Targeted FMD Vaccines for Eastern Africa: The AgResults Foot and Mouth Disease Vaccine Challenge Project

As one of the most infectious livestock diseases in the world, foot and mouth disease (FMD) presents a constant global threat to animal trade and national economies. FMD remains a severe constraint on development and poverty reduction throughout the developing world due to many reasons, including the cost of control measures, closure of access to valuable global FMD-free markets for livestock products, production losses through reduced milk yield, reduced live weight gain, and the inability of infected livestock to perform traction. FMD virus infects a variety of cloven-hoofed animals, including cattle, sheep, goats, swine, all wild ruminants, and suidae, with high morbidity in adult animals. High mortality can occur in young animals due to myocarditis. FMD is endemic in Africa, most of Asia, the Middle East, and parts of South America. The global clustering of FMD viruses has been divided into seven virus pools, where multiple serotypes occur but within which are topotypes that remain mostly confined to that pool. Three pools cover Europe, the Middle East, and Asia; three pools cover Africa; and one pool covers the Americas. The highly infectious nature of FMDV, the existence of numerous continually circulating serotypes and associated topotypes, the potential for wildlife reservoirs, and the frequent emergence of new strains that are poorly matched to existing vaccines all serve to compound the difficulties faced by the governments of endemic countries to effectively control and reduce the burden of the disease at the national and regional levels. This clustering of viruses suggests that if vaccination is to be a major tool for control, each pool could benefit from the use of tailored or more specific vaccines relevant to the topotypes present in that pool, rather than a continued reliance on the currently more widely available vaccines. It should also be noted that, currently, there are varying degrees of effort to identify improved vaccines in different regions. There are relatively few targeted for use in Africa, while the developed world's vaccine banks have a good stock of vaccines destined for emergency outbreak use in FMDV-free countries. The AgResults Foot and Mouth Disease (FMD) Vaccine Challenge Project (the "Project") is an eight-year, US $17.68 million prize competition that supports the development and uptake of high-quality quadrivalent FMD vaccines tailored to meet the needs of Eastern Africa (EA). The Project targets the following Pool Four countries: Burundi, Ethiopia, Kenya, Rwanda, Tanzania and Uganda. The Project is being run in two phases: a development phase, which will encourage the production of regionally relevant vaccines, and a cost-share phase, designed to help to reduce the price of these vaccines in the marketplace to the end users, which is hoped will encourage broader uptake. Manufacturers can submit quadrivalent FMD vaccines containing serotypes A, O, SAT1, and SAT2, which will be assessed as relevant for use in the region through a unique component of the Project requiring the screening of vaccines against the Eastern Africa Foot and Mouth Disease Virus Reference Antigen Panel assembled by the World Reference Laboratory for FMD (WRLFMD), at the Pirbright Institute, UK, in collaboration with the OIE/FAO FMD Reference Laboratory Network. To be eligible for the Project, sera from vaccinated cattle will be used to evaluate serological responses of FMD vaccines for their suitability for use in Eastern African countries. If they pass a determined cut-off threshold, they will be confirmed as relevant for use in the region and will be entered into the Project's cost-share phase.

Epidemiological study on foot-and-mouth disease in small ruminants: Sero-prevalence and risk factor assessment in Kenya.

Foot-and-mouth disease (FMD) is endemic in Kenya affecting cloven-hoofed ruminants. The epidemiology of the disease in small ruminants (SR) in Kenya is not documented. We carried out a cross-sectional study, the first in Kenya, to estimate the sero-prevalence of FMD in SR and the associated risk factors nationally. Selection of animals to be sampled used a multistage cluster sampling approach. Serum samples totaling 7564 were screened for FMD antibodies of non-structural-proteins using ID Screen® NSP Competition ELISA kit. To identify the risk factors, generalized linear mixed effects (GLMM) logistic regression analysis with county and villages as random effect variables was used. The country animal level sero-prevalence was 22.5% (95% CI: 22.3%-24.3%) while herd level sero-prevalence was 77.6% (95% CI: 73.9%-80.9%). The risk factor that was significantly positively associated with FMD sero-positivity in SR was multipurpose production type (OR = 1.307; p = 0.042). The risk factors that were significantly negatively associated with FMD sero-positivity were male sex (OR = 0.796; p = 0.007), young age (OR = 0.470; p = 0.010), and sedentary production zone (OR = 0.324; p<0.001). There were no statistically significant intra class correlations among the random effect variables but interactions between age and sex variables among the studied animals were statistically significant (p = 0.019). This study showed that there may be widespread undetected virus circulation in SR indicated by the near ubiquitous spatial distribution of significant FMD sero-positivity in the country. Strengthening of risk-based FMD surveillance in small ruminants is recommended. Adjustment of husbandry practices to control FMD in SR and in-contact species is suggested. Cross-transmission of FMD and more risk factors need to be researched.

