not able to distinguish between a natural infection and vaccination with an inactivated virus. Therefore, an ELISA was developed that is specific for antibodies to the nonstructural protein 3C of HAV and thus serves to document the occurrence of viral replication. Antibodies to the proteinase were not detected by this assay in serum from HAVAB-seropositive primates that were immunized with inactivated HAV. However, antibodies to the proteinase were detected in the serum of all primates experimentally infected with virulent HAV and in the serum of naturally infected humans.     

Viral antibodies in bovine fetuses in Argentina

In order to establish the prevalence of viral infections of the bovine fetus in Argentina, a serological survey for antibodies against viral agents currently affecting cattle in this country was conducted. Antibodies against foot-and-mouth disease virus (FMDV), bovine herpesvirus-1 (BHV-1), bovine leukaemia virus (BLV), bovine rotavirus (BRV), bovine coronavirus (BCV), bovine viral diarrhoea virus (BVDV) and parainfluenza-3 (PI-3) were investigated in a total of 315 fetal serum samples. Conventional techniques were used: indirect immunofluorescence (FMDV, BHV-1, BVDV and BCV), radial immunodiffusion (BLV), ELISA (BRV) and haemagglutination inhibition (PI-3). Antibodies against BHV-1, BVDV and PI-3 were detected in samples from fetuses in the second and third trimester of gestation, with a prevalence of 1.21 per cent (two of 165), 2.03 per cent (four of 197) and 5.08 per cent (nine of 177), respectively. Either antibodies or non-antibody factors able to bind to BRV and BCV antigens were detected with a prevalence of 2.44 per cent (five of 205) and 4.54 per cent (five of 110), respectively. In addition, 14.68 per cent of non-specific inhibitors of PI-3 mediated haemagglutination were found. No seropositives against FMDV and BLV were detected.     

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.     

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.     

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.     

Sera from HIV-1 infected individuals in all stages of disease preferentially recognize the V3 loop of the prototypic macrophage-tropic glycoprotein gp120 ADA

The outer membrane glycoprotein gp120 and the transmembrane glycoprotein gp41 are predominant targets of the humoral immune response to infection by human immunodeficiency virus type 1. The third hypervariable region (V3 loop) is the principal neutralizing domain and is the primary target of neutralizing antibodies directed against the envelope proteins of HIV-1. The V3 loop is also the major determinant for HIV-1 cell-specific tropism. To further characterize the humoral immune response directed against the gp120 envelope proteins, we expressed two prototypic gp120 envelope proteins (LAI/HXB2 and ADA) and chimeric gp120 envelope proteins in stable transfected Drosophila melanogaster Schneider 2 cells. Sera from four infected adults over the course of infection [McNearney et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, p. 10,242] were assayed for reactivity with the respective envelope proteins. Sera obtained at early stages preferentially recognized the gp120 envelope protein ADA, whereas in later stages of infection the sera showed diminished reactivity with both gp120 LAI/HXB2 and gp120 ADA. Chimeric envelope proteins revealed that the humoral response was directed primarily against the V3 loop of gp120 ADA. Furthermore, 22 sera from HIV-1 infected individuals in different stages of the disease were tested. Reactivity of sera with the gp120 envelope protein ADA was seven-fold higher than with the gp120 envelope protein LAI/HXB2. Our results suggest that the humoral immune response is preferentially elicited against the V3 loop of the prototypic macrophage-tropic gp120 envelope protein ADA.     

Evaluation of serological tests for the detection of pseudorabies gE antibodies during early infection

