Serum Neutralization Test Research Articles

Outbreak of Type C Botulism in Backyard Poultry in Santa Catarina, Brazil

Background: Botulism is a disease caused by the ingestion of neurotoxin produced by Clostridium botulinum, characterized by flaccid paralysis, which can lead to high mortality. They have seven types of neurotoxins (A, B, C, D, E, F, and G) and, in birds, most cases are attributed to type C. They are considered sources of botulinum toxins where the decomposition of organic matter occurs, like stagnant water and rotting food. The main feature of the disease in birds is ascending symmetric flaccid paralysis. The present study aims to describe an outbreak of type C botulism in backyard poultry in the state of Santa Catarina, Southern Brazil.Case: A visit was made to the property with 160 backyard poultry with a history of high mortality in the municipality of Agrolândia, Santa Catarina. Clinical signs were characterized by paralysis of the pelvic limbs, neck and pendular wings, which progressed to death within 48 h. There was a mortality rate of 37.5% (60/160) between March and May 2019. These birds were kept in an overcrowded environment, with different species (chickens, ducks, teals, and turkeys) fed irregularly. The water supplied was provided from kitchen exhaust, accumulating in puddles on the floor that contained organic matter residues such as animal feces, food waste and bone fragments. The disposal of the carcasses of birds that died was in the same enclosure, buried superficially, facilitating the access of other birds to dig them up and consume them. Necropsy was performed on 2 chickens and one duck, no macroscopic or histopathological lesions were observed. Blood, liver, and gastrointestinal content samples were sent for research and identification of botulinum toxin through the serum neutralization test in mice. The presence of type C botulinum toxin was confirmed in the liver chicken of one sampled animals.Discussion: The identification of type C botulism toxin enabled the characterization of the outbreak, which is the toxin most associated with episodes of botulism in birds. It is not always possible to identify the origin of the infection, as intoxication can occur by ingestion of water contaminated with organic waste, however, in this outbreak, as sources of poisoning in birds, it was specified and occurred due to the ingestion of water with organic matter that was stagnant in the floor of the enclosure, and also by ingesting contaminated carcasses present in the area. In subsistence farming, botulism outbreaks are reported with greater frequency in the Northeast and Southeast of Brazil generally, cases in which sanitary conditions and incorrect carcass disposal favor the occurrence of the disease. As noted in the present study, high mortality is a common feature of botulism. The evaluated signs and developed evolution were similar to previous studies, which ranged from 14 to 72 h. The absence of macroscopic and histopathological changes is commonly reported in cases of botulism in domestic animals, since botulinum toxin only causes functional changes, with no tissue damage. The association of clinical signs, epidemiology, post mortem evaluation and detection of type C botulinum toxin concludes the diagnosis of botulism. Avian influenza and Newcastle disease are important diseases that have neurological conditions and high mortality that should be distinguished from botulism. This report confirms the need to associate history, clinical signs, absence of lesions with laboratory research for the diagnosis of botulism in birds. In addition, it reinforces the importance of disclosing basic management measures to prevent the occurrence of outbreaks such as presented in this report.Keywords: Clostridium botulinum, neurotoxin, bird disease, paralysis.Descritores: Clostridium botulinum, neurotoxina, doença de ave, paralisia.

Development and evaluation of NS1 protein based i-ELISA for serodiagnosis of Bluetongue disease

Bluetongue (BT) is an important viral disease of small ruminants and causes major economic losses in India. For serodiagnosis of BT, recombinant antigen-based assays are considered as alternatives to conventional approaches such as serum neutralization test. The present study aimed at the development of an indirect- enzyme-linked immunosorbent assay (i-ELISA) using bluetongue (BTV) recombinant NS1. NS1 gene of BTV encoding a mature NS1 protein was produced as a recombinant protein (rNS1) in Escherichia coli. The immunogenic potential of purified rNS1 was confirmed by detecting specific NS1 antibody responses in sera collected from immunized sheep using ELISA. An i-ELISA based on rNS1 was developed and optimized. In comparison to a commercially available c-ELISA kit, in the detection of BTV specific antibodies, this new assay exhibited a diagnostic sensitivity of 91.2%. This study highlighted the potential utility of rNS1 protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of serodiagnostic assay for surveillance of BTV infection in sheep and goats.

