Evaluation of novel inactivated vaccines for the SAT 1, SAT 2 and SAT 3 serotypes of foot-and-mouth disease in pigs.

BACKGROUND: The foot-and-mouth disease (FMD) virus is classified into seven serotypes, of which the South African types have South African Territories (SAT)1, SAT2, and SAT3 that are prevalent in Africa. Especially SAT2 have spread to Arabian Peninsula and the Palestinian Autonomous Territories. Of these viruses, the incidence of SAT2 is the highest. It is important to prepare for the spread of the virus to other continents, even though most FMD viruses are bovine-derived. In particular, due to the high breeding density of pigs in Asia, more attention is usually paid to the immunity and protection of pigs than cattle. For this reason, this study investigated the immunity and protection of pigs against the SAT viruses. METHODS: Specific vaccines were developed for SAT1, SAT2, and SAT3 serotypes. These vaccine viruses were designed to be distinguished from the wild-type strain. An immunogenicity test was conducted using these vaccines in both cattle (n = 5/group) and pigs (n = 20/group). RESULTS: High virus-neutralizing titer of antibodies (> 1:100) was induced in only 2 weeks after the immunization of cattle with the individual vaccine for SAT1, SAT2 or SAT3, and a clear immune response was induced after the second immunization in pigs. When the vaccinated pigs (n = 4-5/group) were challenged by the homologous wild-type virus strain 4 weeks after immunization, all the pigs were protected from the challenge. CONCLUSIONS: This study confirmed that these vaccines can be used against SAT1, SAT2, and SAT3 viruses in cattle and pigs. The vaccine strains developed in this study are expected to be used as vaccines that can protect against FMD in the event of a future FMD outbreak in pigs in consideration of the situation in Asia.

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.

Long-term study (2005-2010) on the vaccination with BoHV-1 glycoprotein E-deleted marker vaccine in selected two dairy herds in Turkey.

A follow-up study from 2005 to 2010 was carried out in two herds where eradication programme for the bovine herpes virus-1 (BoHV-1) infection depends on the vaccination with inactivated glycoprotein E-deleted vaccine that was started in 2001 following the vaccination with inactivated conventional vaccine between 1999 and 2001. For serological screening, a total of 12,976 sera sampled over several sampling times approximately 6 months of interval during 5 years (2005-2010) were tested for glycoprotein E (gE)- and glycoprotein B-specific antibodies using ELISA. According to the serological evidence, the long-term persistence of BoHV-1 antibodies, success of marker vaccine, first vaccination time of the calves in herds regularly vaccinated, etc. were discussed in this paper. In conclusion, the vaccination programme using gE (-) marker vaccines, with making efforts to prevent the other factors about transmission of infection, was suggested for the eradication of BoHV-1 infection in Turkey as many EU countries. This is the first report on the BoHV-1 eradication programme in some dairy cattle in Turkey.

Classical swine fever virus marker vaccine strain CP7_E2alf: genetic stability in vitro and in vivo.

Recently, CP7_E2alf (SuvaxynCSF Marker), a live marker vaccine against classical swine fever virus, was licensed through the European Medicines Agency. For application of such a genetically engineered virus under field conditions, knowledge about its genetic stability is essential. Here, we report on stability studies that were conducted to assess and compare the mutation rate of CP7_E2alf in vitro and in vivo. Sequence analyses upon passaging confirmed the high stability of CP7_E2alf, and no recombination events were observed in the experimental setup. The data obtained in this study confirm the genetic stability of CP7_E2alf as an important safety component.

Evaluation of a 3A-truncated foot-and-mouth disease virus in pigs for its potential as a marker vaccine.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A93-143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD50 (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A.

Assessment of the Safety Profile of Chimeric Marker Vaccine against Classical Swine Fever: Reversion to Virulence Study.

Chimeric marker vaccine candidates, vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, have been generated and their efficacy and capability to differentiate infected from vaccinated animals were confirmed in previous studies. The safety profile of the two chimeric marker vaccine candidates, particularly in the potential reversion to virulence, was evaluated. Each virus was administered to pigs with a dose equivalent to the vaccination dose, and pooled tonsil homogenates were subsequently inoculated into further pigs. Chimeric virus vGPE-/PAPeV Erns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE-/PhoPeV Erns was detectable until the third passage and disappeared entirely by the fourth passage. The vGPE- strain, assessed alongside, consistently exhibited stable virus recovery across each passage without any signs of increased virulence in pigs. In vitro assays revealed that the type I interferon-inducing capacity of vGPE-/PAPeV Erns was significantly higher than that of vGPE-/PhoPeV Erns and vGPE-. In conclusion, the safety profile of the two chimeric marker vaccine candidates was affirmed. Further research is essential to ensure the stability of their attenuation and safety in diverse pig populations.