The role of viral particle integrity in the serological assessment of foot-and-mouth disease virus vaccine-induced immunity in swine.

The efficacy of foot-and-mouth disease virus (FMDV) inactivated vaccines is mainly dependent on the integrity of the whole (146S) viral particles. If the intact capsids disassemble to 12S subunits, antibodies against internal-not protective epitopes, may be induced. Serological correlates with protection may be hampered if antibodies against internal epitopes are measured. Here we compared the performance of different ELISAs with the virus-neutralization test (VNT) that measures antibodies against exposed epitopes. Sera from pigs immunized with one dose of an expired commercial FMDV vaccine were used. This vaccine contained about 50% of O1/Campos and over 90% of A24/Cruzeiro strains total antigen as whole 146S particles. Specific-total antibodies were measured with the standard liquid-phase blocking ELISA (LPBE). We also developed an indirect ELISA (IE) using sucrose gradient purified 146S particles as capture antigen to titrate total antibodies, IgM, IgG1 and IgG2. A good correlation was found between VNT titers and IgG-ELISAs for A24/Cruzeiro, with the lowest correlation coefficient estimated for IgG2 titers. For O1/Campos, however, the presence of antibodies against epitopes different from those of the whole capsid, elicited by the presence of 12S particles in the vaccine, hampered the correlation between LPBE and VNT, which was improved by using purified O1/Campos 146S-particles for the liquid-phase of the LPBE. Interestingly, 146S particles but not 12S were efficiently bound to the ELISA plates, confirming the efficiency of the IE to detect antibodies against exposed epitopes. Our results indicate that any serological test assessing total antibodies or IgG1 against epitopes exposed in intact 146S-particles correlate with the levels of serum neutralizing antibodies in vaccinated pigs, and might potentially replace the VNT, upon validation. We recommend that antigen used for serological assays aimed to measure protective antibodies against FMDV should be controlled to ensure the preservation of 146S viral particles.

A vaccine-matching assessment of different genetic variants of serotype O foot-and-mouth disease virus isolated in Ethiopia between 2011 and 2014.

The aim of this study was to assess the vaccine-matching and antigenic properties of foot-and-mouth disease virus (FMDV) isolates collected from Ethiopia between 2011 and 2014. Samples (n = 51) were collected from cattle and pigs with clinical signs consistent with foot-and-mouth disease (FMD) on farms in Debre-Berhan, Debre-Zeit/Bishoftu, Sidamo, Mekelle, and Addis Ababa. Infectious FMDV was isolated using BHK-21 cell cultures from 38 of the 51 field samples (74.5%). All of these FMDV-positive samples were characterized as serotype O, belonging to two East Africa topotypes (EA-3 and EA-4), and their VP1-encoding sequences demonstrated amino acid sequence variability encompassing 27 positions in comparison to the vaccine strain (O/ETH/38/2005) currently provided by the National Veterinary Institute of Ethiopia. One-dimensional virus neutralization test (1 dm VNT) results showed that O/ETH/38/2005 was antigenically matched to 10 of the 16 serotype O viruses. These findings indicate that the O/ETH/38/2005 vaccine strain can provide protection against outbreaks caused by the O/EA-3 topotype, although poorer vaccine-matching results for the O/EA-4 topotype reinforce the importance of using a good-quality vaccine with high coverage in the susceptible herds with supporting post-vaccination serosurveillance to ensure that sufficient antibody titers are generated in the vaccinated animals.

Real-time decision-making during emergency disease outbreaks.