Co-circulation of Orthobunyaviruses and Rift Valley Fever Virus in Mauritania, 2015.

Ngari virus (NRIV) has been mostly detected during concurrent outbreaks of Rift Valley fever virus (RVFV). NRIV is grouped in the genus Orthobunyavirus within the Bunyaviridae family and RVFV in the genus Phlebovirus in the family Phenuiviridae. Both are zoonotic arboviruses and can induce hemorrhagic fever displaying the same clinical picture in humans and small ruminants. To investigate if NRIV and its parental viruses, Bunyamwera virus (BUNV) and Batai virus (BATV), played a role during the Mauritanian RVF outbreak in 2015/16, we analyzed serum samples of sheep and goats from central and southern regions in Mauritania by quantitative real-time RT-PCR, serum neutralization test (SNT) and ELISA. 41 of 458 samples exhibited neutralizing reactivity against NRIV, nine against BATV and three against BUNV. Moreover, complete virus genomes from BUNV could be recovered from two sheep as well as two NRIV isolates from a goat and a sheep. No RVFV-derived viral RNA was detected, but 81 seropositive animals including 22 IgM-positive individuals were found. Of these specimens, 61 samples revealed antibodies against RVFV and at least against one of the three orthobunyaviruses. An indirect ELISA based on NRIV/BATV and BUNV derived Gc protein was established as complement to SNT, which showed high performance regarding NRIV, but decreased sensitivity and specificity regarding BATV and BUNV. Moreover, we observed high cross-reactivity among NRIV and BATV serological assays. Taken together, the data indicate the co-circulation of at least BUNV and NRIV in the Mauritanian sheep and goat populations.

A simple method for purification of epsilon toxin of Clostridium perfringens type D for serum neutralization assay

Enterotoxaemia, a disease that affects domestic ruminants, is caused by the epsilon toxin of Clostridium perfringens type D and B. Control and prophylaxis are based on systemic vaccination of small ruminant herds with epsilon toxoid. Purified epsilon toxin is an essential material for vaccine evaluation. It is also necessary for diagnosis of enterotoxaemia disease in the field by in vitro tests including ELISA. The aim of this study was to set up a method for preparation of functional purified epsilon toxin of C. perfringens type D to be used in serum neutralization test. In this study, epsilon toxin was prepared from C. perfringens type D culture precipitated with ammonium sulfate, dialyzed against phosphate buffered saline (PBS) buffer and then, purified using chromatography system. Then, the purified epsilon toxin was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Toxin function was confirmed by cell culture and minimum lethal dose (MLD) assays. Also rabbits were immunized by vaccine in two turns with a 28-day interval. Then, blood samples were collected, and serum neutralization (SN) test was carried out. Results showed that the purified toxin was suitable for SN assay. Our purification method was simple, fast and cost-effective for preparation of epsilon toxin.

Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus.

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for “European” serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