Safety and DIVA Capability of Novel Live Attenuated Classical Swine Fever Marker Vaccine Candidates in Pregnant Sows.

Classical Swine Fever (CSF), a highly contagious viral disease affecting pigs and wild boar, results in significant economic losses in the swine industry. In endemic regions, prophylactic vaccination and stamping-out strategies are used to control CSF outbreaks. However, sporadic outbreaks and persistent infections continue to be reported. Although the conventional attenuated CSF vaccines protect pigs against the disease, they do not allow for the differentiation of infected from vaccinated animals (DIVA), limiting their use as an eradication tool. In this study, three targeted attenuation strategies were employed to generate vaccine candidates based on the current prevalent CSFV group 2 strains GD18 and QZ07: a single deletion of H79 in Erns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in Erns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in Erns combined with a 5-168 amino acids deletion of Npro (GD18-ddNpro-ErnsH-KARD). Additionally, a negative serological marker with four substitutions in a highly conserved epitope in E2 recognized by the monoclonal antibody 6B8 was introduced in each candidate for DIVA purposes. The safety of these four resulting vaccine candidates was evaluated in pregnant sows. Two candidates, GD18-ddErnsHC-KARD and QZ07-sdErnsH-KARD were found to be safe for pregnant sows and unlikely to cause vertical transmission. Both candidates also demonstrated potential to be used as DIVA vaccines, as was shown using a proprietary blocking ELISA based on the 6B8 monoclonal antibody. These results, together with our previous work, constitute a proof-of-concept for the rational design of CSF antigenically marked modified live virus vaccine candidates.

Investigation of the safety and protective efficacy of an attenuated and marker M. bovis-BoHV-1 combined vaccine in bovines.

Bovine respiratory disease (BRD) is one of the most common diseases in the cattle industry worldwide; it is caused by multiple bacterial or viral coinfections, of which Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the most notable pathogens. Although live vaccines have demonstrated better efficacy against BRD induced by both pathogens, there are no combined live and marker vaccines. Therefore, we developed an attenuated and marker M. bovis-BoHV-1 combined vaccine based on the M. bovis HB150 and BoHV-1 gG-/tk- strain previously constructed in our lab and evaluated in rabbits. This study aimed to further evaluate its safety and protective efficacy in cattle using different antigen ratios. After immunization, all vaccinated cattle had a normal rectal temperature and mental status without respiratory symptoms. CD4+, CD8+, and CD19+ cells significantly increased in immunized cattle and induced higher humoral and cellular immune responses, and the expression of key cytokines such as IL-4, IL-12, TNF-α, and IFN-γ can be promoted after vaccination. The 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- combined strain elicited the most antibodies while significantly increasing IgG and cellular immunity after challenge. In conclusion, the M. bovis HB150 and BoHV-1 gG-/tk- combined strain was clinically safe and protective in calves; the mix of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- strain was most promising due to its low amount of shedding and highest humoral and cellular immune responses compared with others. This study introduces an M. bovis-BoHV-1 combined vaccine for application in the cattle industry.

A safe foot-and-mouth disease vaccine platform with two negative markers for differentiating infected from vaccinated animals.