In the event of a new infectious disease outbreak, mathematical and simulation models are commonly used to inform policy by evaluating which control strategies will minimize the impact of the epidemic. In the early stages of such outbreaks, substantial parameter uncertainty may limit the ability of models to provide accurate predictions, and policymakers do not have the luxury of waiting for data to alleviate this state of uncertainty. For policymakers, however, it is the selection of the optimal control intervention in the face of uncertainty, rather than accuracy of model predictions, that is the measure of success that counts. We simulate the process of real-time decision-making by fitting an epidemic model to observed, spatially-explicit, infection data at weekly intervals throughout two historical outbreaks of foot-and-mouth disease, UK in 2001 and Miyazaki, Japan in 2010, and compare forward simulations of the impact of switching to an alternative control intervention at the time point in question. These are compared to policy recommendations generated in hindsight using data from the entire outbreak, thereby comparing the best we could have done at the time with the best we could have done in retrospect. Our results show that the control policy that would have been chosen using all the data is also identified from an early stage in an outbreak using only the available data, despite high variability in projections of epidemic size. Critically, we find that it is an improved understanding of the locations of infected farms, rather than improved estimates of transmission parameters, that drives improved prediction of the relative performance of control interventions. However, the ability to estimate undetected infectious premises is a function of uncertainty in the transmission parameters. Here, we demonstrate the need for both real-time model fitting and generating projections to evaluate alternative control interventions throughout an outbreak. Our results highlight the use of using models at outbreak onset to inform policy and the importance of state-dependent interventions that adapt in response to additional information throughout an outbreak.

Full protection of swine against foot-and-mouth disease by a bivalent B-cell epitope dendrimer peptide.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have reported (Cubillos et al., 2008) that a synthetic dendrimeric peptide consisting of four copies of a B-cell epitope [VP1(136-154)] linked through thioether bonds to a T-cell epitope [3A(21-35)] of FMDV [B4T(thi)] elicits potent B- and T-cell specific responses and confers solid protection in pigs to type C FMDV challenge. Herein we show that downsized versions of this peptide bearing two copies of a B-cell epitope from a type O isolate and using thioether [B2T(thi)] or maleimide [B2T(mal)] conjugation chemistries for their synthesis elicited in swine similar or higher B and T-cell specific responses than tetravalent B4T(thi). Moreover, while partial protection was observed in animals immunized with B4T(thi) (60%) and B2T(thi) (80%), B2T(mal) conferred full (100%) protection against FMDV challenge, associated to high levels of circulating IgG2 and mucosal IgGA, and entirely prevented virus shedding. Interestingly, B2T(mal) is also the most advantageous option in terms of synthetic practicality. Taken together, the results reported here point out to B2T(mal) as a highly valuable, cost-effective FMDV candidate vaccine.

A colorimetric bioassay for high-throughput and cost-effectively assessing anti-foot-and-mouth disease virus activity.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses. This virus is very sensitive to inhibition by type I interferons. Currently, a bioassay based on plaque reduction is used to measure anti-FMDV activity of porcine IFNs. The plaque reduction assay is tedious and difficult to utilize for high-throughput analysis. Using available FMDV susceptible bovine and porcine cells, we developed and tested a colorimetric assay based on cytopathic effect reduction for its ability to quantify FMDV-specific antiviral activity of bovine and porcine type I interferons. Our results show that this new method has significant advantages over other assays in terms of labor intensity, cost, high-throughput capability and/or anti-FMDV specific activity because of simpler procedures and direct measurement of antiviral activity. Several assay conditions were tested to optimize the procedures. The test results show that the assay can be standardized with fixed conditions and a standard or a reference for measuring antiviral activity as units. This is an excellent assay in terms of sensitivity and accuracy based on a statistical evaluation. The results obtained with this assay were highly correlated with a conventional virus titration method.

[Validation of a real time RT-PCR assay to detect foot-and-mouth disease virus and assessment of its performance in acute infection]. / Validación de una técnica de RT-PCR en tiempo real para la detección del virus de fiebre aftosa y evaluación de su desempeño en la infección aguda.

A specific real time reverse transcription polymerase chain reaction (RT-PCRrt) for the detection of foot-and-mouth disease virus was validated using the LightCycler thermocycler 2.0 and its reagents as recommended by the World Organization for Animal Health and was assessed for the detection of the virus in acute infection of cattle experimentally vaccinated and challenged with virus A Argentina/2001 or A24 Cruzeiro. The technique proved to be robust, showing coefficients of variation lower than 4% for different ARN extractions, days or repetitions and was able to detect up to 0,4 TCID 50%, and/or up to 100 RNA molecules. In probang samples, diagnostic sensitivity was 93.1 (95% CI 86.5-96.6) and diagnostic specificity 100 (95% CI 96.3-100). The results of the challenge in vaccinated or multivaccinated bovines showed that although there were high levels of clinical protection in the vaccinated group, FMDV could be detected in all challenged groups. However, detection was 100 times lower in immunized animals.

Comparing control strategies against foot-and-mouth disease: will vaccination be cost-effective in Denmark?