COMPARATIVE STUDY BETWEEN ROTA VIRUSES ISOLATED FROM DIARRHEATIC INFANT BABIES AND NEWLY BORN CALVES

Rota viruses were isolated in vero continuous cell line from infant babies and newly borne calves affected by diarrhea after treatment in every passage with 10 mg/ml of trypsin and adding 0.5 mg/ml of trypsin in the maintenance media. The isolated viruses induced distinctive and progressive type of cytopatheic effect in infected cells and highest titer was 2x10 6. TCID,50/0.1 for bovine viral isolate. The isolated 50/0.1 for human viral isolate and 2x10 » viruses were identified by indirect fluorescent antibody technique by using reference calf rotavirus antisera. Comparative study were conducted on both human and bovine viral isolate including growth in different cell culture, vero cell line was very sensitive to support growth of both viruses than secondary embryonic calf kidney cell culture, lamb testes and primary embryonic chicken fibroblast cell culture. Both viruses induced morphologically similar kind or plaques in vero cell line but plaqes formed by human isolate were larger in size about 1.5 - 2.5 mm in diameter than those of bovine isolate 1.5 - 2 mm. Cross reactive viral antigens were detected between the isolated viruses in indirect fluorescent antibody technique but not in serum neutralization test by using reference bovine rotavirus antisera.

Proteome Modulation in Peripheral Blood Mononuclear Cells of Peste des Petits Ruminants Vaccinated Goats and Sheep.

In the present study, healthy goats and sheep (n = 5) that were confirmed negative for peste des petits ruminants virus (PPRV) antibodies by monoclonal antibody-based competitive ELISA and by serum neutralization test and for PPRV antigen by s-ELISA were vaccinated with Sungri/96. A quantitative study was carried out to compare the proteome of peripheral blood mononuclear cells (PBMCs) of vaccinated goat and sheep [5 days post-vaccination (dpv) and 14 dpv] vs. unvaccinated (0 day) to divulge the alteration in protein expression following vaccination. A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep. Network generated by Ingenuity Pathway Analysis was “infectious diseases, antimicrobial response, and inflammatory response,” which includes the highest number of focus molecules. The bio functions, cell-mediated immune response, and humoral immune response were highly enriched in goats at 5 dpv and at 14 dpv. At the molecular level, the immune response produced by the PPRV vaccine virus in goats is effectively coordinated and stronger than that in sheep, though the vaccine provides protection from virulent virus challenge in both. The altered expression of certain PBMC proteins especially ISG15 and IRF7 induces marked changes in cellular signaling pathways to coordinate host immune responses.

Attenuated infection by a Pteropine orthoreovirus isolated from an Egyptian fruit bat in Zambia.

BackgroundPteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia.MethodsEgyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017–2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively.ResultsAn PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient.ConclusionsA high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans.

Antibody seroprevalence and rate of asymptomatic infections with SARS-CoV-2 in Austrian hospital personnel

BackgroundThe aims of this study are to determine (i) SARS-CoV-2 antibody positive employees in Austrian trauma hospitals and rehabilitation facilities, (ii) number of active virus carriers (symptomatic and asymptomatic) during the study, (iii) antibody decline in seropositive subjects over a period of around 6 months, (iv) the usefulness of rapid antibody tests for outpatient screening.MethodA total of 3301 employees in 11 Austrian trauma hospitals and rehabilitation facilities of the Austrian Social Insurance for Occupational Risks (AUVA) participated in this open uncontrolled prospective cohort study.Rapid lateral flow tests, detecting a combination of IgM and IgM against SARS-CoV-2), two different types of CLIA (Diasorin, Roche), RT-PCR tests and serum neutralization tests (SNTs) were performed. The tests were conducted twice, with an interval of 42.4 ± 7.7 (Min = 30, Max = 64) days. Positive participants were re-tested with CLIA/SNT at a third time point after 188.0 ± 12.8 days.ResultsOnly 27 out of 3301 participants (0.82%) had a positive antibody test at any time point during the study confirmed via neutralization test. Among positively tested participants in either test, 50.4% did not report any symptoms consistent with common manifestations of COVID-19 during the study period or within the preceding 6 weeks. In the group who tested positive during or prior to study inclusion the most common symptoms of an acute viral illness were rhinitis (21.9%), and loss of taste and olfactory sense (21.9%).Based on the neutralization test as the true condition, the rapid antibody test performed better on serum than whole blood as 84.6% instead of 65.4% could be detected correctly. Concerning both CLIA tests overall the Roche test detected 24 (sensitivity = 88.9%) and the Diasorin test 22 positive participants (sensitivity = 81.5%).In participants with a positive SNT result, a significant drop in neutralizing antibody titre from 31.8 ± 22.9 (Md = 32.0) at T1 to 26.1 ± 17.6 (Md = 21.3) at T2 to 21.4 ± 13.4 (Md = 16.0) at T3 (χ2 = 23.848, df = 2, p < 0.001) was observed (χ2 = 23.848, df = 2, p < 0.001)—with an average time of 42.4 ± 7.7 days between T1 and T2 and 146.9 ± 13.8 days between T2 and T3.ConclusionsDuring the study period (May 11th–August 3rd) only 0.82% were tested positive for antibodies in our study cohort. The antibody concentration decreases significantly over time with 14.8% (4 out of 27) losing detectable antibodies.