Vaccination of domestic animals with chemically inactivated foot-and-mouth disease virus (FMDV) is widely practiced to control FMD. Currently, FMD vaccine manufacturing requires the growth of large volumes of virulent FMDV in biocontainment-level facilities. Here, two marker FMDV vaccine candidates (A(24)LL3D(YR) and A(24)LL3B(PVKV)3D(YR)) featuring the deletion of the leader coding region (L(pro)) and one of the 3B proteins were constructed and evaluated. These vaccine candidates also contain either one or two sets of mutations to create negative antigenic markers in the 3D polymerase (3D(pol)) and 3B nonstructural proteins. Two mutations in 3D(pol), H(27)Y and N(31)R, as well as RQKP(9-12)→PVKV substitutions, in 3B(2) abolish reactivity with monoclonal antibodies targeting the respective sequences in 3D(pol) and 3B. Infectious cDNA clones encoding the marker viruses also contain unique restriction endonuclease sites flanking the capsid-coding region that allow for easy derivation of custom designed vaccine candidates. In contrast to the parental A(24)WT virus, single A(24)LL3D(YR) and double A(24)LL3B(PVKV)3D(YR) mutant viruses were markedly attenuated upon inoculation of cattle using the natural aerosol or direct tongue inoculation. Likewise, pigs inoculated with live A(24)LL3D(YR) virus in the heel bulbs showed no clinical signs of disease, no fever, and no FMD transmission to in-contact animals. Immunization of cattle with chemically inactivated A(24)LL3D(YR) and A(24)LL3B(PVKV)3D(YR) vaccines provided 100% protection from challenge with parental wild-type virus. These attenuated, antigenically marked viruses provide a safe alternative to virulent strains for FMD vaccine manufacturing. In addition, a competitive enzyme-linked immunosorbent assay targeted to the negative markers provides a suitable companion test for differentiating infected from vaccinated animals.

Chimeric porcine circoviruses (PCV) containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2 neutralizing antibodies in pigs.

A chimeric porcine circovirus (PCV1-2) with the capsid gene of pathogenic PCV2 cloned into the genomic backbone of nonpathogenic PCV1 is attenuated in pigs but elicits protective immunity against PCV2. In this study, short epitope tags were inserted into the C terminus of the capsid protein of the chimeric PCV1-2 vaccine virus, resulting in a tractable marker virus that is infectious both in vitro and in vivo. Pigs experimentally infected with the epitope-tagged PCV1-2 vaccine viruses produced tag-specific antibodies, as well as anti-PCV2 neutralizing antibodies, indicating that the epitope-tagged viruses could potentially serve as a positive-marker modified live-attenuated vaccine.

Genetically distinct pestiviruses pave the way to improved classical swine fever marker vaccine candidates based on the chimeric pestivirus concept.

Classical swine fever (CSF) is one of the most important viral diseases of pigs. In many countries, the use of vaccines is restricted due to limitations of subunit vaccines with regard to efficacy and onset of protection as well as failure of live vaccines to differentiate infected from vaccinated animals (DIVA principle). Chimeric pestiviruses based on CSF virus (CSFV) and the related bovine viral diarrhea virus (BVDV) have been licensed as live marker vaccines in Europe and Asia, but cross-reactive antibodies can cause problems in DIVA application due to close antigenic relationship. To develop marker vaccine candidates with improved DIVA properties, three chimeric viruses were generated by replacing Erns of CSFV Alfort-Tübingen with homologue proteins of only distantly related pestiviruses. The chimeric viruses "Ra", "Pro", and "RaPro" contained Erns sequences of Norway rat and Pronghorn pestiviruses or a combination of both, respectively. In porcine cells, the "Pro" chimera replicated to high titers, while replication of the "Ra" chimera was limited. The "RaPro" chimera showed an intermediate phenotype. All vaccine candidates were attenuated in a vaccination/ challenge trial in pigs, but to different extents. Inoculation induced moderate to high levels of neutralizing antibodies that protected against infection with a genetically heterologous, highly virulent CSFV. Importantly, serum samples of vaccinated animals did not show any cross-reactivity in a CSFV Erns antibody ELISA. In conclusion, the Erns antigen from distantly related pestiviruses can provide a robust serological negative marker for a new generation of improved CSFV marker vaccines based on the chimeric pestivirus concept.

Assessment of the efficacy of an attenuated live marker classical swine fever vaccine (Flc-LOM-BErns) in pregnant sows.

Here, we constructed an attenuated live marker classical swine fever (CSF) vaccine (Flc-LOM-BErns) to eradicate CSF. This was done by taking infectious clone Flc-LOM, which is based on an attenuated live CSF vaccine virus (LOM strain), and removing the full-length classical swine fever virus (CSFV) Erns sequences and the 3' end (52 base pairs) of the CSFV capsid. These regions were substituted with the full-length bovine viral diarrhoea virus (BVDV) Erns gene sequence and the 3' end (52 base pairs) of the BVDV capsid gene. Sows were vaccinated with the Flc-LOM-BErns vaccine 3 weeks before insemination and then challenged with virulent CSFV at the early, mid- or late stages of pregnancy. We then examined transplacental transmission to the foetuses. Piglets born to sows vaccinated with Flc-LOM-BErns did not show vertical infection, regardless of challenge time. In addition, CSFV challenge did not affect the delivery date, weight or length of the foetus. Pregnant sows inoculated with the Flc-LOM-BErns vaccine were anti-CSF Erns antibody-negative and anti-BVDV Erns antibody-positive. Challenge of pregnant sows with virulent CSFV resulted in anti-CSF Erns antibody positivity. These results strongly indicate that differential diagnosis can be conducted between the Flc-LOM-BErns vaccinated animal and virulent CSFV affected animal by detecting antibody against BVDV Erns or CSF Erns gene. Therefore, the Flc-LOM-BErns vaccine may fulfil the function of differential diagnosis which required for DIVA vaccine.