Recent outbreaks of foot-and-mouth disease (FMD) in Europe have highlighted the need for assessment of control strategies to optimise control of the spread of FMD. Our objectives were to assess the epidemiological and financial impact of simulated FMD outbreaks in Denmark and the effect of using ring depopulation or emergency vaccination to control these outbreaks. Two stochastic simulation models (InterSpreadPlus (ISP) and the modified Davis Animal Disease Simulation model (DTU-DADS)) were used to simulate the spread of FMD in Denmark using different control strategies. Each epidemic was initiated in one herd (index herd), and a total of 5000 index herds were used. Four types of control measures were investigated: (1) a basic scenario including depopulation of detected herds, 3 km protection and 10 km surveillance zones, movement tracing and a three-day national standstill, (2) the basic scenario plus depopulation in ring zones around detected herds (Depop), (3) the basic scenario plus protective vaccination within ring zones around detected herds, and (4) the basic scenario plus protective vaccination within ring zones around detected herds. Disease spread was simulated through direct animal movements, medium-risk contacts (veterinarians, artificial inseminators or milk controllers), low-risk contacts (animal feed and rendering trucks, technicians or visitors), market contacts, abattoir trucks, milk tanks, or local spread. The two simulation models showed different results in terms of the estimated numbers. However, the tendencies in terms of recommendations of strategies were similar for both models. Comparison of the different control strategies showed that, from an epidemiological point of view, protective vaccination would be preferable if the epidemic started in a cattle herd in an area with a high density of cattle, whereas if the epidemic started in an area with a low density of cattle or in other species, protective vaccination or depopulation would have almost the same preventive effect. Implementing additional control measures either 14 days after detection of the first infected herd or when 10 herds have been diagnosed would be more efficient than implementing additional control measures when more herds have been diagnosed. Protective vaccination scenarios would never be cost-effective, whereas depopulation or suppressive vaccination scenarios would most often be recommended. Looking at the median estimates of the cost-benefit analysis, depopulation in zones would most often be recommended, although, in extreme epidemics, suppressive vaccination scenarios could be less expensive. The vast majority of the costs and losses associated with a Danish epidemic could be attributed to export losses.

Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle.

Serological assessment of the heterologous response among Foot-and-Mouth Disease Virus (FMDV) strains is mainly performed by virus neutralization test (VNT), liquid phase blocking ELISA and complement fixation assay. In this study two high-throughput ELISA techniques, avidity and IgG subtype ELISA, were developed and used to further characterize heterologous antibody responses in cattle during vaccination and challenge. Both assays were applied to a set of previously characterized sera from animals immunized with an inactivated A24 Cruzeiro/Brazil/55 (A24 Cruzeiro) strain monovalent FMDV vaccine and challenged with the heterologous A/Argentina/2001 (A/Arg/01) strain. Single dilution avidity ELISA assessment showed that animals that were protected against A/Arg/01 challenge had higher avidity antibodies to this heterologous strain than non-protected cattle. Animals with low or even undetectable anti-A/Arg/01 serum-neutralizing titers that passed the heterologous challenge presented higher IgG1/IgG2 ratio than non-protected animals. In this study, the three assessments (VNT and both ELISAs) discriminated between protected and not protected animals against a heterologous challenge. The combination of these techniques may be applied to complement current indirect serological vaccine-matching assessments. The measurement of these qualitative parameters may provide additional information to understand the mechanisms underlying FMD heterologous responses and the induction of cross-protection in cattle.

Modelling the within-host dynamics of the foot-and-mouth disease virus in cattle.

In this paper we investigate the within-host dynamics of the foot-and-mouth disease virus (FMDV) in cattle using previously published data for 8 experimentally infected cows. An 8-compartment, 14-parameter differential equation model was fitted to data collected from each cow every 24 h over the course of an infection on: (i) the concentration of FMDV genomes in the blood, (ii) the concentration of infectious virus in the blood, (iii) antibody levels, and (iv) interferon levels. Model parameters were estimated using maximum-likelihood methods. The likelihood surface was sampled using Markov chain Monte Carlo methods giving credible intervals for each of the model parameters. The model was able to capture the within-host dynamics well for 6 of the infections, with both the innate (type 1 interferon) and antibody responses playing key roles in determining the height and duration of peak levels of virus. There was considerable variation between virus dynamics in individual cattle which was only partly accounted for by inferred differences in the dose of virus received. A better understanding of the within-host dynamics also provides insights into the dynamics of infectiousness and the transmission of virus to new hosts.