Duration of Protective Immunity in Sheep Vaccinated with a Combined Vaccine against Peste des Petits Ruminants and Sheep Pox.

In this study, the ability of the combined vaccine against peste des petits ruminants (PPR) (Nigeria strain 75/1) and sheep pox (SPP) (NISKhI strain) to form a protective immune response for 12 months in Kazakh breed fine-fleeced sheep aged 6–12 months was demonstrated. The duration of the protective immunity of immunized sheep from PPR and from SPP was evaluated using a serum neutralization test (SNT), followed by testing of the resistance of vaccinated sheep to infection with the field strain Kentau-7 of the PPRV and the virulent strain A of the SPPV. The PPR antibody response was additionally measured by c-ELISA. A single immunization of sheep with a combined vaccine in a volume of 2.0 mL, containing the PPR and SPP vaccine viruses in the titers of 103.0 TCID50/mL, provided reliable protection of animals from two infections simultaneously for 12 months (observation period). At the same time, in sheep immunized with the combined vaccine, antibodies of PPRV persisted for up to 12 months, with slight fluctuations. The combined vaccine induced 100% clinical protection against the field strain of PPRV and the virulent strain of SPPV in immunized sheep for up to 12 months, while unvaccinated animals became ill with the manifestation of clinical signs specific to PPRV and SPPV.

Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three ‘novel’ serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same ‘VP2 nucleotype’.

Long-term determinants of the seroprevalence of the bluetongue virus in deer species in southern Spain

Bluetongue is a vector-borne disease affecting domestic and wild ruminants, with a major socioeconomic impact. Endemic circulation of the bluetongue virus serotype 4 (BTV-4) and BTV-1 have occurred in Spain since 2004 and 2007, respectively. However, epidemiological studies have seldom been approached from a long-term perspective in wild ruminants. A total of 881 deer (red deer and fallow deer) were necropsied from 2005 to 2018 as part of the DNP health-monitoring program. Serum samples were tested for antibodies against BTV with the aims of assessing the temporal trend and to evaluate the modulating factors: individual, populational, environmental, and stochastic. Red deer displayed statistically significant higher seroprevalences of BTV (SBT; 78.6±3.8%) than fallow deer (53.1±4.7%). The detection of BTV-1 and BTV-4 by the serum neutralization test in calves suggested the circulation of both serotypes over the study period. For red deer, wet years together with high densities could provide suitable conditions for vector borne BTV transmission. Moreover, proximity to high suitability habitat for Culicoides, permanent pasturelands, was associated with higher SBT. The differences in the ecology and behaviour of deer species influencing the exposure to the vectors could determine the differences found in the SBT patterns. This study evidences the role that deer species may play in the maintenance of BTV, however, elucidating the epidemiological role of host in different contexts as well as the consequences of climate change on the competent vector populations and its potential effect on the dynamics of BTV infection in hosts communities deserve further research.