Cedivac-FMD can be used according to a marker vaccine principle.

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine. All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD(50) vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7-14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of 'marker vaccine' applies.

Protection against transplacental transmission of moderately virulent classical swine fever virus using live marker vaccine "CP7_E2alf".

Classical swine fever (CSF) remains as one of the most important infectious diseases of swine. While prophylactic vaccination is usually prohibited in free countries with industrialized pig production, emergency vaccination is still foreseen. In this context, marker vaccines are preferred as they can reduce the impact on trade. The live-attenuated Suvaxyn® CSF Marker vaccine by Zoetis (based on pestivirus chimera "CP7_E2alf"), was recently licensed by the European Medicines Agency. Its efficacy for the individual animal had been shown in prior studies, but questions remained regarding protection against transplacental transmission. To answer this question, a trial with eight pregnant sows and their offspring was performed as prescribed by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Six of the sows were intramuscularly vaccinated on day 44 of gestation, while the other two remained as unvaccinated controls. All sows were challenged with the moderately virulent CSFV strain "Roesrath" and euthanized shortly before the calculated farrowing date. Sows and piglets were grossly examined and necropsied. Organs (spleen, tonsil, lymph node, and kidney), EDTA-blood and serum were collected from all animals. All samples were tested for antibodies against CSFV glycoproteins E2 and Erns as well as CSFV (virus, antigen and genome). It could be demonstrated that the vaccine complies with all requirements, i.e. no virus was found in the blood of vaccinated sows and their fetuses, and no antibodies were found in the serum of the fetuses from the vaccinated sows. All controls were valid. Thus, it was demonstrated that a single dose vaccination in the sows efficiently protected the offspring against transplacental infection with a moderately virulent CSFV strain.

Immunity against NS3 protein of classical swine fever virus does not protect against lethal challenge infection.

Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. In the European Union the virus has been eradicated from the domestic pig population and prophylactic immunization has been banned. Nevertheless, intervention immunizations using marker vaccines are one possibility to deal with reintroduced CSFV. At present, baculovirus-expressed E2 protein is used as such a marker vaccine. However, this vaccine cannot fully protect against viral spread; hence the use of another subunit, or of a combination of two or more subunits, might be beneficial. Therefore the immunological effects of nonstructural protein 3 (NS3) on the humoral as well as the cellular arms of the immune system were investigated. Although effectors of both sides of the immune system were stimulated by application of recombinant NS3 protein, no protection against lethal CSFV challenge could be achieved.

Characterization of three commercial ELISA kits for detection of BOHV-1 gE specific antibodies in serum and milk samples and applicability of bulk milk for determination of herd status.

Vaccination of animals with gE-deleted vaccine strains (gE- marker vaccines) and differential detection of vaccinated vs infected animals with antibody ELISA targeting the gE or the gB proteins have been proved to be useful tools in programs for control and eradication of the bovine herpesvirus 1 (BoHV-1) responsible for infectious bovine rhinotracheitis (IBR), a major pathogen of cattle. The diagnostic sensitivity (DSe) and specificity (DSp) of three commercial gE ELISA kits from IDEXX, IDVet and CIV-HIPRA were compared for serum and milk matrices. Limiting the analysis to 198 individual with concordant ELISA results in serum (91 naïve, 37 vaccinated and 70 infected) the DSe of gE kits was estimated to 0,97 for IDEXX, 0,93 for CIV-HIPRA and 0,53 for IDVet using milk samples and the DSp to 0,95 for IDEXX, 1,00 for IDVet and CIV-HIPRA. The applicability of gE ELISA for individual or bulk milk testing as an additional tool in control programs dedicated to the certification and control of vaccinated herds was evaluated. Two of the three evaluated gE ELISA kits presented substantial to good agreement individual milk and serum samples. The bulk-tank milk also proved to be suitable for the detection of BoHV-1 in vaccinated herds provided that gE prevalence is superior to 10% as false negative results are often observed at lower gE herd prevalence. This limitation could be reduced to 8% of prevalence when a prior concentration step was applied to bulk milk samples.

Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine.

Foot-and-mouth disease (FMD) is a devastating animal disease. Strategies for differentiation of infected from vaccinated animals (DIVA) remain very important for controlling disease. Development of an epitope-deleted marker vaccine and accompanying diagnostic method will improve the efficiency of DIVA. Here, a monoclonal antibody (Mab) was found to recognize a conserved "AEKNPLE" epitope spanning amino acids 109-115 of non-structural protein (NSP) 3A of foot-and-mouth disease virus (FMDV; O/Tibet/CHA/99 strain), which could be deleted by a reverse-genetic procedure. In addition, a blocking ELISA was developed based on this Mab against NSP 3A, which could serve as a matching test for a negative-marker vaccine. The criterion of this blocking ELISA was determined by detecting panels of sera from different origins. The serum samples with a percentage inhibition (PI) equal or greater than 50% were considered to be from infected animals, and those with <50% PI were considered to be from non-infected animals. This test showed similar performance when compared with other 2 blocking ELISAs based on an anti-NSP 3B Mab. This is the first report of the DIVA test for an NSP antibody based on an Mab against the conserved and predominant "AEKNPLE" epitope in NSP 3A of FMDV.

The double-antigen ELISA concept for early detection of Erns -specific classical swine fever virus antibodies and application as an accompanying test for differentiation of infected from marker vaccinated animals.

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of Erns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel Erns -specific prototype ELISA (pigtype CSFV Erns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect Erns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other Erns -based antibody ELISAs were identified correctly by the novel prototype Erns ELISA and vice versa. In conclusion, the pigtype CSFV Erns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional Erns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.

Classical swine fever vaccines-State-of-the-art.

Due to its impact on animal health and pig industry, classical swine fever (CSF) is still one of the most important viral diseases of pigs. To control the disease, safe and highly efficacious live attenuated vaccines exist for decades. These vaccines have usually outstanding efficacy and safety but lack differentiability of infected from vaccinated animals (DIVA or marker strategy). In contrast, the first generation of E2 subunit marker vaccines shows constraints in efficacy, application, and production. To overcome these limitations, new generations of marker vaccines are developed. A wide range of approaches have been tried including recombinant vaccines, recombinant inactivated vaccines or subunit vaccines, vector vaccines, and DNA/RNA vaccines. During the last years, especially attenuated deletion vaccines or chimeric constructs have shown potential. At present, especially two new constructs have been intensively tested, the adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine candidate "rAdV-SFV-E2" and the pestivirus chimera "CP7_E2alf". The later was recently licensed by the European Medicines Agency. Under field conditions, all marker vaccines have to be accompanied by a potent test system. Particularly this point shows still weaknesses and it is important to embed vaccination in a well-established vaccination strategy and a suitable diagnostic workflow. In summary, conventional vaccines are a standard in terms of efficacy. However, only vaccines with DIVA will allow improved eradication strategies e.g. also under emergency vaccination conditions in free regions. To answer this demand, new generations of marker vaccines have been developed and add now to the tool box of CSF control.

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock.

Paratuberculosis (pTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) in a wide variety of domestic and wild animals. Control of pTB is difficult due to the lack of sensitive, efficacious and cost-effective diagnostics and marker vaccines. Microscopy, culture, and PCR have been used for the screening of MAP infection in animals for quite a long time. Besides, giving variable sensitivity and specificity, these tests have not been considered ideal for large-scale screening of domestic livestock. Serological tests like ELISA easily detects anti-MAP antibodies. However, it cannot differentiate between the vaccinated and infected animals. Nanotechnology-based diagnostic tests are underway to improve the sensitivity and specificity. Newer generation diagnostic tests based on recombinant MAP secretory proteins would open new paradigm for the differentiation between infected and vaccinated animals and for early detection of the infection. Due to higher seroreactivity of secretory proteins vis-à-vis cellular proteins, the secretory proteins may be used as marker vaccine, which may aid in the control of pTB infection in animals. Secretory proteins can be potentially used to develop future diagnostics, surveillance and monitoring of the disease progression in animals and the marker vaccine for the control and eradication of pTB.