Two enzyme-linked immunosorbent assays (ELISAs) and a particle concentration fluorescence immunoassay (PCFIA) were compared for their ability to detect antibodies against pseudorabies virus (Aujeszky's disease virus) glycoprotein E (gE) in the early stages of infection in pigs previously vaccinated with gE-deleted pseudorabies vaccines. Seventy pigs were included in the study. Five groups of 6 pigs each were vaccinated with one of 5 different pseudorabies virus (PRV) gE-deleted vaccines, and subsequently infected intranasally with 10(5.6) TCID50 of the Iowa 4892 pneumotropic strain of PRV. This entire procedure was repeated using 10(4.6) TCID50 of the Rice strain of PRV. Five unvaccinated control pigs were also challenged with each virus strain. Three control pigs died before seroconverting, leaving 67 pigs for comparison. Blood samples were drawn from experimentally inoculated pigs on the day of vaccination, the day of challenge, and on 4-10, 14, and 21 days postchallenge (DPC). Serology test sensitivity estimates and comparisons among tests were made for each sampling day. Results of this study demonstrated differences among the tests in the time from inoculation to initial antibody detection, and the time to detect 50% and 75% of the infected pigs. The average time until first detection of pigs as seropositive for gE antibodies by PCFIA was 7.5 DPC. The blocking ELISA detected pigs as seropositive an average of 8.8 DPC, and the indirect ELISA first detected gE antibodies by 9.3 DPC. Fifty percent of the pigs were detected as seropositive by days 7, 8, and 9 for the PCFIA, blocking ELISA, and indirect ELISA, respectively. Similarly, 75% of the pigs were detected as seropositive by days 8, 9, and 10 for the PCFIA, blocking ELISA, and indirect ELISA, respectively. All pigs were detected as seropositive by 14 DPC for all 3 tests.     

The outbreak of equine influenza (H3N8) in the United Kingdom in 1989: diagnostic use of an antigen capture ELISA

In July 1989 influenza A/equine-2 (H3N8) was isolated from a nasopharyngeal swab taken from a non-thoroughbred horse exhibiting acute clinical respiratory disease. This was the first isolation of equine influenza virus in the United Kingdom since 1981. Subsequent investigations of acute respiratory disease in horses indicated that the infection was dispersed throughout the UK. However, unlike the previous epidemic of 1979, the first horses from which the virus was isolated had been vaccinated. This outbreak of influenza provided an opportunity to evaluate an antigen capture ELISA, directed against the influenza virus nucleoprotein, as a rapid method for detecting virus in the nasopharyngeal secretions of naturally infected horses.     

Neospora caninum causes abortions in a cattle herd in North Rhine Westphalia]

In a cattle herd in North Rhine-Westphalia a series of eight abortions occurred between 15th August and 14th October, 1996. Four of the aborted fetuses were histopathologically, virologically, bacteriologically and parasitologically examined. A multifocal necrotising encephalitis was observed in two fetuses. In addition, the liver of two fetuses and the placenta of one fetus contained infected foci. In three fetuses protozoan stages were detected which were identified as N. caninum by immunohistochemistry. A polymerase chain reaction conducted with histological material revealed N. caninum-DNA in the placenta of an aborted fetus. When seven cows that had aborted were tested for antibodies directed against N. caninum, six animals were positive by immunofluorescence and all seven by immunoblotting. The serological examination of the entire herd revealed a high herd seroprevalence of antibodies directed against N. caninum. Potential modes of infection with N. caninum in the herd are discussed, in particular the possibility of exposure to a point source (definitive host).     

Bovine mastitis caused by Listeria monocytogenes: kinetics of antibody responses in serum and milk after experimental infection

The kinetics of antibodies in serum and milk directed against proteins from Listeria monocytogenes were studied using 4 lactating cows after infection was experimentally induced in the udder with four strains of serotypes 4b or 1/2a. Antibodies (IgG and IgA) in samples of composite quarter milk and serum of the cow were measured by indirect ELISA. Microtiter plates were coated with proteins obtained from the culture supernatant of L. monocytogenes 4b. After challenge, an IgG response in serum and milk to listerial infections in the udder occurred for all cows, although the response varied among cows. In sera, the IgG titers reached a peak at 9 to 13 wk after challenge and remained elevated until 21 to 33 wk after challenge. In milk, the IgG titer increased significantly 3 wk after the challenge for all cows. A weak and nonpersistent increase in IgA antibodies also occurred. These results indicate that IMI by L. monocytogenes induced an increase of antibodies in milk, which could be detected with an ELISA test using our antigenic preparation. Therefore, this antigenic preparation could be used for the evaluation of a new method of diagnosis for bovine mastitis caused by L. monocytogenes.