Monoclonal antibody-based blocking enzyme-linked immunosorbent assay for specific detection and titration of peste-des-petits-ruminants virus antibody in caprine and ovine sera

A blocking enzyme-linked immunosorbent assay (B-ELISA), using two neutralizing monoclonal antibodies (MAbs), was established and compared with the virus neutralization test (VNT) for detecting specific peste-des-petits-ruminants virus (PPRV) antibody in caprine and ovine sera. This technique was developed because VNT, the only available specific serological test for PPRV and the cross-reactive rinderpest virus (RPV), is time-consuming and unaffordable for most laboratories in regions where both peste des petits ruminants and rinderpest occur. The test depends on the blocking of the binding of the MAb to a specific epitope in the presence of positive serum. Test conditions were optimized by using peste-des-petits-ruminants and rinderpest sera that were known to be VNT positive and negative. A blocking format, in which serum is preincubated with a solid-phase PPRV antigen and then incubated with the MAb, yielded levels of sensitivity and specificity superior to those of a competitive format, in which the two reagents are added simultaneously. A threshold value of 45% inhibition, representing the mean for a negative population (n = 277) plus 2.7 standard deviations, was adopted for routine screening. A total of 605 serum samples were screened by B-ELISA and the VNT. The sensitivity and specificity of B-ELISA relative to the VNT were 90.4 and 98.9%, respectively. Of 264 field serum samples tested, 11 (4.2%) could not be assayed by the VNT because of contamination or cytotoxicity; the overall agreement quotient between results of the two tests (n = 253) was 0.91. A high correlation (r>/=0.98) was observed between B-ELISA and the VNT for endpoint titration of sera (n=57). Because B-ELISA proved to be nearlyas sensitive and specific as the VNT while being simpler and more rapid, it would be an adequate substitute for the VNT for assessing herd immune status and for epidemiologic surveillance.     

Detection of peste des petits ruminants virus antigen in conjunctival smears of goats by indirect immunofluorescence

Peste des petits ruminants virus (PPRV) antigen was detected in conjunctival epithelial cells obtained from goats in the early or late stage of the disease by the use of a specific monoclonal antibody (mAb) to PPRV in an immunofluorescent antibody test (IFAT). The affected goats were sampled during an outbreak of peste des petits ruminants in Eritrea. Syncytia were also observed in some smears, consistent with a morbillivirus infection, but the IFAT was more sensitive than staining for syncytia in the detection of viral antigen, the two tests giving 63 per cent and 40 per cent of animals, respectively, with positive tests. Positive immunofluorescence was observed in samples from goats in the early and late stages of the disease, but was not observed with a specific rinderpest mAb or with conjunctival smears from uninfected animals. It is concluded that preparation of conjunctival smears for staining for syncytia is a simple procedure which can be applied in the field, and by use of a specific mAb PPRV infection can be rapidly confirmed and differentiated from rinderpest.     

Morbillivirus downregulation of CD46

There is evidence that CD46 (membrane cofactor protein) is a cellular receptor for vaccine and laboratory-passaged strains of measles virus (MV). Following infection with these MV strains, CD46 is downregulated from the cell surface, and consequent complement-mediated lysis has been shown to occur upon infection of a human monocytic cell line. The MV hemagglutinin (H) protein alone is capable of inducing this downregulation. Some wild-type strains of MV fail to downregulate CD46, despite infection being prevented by anti-CD46 antibodies. In this study we show that CD46 is also downregulated to the same extent by wild-type, vaccine, and laboratory-passaged strains of rinderpest virus (RPV), although CD46 did not appear to be the receptor for RPV. Expression of the RPV H protein by a nonreplicating adenovirus vector was also found to cause this downregulation. A vaccine strain of peste des petits ruminants virus caused slight downregulation of CD46 in infected Vero cells, while wild-type and vaccine strains of canine distemper virus and a wild-type strain of dolphin morbillivirus failed to downregulate CD46. Downregulation of CD46 can, therefore, be a function independent of the use of this protein as a virus receptor.     