Foot-and-mouth disease in pigs: current epidemiological situation and control methods.

Foot-and-mouth disease (FMD) is the paradigm of a transboundary animal disease. Beyond any doubt, it is the most serious challenge for livestock's health. Official Veterinary Services from free countries invest considerable amount of money to prevent its introduction, whereas those from endemic countries invest most of their resources in the control of the disease. A very important volume of scientific production is developed every year in different aspects of FMD, and for that reason, the current knowledge makes the diagnosis of the disease easier to a great extent. However, FMD is still endemic in about two-thirds of the countries, and periodically re-emergent in several countries. This paper is a review of recent publications, focusing mainly on control measures and current world epidemiological situation, emphasizing primarily pigs.

Study on seroprevalence, risk factors, and economic impact of foot-and-mouth disease in Borena pastoral and agro-pastoral system, southern Ethiopia.

Cross-sectional serological study and questionnaire survey were conducted in Borana pastoral and agro-pastoral area to determine seroprevalence and risk factors associated with foot-and-mouth disease (FMD) infection and to assess community perceptions as to importance of the disease. A multistage random sampling was carried out to select cattle for seroprevalence and households for interviews. Totally, 768 sera were collected from 111 herds. The overall individual level seroprevalence of 23.0% (n = 177) and herd level seroprevalence of 58.6% (n = 65) were recorded using 3ABC ELISA test. The variation of individual level seroprevalence in districts were statistically significant (P < 0.05) which was 29.9% in Arero, 24.0% in Yabello, and 15.7% in Teltele. From multivariate logistic regression analysis, herd size and age were seen to be significantly (P < 0.05) associated with FMD seroprevalence. The result of the questionnaire survey based on 120 respondents indicated that, the daily milk yield of cows infected with FMD during outbreaks is reduced to an average of 0.5 L for 25.5 days while cows developing heat-intolerance syndrome after acute infection gave an average 0.67 L for 3.8 months and their calving interval prolonged about 12 months. The questionnaire survey in agro-pastoral area of Borena also indicated that FMD-infected oxen remained off-plough for one season when outbreaks occur in cropping time, whereas heat-intolerant oxen were no longer used for traction. These findings of the present study indicated that FMD is a highly prevalent and economically important disease in the Borana pastoral and agro-pastoral production systems which need effective control strategy for the disease.

Foot-and-mouth disease: the question of implementing vaccinal control during an epidemic.

The question of whether or not to use vaccines during an epidemic of foot-and-mouth disease (FMD) has interested veterinary administrators for many decades. This review assesses the historical uses, successes and failures of vaccinal control, and addresses the questions of where, how, and when to use vaccination against FMD. Approaching the problem in this manner can aid in identifying which tools are likely to be most effective during an epidemic, and how successful a given contingency plan might be. The infection status (endemic, semi-endemic, disease-free) of a region has historically mapped where global vaccination has been implemented according to the generality: endemic>semi-endemic>disease-free. More specifically, biomodels and cost-benefit analyses can indicate when vaccination should be implemented for optimal disease control. Finally, numerous local epidemiological factors will provide useful insights into how vaccinal controls can be used effectively.

Risk evaluation of nonvaccinated, weaned calves transported through areas under systematic foot and mouth disease (FMD) vaccination.

The recurrence and persistence of foot and mouth disease (FMD) could be the consequence of cyclic and massive transportation of calves. For this reason, in South America, vaccination strategies related to livestock dynamic are being promoted. In order to aid the evaluation of such strategies, a method for predicting the risk of transportation of nonvaccinated weaned calves was developed; this method combines expert opinion and empirical evidence using Bayesian estimators. It was applied through Monte Carlo simulation to data of Argentina under four hypothetical vaccination schemes: E1, extended vaccination season of 1/6 of the population of calves each month from July to December without second round vaccination (SRV); E2, extended irregular vaccination from July to December with SRV applied to 70% of the calves resembling the scheme applied in Argentina in 2001; E3, vaccination in November and December without SRV; and E4, vaccination concentrated in November. E1 resulted in probability of transporting non vaccinated calves (tnvc) reaching its maximum in the following year in May with mean=0.0250 and percentile 95% (P95)=0.0404; for the same month tnvc estimates for the other schemes were E2: mean=0.0071; P95=0.0162; E3: mean=0.0017; P95=0.0042 and E4: mean=0.0001; P95=0.0004. Bonferroni multiple comparison for simultaneous assertions for May showed that E4 resulted the best scheme, E1 the worst, and E2 and E3 are intermediate with nonsignificant difference observed between overall (p<0.05). Results were consistent with historical records and quantification for future needs for re-vaccination was made possible. While the ratio "total vaccinated"/"total estimated existences" will give a biased vision of vaccination coverage under the situation of extended vaccination campaigns, a model as the one developed here could allow a more accurate assessment and the design of mitigation plans.