Sero-Monitoring of Horses Demonstrates the Equivac® HeV Hendra Virus Vaccine to Be Highly Effective in Inducing Neutralising Antibody Titres

Hendra virus (HeV) is a high consequence zoonotic pathogen found in Australia. The HeV vaccine was developed for use in horses and provides a One Health solution to the prevention of human disease. By protecting horses from infection, the vaccine indirectly protects humans as well, as horses are the only known source of infection for humans. The sub-unit-based vaccine, containing recombinant HeV soluble G (sG) glycoprotein, was released by Pfizer Animal Health (now Zoetis) for use in Australia at the end of 2012. The purpose of this study was to collate post-vaccination serum neutralising antibody titres as a way of assessing how the vaccine has been performing in the field. Serum neutralization tests (SNTs) were performed on serum samples from vaccinated horses submitted to the laboratory by veterinarians. The SNT results have been analysed, together with age, dates of vaccinations, date of sampling and location. Results from 332 horses formed the data set. Provided horses received at least three vaccinations (consisting of two doses 3–6 weeks apart, and a third dose six months later), horses had high neutralising titres (median titre for three or more vaccinations was 2048), and none tested negative.

Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

The Carboxy Terminal Region on Spike Protein of Porcine Epidemic Diarrhea Virus (PEDV) Is Important for Evaluating Neutralizing Activity.

In this study, we evaluated 62 sow sera samples from PED-vaccinated sows to compare the serum neutralizing test (SNT) and enzyme-linked immunosorbent assay (ELISA). We performed protein ELISA (pELISA) using fragments of spike proteins S1, S2, S3 and entire nucleocapsid proteins, and found a correlation between the SNT and ELISA in PEDV-vaccinated sera. Sera with higher neutralizing activity showed higher titers of IgG. In the antibody profiling, the neutralizing activities are correlated with the levels of the spike antibody, especially the S3 region. We confirmed that the carboxy-terminal region, including the endodomain of the S protein, induced stronger neutralizing activity than the ectodomain. This region of the S protein could be useful for evaluating PED vaccine efficacy, and it is a strong neutralizing epitope of PEDV. The S3 protein could be useful for evaluating PED vaccine efficacy, and it is a strong neutralizing epitope of PEDV.

Development of Foot and Mouth Disease Vaccine Formulation in Egypt by Using Silver Nanoparticles as Immunogenic and Adjuvant Corresponding author: Name: Rania Dweek, Tel: 01009377501 E-mail : raniadweek@yahoo.com

ABSTRACTObjective: To evaluate the effect of silver nanoparticles (AgNPs) with FMD inactivated oil adjuvant vaccine on immunological response of cattle.Study Design: Randomized clinical trial. Procedures: Silver nanoparticles (AgNPs) were prepared then added to locally produced inactivated trivalent FMD vaccine used in Egypt. Twenty cattle were classified into 4 groups (5 in each group).The first group was vaccinated by FMD trivalent inactivated oil adjuvant vaccine plus AgNPs (3ml subcutaneously), the second group was vaccinated by FMD trivalent inactivated oil adjuvant vaccine (3ml subcutaneously). The third group was vaccinated by FMD trivalent inactivated vaccine plus AgNPs as adjuvant (2ml subcutaneous). The fourth group was injected by AgNPs only (1 ml subcutaneous). Sera samples were collected from all animals groups for measuring humeral immunity by using serum neutralization test (SNT) and cell mediated immunity by using bovine Interleukin 6 (IL-6) at first ,second, third and fourth week for (IL-6 and SNT) and at 2 months and 4 months for SNT.Results: The size of AgNPs by TEM (Transmission Electron Microscope) is ranged from 5.8 to 28.51 nm. Antibody titer by SNT revealed that first group was the highest group reach to protective value at 2 weeks (1.538 log10) and reach to peak at 2 months (2.77 log10). Bovine IL-6 was revealed that animals vaccinated by AgNPs appear more quickly than that vaccinated by FMD inactivated oil adjuvant vaccine. Conclusion: The beneficial effect of AgNPs to potentiate both cellular and humeral immunity against FMD.