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Cowdria ruminantium is the etiologic agent of heartwater, a tick-transmitted foreign animal disease with considerable potential for entrance into the USA. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect serologic responses to C. ruminantium infection. The cELISA utilized a recombinant form of the C. ruminantium major antigenic protein (MAP-1) as the antigen and an anti-MAP-1 monoclonal antibody as the competing indicator reagent. Experimental antisera to C. ruminantium and a wide variety of related ehrlichial organisms were used to evaluate cELISA reactivity. Only sera against C. ruminantium, Ehrlichia canis, E. chaffeensis, and a recently discovered cervine ehrlichia-like organism reacted positively in the cELISA. Specificity of the cELISA was > or = 99.5% in a survey of 1,774 southeastern US and Puerto Rican slaughter cattle sera but was only 85% in a group of 79 hunter-killed white-tailed deer (Odocoileus virginianus) from the southeastern USA. Reference true-positive and cELISA false-positive sera were further analyzed by end point titrations using the cELISA and by indirect fluorescent antibody (IFA) tests for reactivity with C. ruminantium, E. canis, and E. chaffeensis antigens. True heartwater-positive sera were significantly more reactive using the cELISA and C. ruminantium IFA procedures (P < 0.05), whereas false-positive sera were significantly more reactive with the antigens used in the E. chaffeensis IFA procedure (P < 0.05). A group of sera from 210 field-origin ruminants residing on known or potentially heartwater-endemic Caribbean islands revealed a substantial (12.4%) prevalence of cELISA-positive specimens. The cELISA is a relatively specific serodiagnostic test for heartwater in cattle and could be used to monitor for possible introduction of the disease into the USA. The cELISA may also be an excellent tool for monitoring the success of an ongoing Caribbean Amblyomma tick eradication program designed to eliminate the biological vector responsible for the perpetuation and spread of this dangerous foreign animal disease.     

Immunogenic and protective properties of haemagglutinin protein (H) of rinderpest virus expressed by a recombinant baculovirus

The hemagglutinin (H) protein of Rinderpest virus expressed by a recombinant baculovirus used as a vaccine produced high titres of neutralizing antibody to Rinderpest virus in the vaccinated cattle, comparable to the levels produced by live attenuated vaccine. The immunized cattle were protected against a vaccine-virus challenge, as demonstrated by the failure of development of antibodies to N protein of the vaccine virus. The lack of replication of vaccine virus in the immunized cattle indicated that they are capable of showing a protective response if challenged with a virulent virus.     

Characterisation of African isolates of rinderpest virus

Isolates of rinderpest virus (RPV) recovered from outbreaks of the disease in Kenya and Southern Sudan between 1986 and 1993 were compared to each other and to earlier isolates from East and West Africa. The recent isolates were mildly pathogenic for susceptible cattle and thus resembled other mild strains of RPV recovered from cattle and wildlife in East Africa more than 30 years ago. Monoclonal antibody analysis using a panel of 12 anti-RPV haemagglutinin protein-specific antibodies (mAbs) revealed that individual isolates were distinguishable. However, the panel of mAbs could not be used to relate the isolates on the basis of their pathogenicity or geographic origin. Immunoprecipitation of the virus-induced proteins from infected Vero cells, followed by SDS-polyacrylamide gel electrophoresis, showed that the recent mild RPV isolates from eastern Africa were closely related to each other and to their contemporary isolates from Nigeria and Egypt, but they were distinct from another mild isolate recovered from the region three decades ago. Two distinct lineages of African RPV isolates were identified by sequencing a region of the genome around the proteolytic enzyme cleavage site of the fusion protein from the old and new isolates. One lineage, which included virus isolates recovered from East and West Africa during the 1960s, showed a closer phylogenetic relationship to Asian and Middle Eastern RPV isolates. The other lineage consisted mainly of isolates recovered from East, West and North Africa between 1983 and 1993. The results showed that there was co-circulation of two different lineages of RPV in Nigeria during the epizootics of the 1980s.     

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'.     

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.     

Morbillivirus infections in aquatic mammals

Morbillivirus infections which were not documented in aquatic mammals until 1988, have caused at least five epizootics in these species during the last 10 years. Affected populations include European harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) in 1998, Baikal seals (Phoca siberica) in Siberia from 1987-1988, striped dolphins (Stenella coeruleoalba) in the Mediterranean Sea from 1990-1992 and bottlenose dolphins (Tursiops truncatus) along the eastern coast of the United States from 1987-1988 and in the Gulf of Mexico from 1993-1994. Clinical signs and lesions in affected animals were similar to those of canine distemper. Lesions were mainly seen in lung, central nervous and lymphoid tissues and included formation of intranuclear and intracytoplasmic inclusion bodies. Syncytia were commonly found in lung and lymphoid tissues of cetaceans but not of pinnipeds. Antigenic and molecular biological studies indicate that a newly discovered morbillivirus, termed phocine distemper virus, and canine distemper virus were responsible for recent pinniped epizootics; cetacean die-offs were caused by strains of a second, newly recognized cetacean morbillivirus. Serological evidence of morbillivirus infection has been identified in a broad range of marine mammal populations and recent epizootics probably resulted from transfer of virus to immunologically-naive populations.     

