Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen

Background

In late 2011, a new Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA.

Results

The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off (S/P value?=?sample value as percentage of positive control value). With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test.

Conclusion

The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV.

     

Serological Evidence of Rift Valley Fever Virus Circulation in Sheep and Goats in Zambézia Province,Mozambique

Rift Valley fever (RVF) is endemic in most parts of Africa and has also been reported to occur in the Arabian Peninsula. It is responsible for significant morbidity and mortality, particularly in livestock, but also in humans. During the last two decades several outbreaks of RVF have been reported in countries in Southern Africa. In contrast to other countries, no clinical disease has been reported in Mozambique during this period. In a serological study conducted in 2007 in five districts of Zambézia Province, Mozambique, of a total of 654 small ruminants sampled (277 sheep and 377 goats), 35.8% of sheep sera and 21.2% of goat sera were positive for RVF virus (RVFV) antibodies in a virus neutralization test (VN) and in an IgG enzyme-linked immunosorbent assay (ELISA). In 2010, a cross-sectional survey was conducted in 313 sheep and 449 goats in two districts of the same province. This study revealed an overall seropositivity rate of 9.2% in sheep and 11.6% in goat and an increased likelihood of being seropositive in older animals (OR = 7.3; p<0.001) using an IgG ELISA. 29 out of 240 animals assessed for RVF specific IgM by ELISA were positive, suggesting recent exposure to RVFV. However, a longitudinal study carried out between September 2010 and April 2011 in a cohort of 125 of these animals (74 sheep and 51 goats) failed to demonstrate seroconversion. The results of the study indicate that RVFV circulates sub-clinically in domestic small ruminants in Zambézia Province.     

Seroprevalence of Sheep and Goat Pox,Peste Des Petits Ruminants and Rift Valley Fever in Saudi Arabia

Sheep and goat pox, peste des petits ruminants and Rift Valley fever are important diseases of small ruminant livestock. Sheep and goat pox, along with peste des petits ruminants, are endemic throughout most of Africa, Asia and the Middle East. Whereas Rift Valley fever is endemic in Africa, outbreaks in the Middle East have been reported over the past decade, including the Arabian Peninsula. Saudi Arabia is a major importer of livestock, and understanding the prevalence of these viral infections would be useful for disease control. In this study, sera from sheep and goats were collected from 3 regions in Saudi Arabia. They were evaluated for antibodies specific to sheep and goat pox, peste des petits ruminants and Rift Valley fever by virus neutralization assays. To the best of our knowledge, this is the first study to evaluate the seroprevalence of these viruses in sheep and goats.     

Serological survey for potential disease agents of free-ranging cervids in six selected national parks from Germany

A total of 164 blood samples, collected from free-ranging red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama) in six German national parks (NP) between 2000 and 2002, were assayed for antibodies against nine viral disease agents. Antibodies were only detected against the alpha-herpesviruses; specifically, bovine herpesvirus-1 (BHV-1) (22 of 157, 14%), cervid herpesvirus-1 (17 of 157, 10.8%), and caprine herpesvirus-1 (11 of 159, 6.9%). Titers ranged from 4 to 102. Most of the seropositive sera, and those with the highest antibody titers, were from red and roe deer in the Harz and Hochharz NP, which are connected and allow migration between the two. The distribution and specificity of antibodies detected in individual deer suggests that the three alpha-herpesviruses are circulating in these deer populations. No antibodies were detected against bovine viral diarrhea virus, epizootic hemorrhagic disease virus, bovine leukemia virus, bluetongue virus, foot-and-mouth disease virus, or sheep and goat poxvirus.     

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

Pestivirus Infections in Norway. Serological Investigations in Cattle,Sheep and Pigs

Serum samples from 1133 dairy cows (187 herds), 3712 ewes (103 flocks) and 1317 adult pigs (877 herds), were tested for neutralizing antibodies against the NADL strain of bovine virus diarrhoea virus. The prevalence rate of seropositive animals was 18.5% in cattle, 4.5% in sheep and 2.2% in pigs, such seroreactors being found in 28 % of the cattle herds and 18 % of the sheep flocks. In all three species the rate showed considerable herd and geographical variation. In cattle the seroreactor rate was similar in herds with normal reproduction and in 62 herds with problems of repeat breeding. Of 31 pig sera containing antibodies against the NADL strain, 27 were also positive in a neutralization test for antibodies against swine fever virus (Baker strain). However, all sera showed a higher titre of antibodies against the bovine strain than against the swine fever virus. It was concluded that the immune response of the pigs had been induced by ruminant pestivirus, and not by swine fever virus.     