First evidence of Schmallenberg virus infection in domestic ruminants in Kosovo and Albania.

Schmallenberg virus (SBV), a novel Orthobunyavirus, emerged in European domestic ruminants in 2011 causing abortions and malformations in newborns and none or mild clinical symptoms in adult animals. Here, a total of 364 bovine, ovine and caprine serum samples were collected in Kosovo and Albania between May 2014 and August 2016 and analyzed for the presence of anti‑SBV antibodies. Sera were tested using an enzyme‑linked immunosorbent assay (ELISA), and 48 ELISA‑positive samples were subsequently analyzed by serum neutralization test (SNT). The overall percentage of ELISA positive results was 17.9%; 23.1% (53/229) was the prevalence observed in Kosovo (cattle 45.5%, sheep 19.2% and goat 6.8%), while 8.9% (12/135) was that observed in Albania (cattle 11.1%, sheep 0% and goat 20.0%). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested. This is the first study reporting SBV circulation in domestic ruminants in Kosovo and Albania, with indication that this virus has been present in Kosovo and Albania at least since 2014 without being detected.

Genetic Characterization of Feline Parvovirus Isolate Fe-P2 in Korean Cat and Serological Evidence on Its Infection in Wild Leopard Cat and Asian Badger.

Feline parvovirus (FPV) is a small, non-enveloped, single-stranded DNA virus that infects cats. We recently isolated a feline parvovirus Fe–P2 strain from a dead stray cat in Iksan, 2017. Its partial genomic sequence (4,643 bases) was obtained, and phylogenetic analysis based on the VP2 nucleotide sequence showed that the FPV Fe-P2 strain was closely related to the FPV isolate Gigucheon in cat, 2017 (MN400978). In addition, we performed a serum neutralization (SN) test with the FPV isolates in various mammalian sera. These were from raccoon dog, water deer, Eurasian otter, Korean hare, leopard cat, and Asian badger, which were kindly provided by Chungnam Wild Animal Rescue Center. Notably, serological evidence of its infection was found in Asian badger, Meles leucurus (2/2) and leopard cat, Prionailurus bengalensis (5/8) through SN tests, whereas there was no evidence in raccoon dog, water deer, Eurasian otter, and Korean hare based on the collected sera in this study. These findings might provide partial evidence for the possible circulation of FPV or its related viruses among wild leopard cat and Asian badger in Korea. There should be additional study to confirm this through direct detection of FPVs in the related animal samples.

Adjuvant effect of saponin in an oil-based monovalent (serotype O) foot-and-mouth disease virus vaccine on the antibody response in guinea pigs and cattle.

To enhance the potency of a foot-and-mouth disease (FMD) vaccine, saponin was included in the vaccine formula. In this study, the combined effect of Montanide ISA 50 and saponin was evaluated. Two experiments were performed in guinea pigs and one in cattle to determine the optimal antigen and saponin doses. Only serotype O of foot-and-mouth disease virus (O/PanAsia-2 of ME-SA topotype) was employed in preparation of the monovalent vaccine. All animals were immunized twice with a four-week interval, except for the negative controls. Blood was collected 10 days after the second booster, and the immune response was evaluated using a serum neutralization test. Oil-based FMD vaccines containing saponin induced higher neutralizing antibody levels than formulations lacking saponin. The addition of saponin to formulations with low antigen payload (2.5 µg of inactivated whole virus particles [146S particles] per dose) gave significantly higher neutralizing antibody levels (p < 0.005) than 5 µg of 146S without saponin, suggesting that it can be used to improve FMD vaccine potency in susceptible animals. No adverse effects were observed in vaccinated cattle or guinea pigs.

Development and qualification of a fast, high-throughput and robust imaging-based neutralization assay for respiratory syncytial virus

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.