Canine distemper virus DNA vaccination induces humoral and cellular immunity and protects against a lethal intracerebral challenge

We have studied the immune responses to the two glycoproteins of the Morbillivirus canine distemper virus (CDV) after DNA vaccination of BALB/c mice. The plasmids coding for both CDV hemagglutinin (H) and fusion protein (F) induce high levels of antibodies which persist for more than 6 months. Intramuscular inoculation of the CDV DNA induces a predominantly immunoglobulin G2a (IgG2a) response (Th1 response), whereas gene gun immunization with CDV H evokes exclusively an IgG1 response (Th2 response). In contrast, the CDV F gene elicited a mixed, IgG1 and IgG2a response. Mice vaccinated (by gene gun) with either the CDV H or F DNA showed a class I-restricted cytotoxic lymphocyte response. Immunized mice challenged intracerebrally with a lethal dose of a neurovirulent strain of CDV were protected. However, approximately 30% of the mice vaccinated with the CDV F DNA became obese in the first 2 months following the challenge. This was not correlated with the serum antibody levels.     

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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.     

Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.     

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Highly purified recombinant gag and env proteins derived from Icelandic strain 1514 of maedi-visna virus were used in an indirect enzyme immunoassay (ELISA) to detect antibodies to small ruminant lentiviruses in sheep and goat sera. The recombinant protein-based ELISA performed very well relative to whole maedi-visna virus and whole caprine arthritis-encephalitis-virus-based ELISAs in its ability to detect anti-maedi visna virus and anti-caprine arthritis-encephalitis virus antibodies, despite the antigenic and genomic variability that is known to exist within and between these two small ruminant lentiviruses. The data suggest that these recombinant maedi-visna virus proteins can be reliably used in an ELISA for the routine serodiagnosis of lentiviral infections in sheep and goats.     

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.     

Morbillivirus infections in wildlife (in relation to their population biology and disease control in domestic animals)

The three members of the morbillivirus genus that infect wildlife in ecosystems where domestic animals occur are rinderpest, peste des petits ruminants (PPR) and canine distemper. Data on the relative susceptibility of species of the Order Artiodactyla for rinderpest have been obtained from historical records of outbreaks. Rinderpest in wildlife has only occurred in equatorial and eastern Africa since the great pandemic of 1889-1897. The distributions, densities and population dynamics of susceptible species in this region are described. There has only been one recorded outbreak of PPR in wildlife but the possibility of its occurrence in the future now that it is present in many parts of west and eastern Africa is discussed. Wild carnivora are not likely to be important maintenance hosts for canine distemper but the disease is of significance in free-ranging carnivores and particularly in small populations of endangered susceptible wildlife species. It is also of great significance in zoo populations.     

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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.     

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.     

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.     

Detection of antibodies to bovine viral diarrhoea virus (BVDV) and characterization of genomes of BVDV from Brazil

An ELISA for the detection of antibodies to bovine viral diarrhea virus (BVDV) was developed based on antigens derived from a genotype I BVDV strain isolated in Switzerland. Using monoclonal antibodies we showed that this antigen contained the conserved non-structural protein NS3 whereas it essentially lacked the more strain-specific E2 surface glycoprotein. This ELISA has a sensitivity of 97.5% and a specificity of 99.2% as compared to the serum neutralization test (SNT). Preliminary experiments showed that this ELISA reliably detects antibodies to BVDV strains circulating in Brazil. Serum samples obtained from 430 adult cattle on 19 farms of the State of Rio Grande do Sul (Brazil) and one farm from Corrientes (Argentina) were tested for antibodies by means of this ELISA. We found antibodies in 56% +/- 15.1% of the cattle sera tested, which indicates that, in Brazil, the prevalence of infection with BVDV is similar to that found in Europe and the USA. Our sequence analysis of two BVDV isolates showed that BVDV of both genotypes I and II circulate in Brazil.