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax and its use in a seroepidemiological survey of the Eastern Caribbean Basin

An enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against a South American (New World) strain of Trypanosoma vivax was developed and used for mass screening of cattle from 20 islands in the Eastern Caribbean Basin. The sensitivity and specificity of antigens prepared from a bovine-derived field strain and a murine-adapted laboratory strain of T. vivax, both of New World origin, were compared using an indirect fluorescent antibody (IFA) test, and an antigen prepared from the murine-adapted strain was subsequently used to develop an ELISA test. The results of the ELISA test were then compared with the results of a concurrently run IFA test. There was no cross-reactivity with either test using serum from a Trypanosoma theileri-infected cow. Both tests were weakly cross-reactive with sera from a T. brucei-infected steer, and the IFA test was moderately cross-reactive with several serum samples from a T. evansi-infected steer. For bovine sera collected from herds on islands in the Eastern Caribbean region, only five of 640 tested positive with the ELISA test. Thirty five of 653 sera tested were positive by IFA although the fluorescence elicited was weak as compared to that elicited by sera from known infected animals. Sera collected from 27 cattle in a region known to be free of T. vivax (OH, U.S.A) were negative with the ELISA test, whereas seven of 30 sera from a herd in French Guiana known to be infected with T. vivax were positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Goat warble fly infestation by Przhevalskiana silenus (Diptera: Oestridae): immunoepidemiologic survey in the Basilicata region (southern Italy)

The demonstration of serological cross-reactivity between the Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies led us to prepare an immunological test (ELISA) for an early diagnosis of goat warble fly infestation. Using the Hypodermosis ELISA-Kit (Vétoquinol Diagnostic, France) produced for the immunodiagnosis of bovine hypodermosis, an epidemiological assay was carried out in Basilicata region where goat breeding is very common and no data are reported with regards to the distribution of goat warble fly infestation. Out of a total of 1,100 flocks and 41,200 goats, 105 randomly extracted flocks proved to be infected and 262 sera out of 1,316 were positive; goat warble fly infestation proved to be present in Basilicata with values similar to those recorded in the surrounding regions (Apulia and Calabria). Statistical evaluation showed highly significant differences between the number of infected flocks in the mountainous areas and hills and those of the mountainous areas and the plain, but no differences between hills and plains. The higher number of positive sera and antibody titres in November-December confirmed that these months are the optimal period for sampling sera in order to perform an early diagnosis. The ELISA test was confirmed to be an easy and economic tool especially when sera sampled within a brucellosis eradication program are used.     

Development of an immunofluorescence method for the detection of antibodies to Ebola virus subtype Reston by the use of recombinant nucleoprotein-expressing HeLa cells

An indirect immunofluorescent assay (IFA) to detect Ebola virus subtype Reston (EBO-R) antibodies was developed by the use of a HeLa cell line stably expressing EBO-R nucleoprotein (NP). This IFA has a high specificity for the detection of EBO-R IgG antibodies in both hyperimmune rabbit sera and monkey sera collected during an EBO-R outbreak in the Philippines in 1996. Furthermore, this IFA showed a higher sensitivity for the detection of EBO-R antibodies than did the IFA using HeLa cells expressing the NP of Ebola virus subtype Zaire. These results suggest that this new IFA is useful for seroepidemiological studies of EBO-R infection among monkeys.     

Significant Low Prevalence of Antibodies Reacting with Simian Virus 40 Mimotopes in Serum Samples from Patients Affected by Inflammatory Neurologic Diseases,Including Multiple Sclerosis

Many investigations were carried out on the association between viruses and multiple sclerosis (MS). Indeed, early studies reported the detections of neurotropic virus footprints in the CNS of patients with MS. In this study, sera from patients affected by MS, other inflammatory (OIND) and non-inflammatory neurologic diseases (NIND) were analyzed for antibodies against the polyomavirus, Simian Virus 40 (SV40). An indirect enzyme-linked immunosorbent assay (ELISA), with two synthetic peptides, which mimic SV40 antigens, was employed to detect specific antibodies in sera from patients affected by MS, OIND, NIND and healthy subjects (HS). Immunologic data indicate that in sera from MS patients antibodies against SV40 mimotopes are detectable with a low prevalence, 6%, whereas in HS of the same mean age, 40 yrs, the prevalence was 22%. The difference is statistically significant (P = 0.001). Significant is also the difference between MS vs. NIND patients (6% vs. 17%; P = 0.0254), whereas no significant difference was detected between MS vs OIND (6% vs 10%; P>0.05). The prevalence of SV40 antibodies in MS patients is 70% lower than that revealed in HS.     

Rift valley fever on the east coast of Madagascar

In March 1990, a Rift Valley fever virus (RVFV) outbreak was suspected in the district of Fenerive on the east coast of Madagascar after an abnormally high incidence of abortions and disease in livestock. Sera from humans and cattle were tested for RVFV antibodies by immunofluorescence assay (IFA) and ELISA-IgM capture. Sera and mosquitoes collected in the same area were tested for virus isolation by tissue culture and suckling mouse intracerebral inoculation, and for antigen detection by an ELISA antigen capture assay. Among cattle from the area, RVFV antibody prevalence was 58.6 % by IFA and 29.6 % by ELISA-IgM. In contrast, human populations in the same area had a lower RVFV antibody prevalence, with 8.01 % IFA and 5.4 % IgM-positive sera. No RVFV antigen was detected and virus isolation was unsuccessful from the sera and mosquito pools tested. Different hypotheses concerning the emergence and diffusion of RVFV in this area and the occurrence of the outbreak are discussed.     

Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera. Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.     

Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

Exposure of Asian Elephants and Other Exotic Ungulates to Schmallenberg Virus

Schmallenberg virus (SBV) is an emerging Orthobunyavirus, first described in 2011 in cattle in Germany and subsequently spread throughout Europe, affecting mainly ruminant livestock through the induction of foetal malformations. To gain a better understanding of the spectrum of susceptible species and to assess the value of current SBV serological assays, screening of serum samples from exotic artiodactyls and perissodactyls collected at the Living Collections from the Zoological Society of London (Whipsnade and London Zoos) and Chester Zoo was carried out. There was compelling evidence of SBV infection in both zoological collections. The competitive ELISA has proved to be applicable for the detection of SBV in exotic Bovidae, Cervidae, Suidae, Giraffidae and most notably in endangered Asian elephants (Elephas maximus), but unreliable for the screening of Camelidae, for which the plaque reduction neutralisation test was considered the assay of choice.     

Development of an Indirect ELISA for Serological Diagnosis of Bovine herpesvirus 5

Bovine herpesviruses 1 and 5 (BoHV-1 and BoHV-5) are economically important pathogens, associated with a variety of clinical syndromes, including respiratory and genital disease, reproductive failure and meningoencephalitis. The standard serological assay to diagnose BoHV-1 and BoHV-5 infections is the virus neutralization test (VNT), a time consuming procedure that requires manipulation of infectious virus. In the present study a highly sensitive and specific single dilution indirect ELISA was developed using recombinant glycoprotein D from BoHV-5 as antigen (rgD5ELISA). Bovine serum samples (n = 450) were screened by VNT against BoHV-5a and by rgD5ELISA. Compared with the VNT, the rgD5ELISA demonstrated accuracy of 99.8%, with 100% sensitivity, 96.7% specificity and coefficient of agreement between the tests of 0.954. The rgD5ELISA described here shows excellent agreement with the VNT and is shown to be a simple, convenient, specific and highly sensitive virus-free assay for detection of serum antibodies to BoHV-5.     

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (N<Superscript>pro</Superscript>) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N^pro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N^pro-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N^pro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N^pro rabbit serum. When rabbits were immunized with the N^pro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N^pro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N^pro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N^pro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.     

Circulation of Crimean-Congo Hemorrhagic Fever Virus in the Former Yugoslav Republic of Macedonia Revealed by Screening of Cattle Sera Using a Novel Enzyme-linked Immunosorbent Assay

BackgroundThere are only few assays available for the detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-specific antibodies in animals, and data about diagnostic sensitivity and specificity are incompletely documented for most of these tests. This is unfortunate since CCHFV antibodies in animals can be used as indicator for virus circulation in a geographic area and therewith potential risk of human exposure. This paper therefore reports on a novel ELISA for the detection of CCHFV-specific antibodies in cattle and on its application for testing ruminant sera from the Former Yugoslav Republic of Macedonia.ConclusionThis article describes a fully validated, highly sensitive and specific ELISA for the detection of CCHFV-specific IgG antibodies in cattle. Using this assay, CCHFV-specific antibodies were detected for the first time in cattle in the Former Yugoslav Republic of Macedonia, giving evidence for an active circulation of this virus in the country. Supporting this conclusion, the occurrence of the main vector of CCHFV was demonstrated in the present work for the first time in Former Yugoslav Republic of Macedonia.     

ELISA for detection of IgM and IgG antibodies to sandfly fever sicilian virus

• &#x02022;An enzyme-linked immunosorbent assay (ELISA) was developed to detect specific human immunoglobulin G and M antibodies to sandfly fever Sicilian (SFS) virus. Acute and early convalescent serum pairs with ⩾ 7 days between the 2 specimens were available from 20 patients and all showed significant optical density (OD) increase and significant titre rise (⩾ 4-fold) by IgG ELISA. However, negative or borderline-positive sera were found as late as 11 days after onset of symptoms when tested by IgG ELISA.
• &#x02022;Specific IgM antibodies were detected during the first week of symptoms, and maximum OD values were obtained during the first 4 weeks after onset of disease. The IgM OD values declined over the following 3–9 months. All sera collected later than 14 months post-onset were negative by IgM ELISA.
• &#x02022;The combination of early antibody response and the need to test only one serum specimen gives IgM ELISA an advantage over IgG ELISA in patient diagnosis.
• &#x02022;The IgG ELISA was also evaluated as a seroepidemiological tool and compared to a plaque reduction neutralization test (PRNT) using sera from a normal Cypriot population. Of 183 sera tested, 34 (19%) were positive in plaque reduction neutralization tests (PRNT) and 113 (62%) by IgG ELISA. A number of PRNT-negative sera were strongly positive by IgG ELISA and also by indirect immunofluorescence test, which may suggest the presence of a virus related to SFS in Cyprus which has not yet been isolated.