[Fusion expression of Asia I type FMDV neutralizing epitope with heavy chain constant region of sheep IgG and the assessment of its immunogenicity].

VP1 is a major antigenic protein of foot-and-mouth disease virus(FMDV), which induces the immune response against FMDV infection, and contains several epitopes of the virus. We designed and chemically synthesized a DNA fragment which encoding a tandem repeat protein of 136-160aa and 198-211aa of a strain of type Asia I FMDV, and cloned the gene of heavy chain constant region of sheep IgG. By using the BamH I, EcoR I and Xho I sites, both genes were cloned into pPROExHTb vector in turn to form a recombinant plasmid pPRO-FshIgG A chimeric protein, named FshIgG, was obtained after transforming the pPRO-FshIgG into Escherichia coli BL21 (DE3) host cell and induced by IPTG. Inoculation with 100 microg FsIgG induced strong neutralizing antibody response in guinea pigs, and FshIgG inoculated guinea pigs were also protected against 200 ID50 FMDV challenge. Our study indicated that the heavy chain constant region of sheep IgG can act as the carrier protein for FMDV peptide epitopes, and FshIgG is a potential multiepitope peptide vaccine candidate to prevent FMDV infection.

Assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide.

To assess the ability of gold nanoparticles (GNPs) to act as a size-dependent carrier, a synthetic peptide resembling foot-and-mouth disease virus (FMDV) protein was conjugated to GNPs ranging from 2 to 50 nm in diameter (2, 5, 8, 12, 17, 37, and 50 nm). An extra cysteine was added to the C-terminus of the FMDV peptide (pFMDV) to ensure maximal conjugation to the GNPs, which have a high affinity for sulfhydryl groups. The resultant pFMDV-GNP conjugates were then injected into BALB/c mice. Immunization with pFMDV-keyhole limpet hemocyanin (pFMDV-KLH) conjugate was also performed as a control. Blood was obtained from the mice after 4, 6, 8, and 10 weeks and antibody titers against both pFMDV and the carriers were measured. For the pFMDV-GNP immunization, specific antibodies against the synthetic peptide were detected in the sera of mice injected with 2, 5, 8, 12, and 17 nm pFMDV-GNP conjugates. Maximal antibody binding was noted for GNPs of diameter 8-17 nm. The pFMDV-GNPs induced a three-fold increase in the antibody response compared to the response to pFMDV-KLH. However, sera from either immunized mouse group did not exhibit an antibody response to GNPs, while the sera from pFMDV-KLH-immunized mice presented high levels of binding activity against KLH. Additionally, the uptake of pFMDV-GNP in the spleen was examined by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscopy (TEM). The quantity of GNPs that accumulated in the spleen correlated to the magnitude of the immune response induced by pFMDV-GNP. In conclusion, we demonstrated the size-dependent immunogenic properties of pFMDV-GNP conjugates. Furthermore, we established that GNPs ranging from 8 to 17 nm in diameter may be ideal for eliciting a focused antibody response against a synthetic pFMDV peptide.

Quantitative single serum-dilution liquid phase competitive blocking ELISA for the assessment of herd immunity and expected protection against foot-and-mouth disease virus in vaccinated cattle.

A single serum-dilution liquid phase ELISA (slpELISA) was standardized to be used for serological evaluation of herd immunity against foot-and-mouth disease. The absorbance value at a dilution 1:64 of each serum sample was interpolated in a standard curve by plotting the antibody titers of six control sera determined by end point dilution liquid phase ELISA (lpELISA), against the absorbance values for the same control sera at 1:64 dilutions. A straight line was obtained by linear regression analysis (r>0.90) in the titer range of 1.40-2.40. The reliability of the antibody titers was confirmed by the simultaneous titration of 60 cattle sera by slpELISA and lpELISA, which showed an acceptable correlation (R(2)>0.87) for viral strains A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial. Titers obtained by both methods were not significantly different (p>0.05), thus confirming that slpELISA could be used successfully to replace the conventional serial dilution ELISA for the assessment of protection status of cattle in epidemiological studies. In addition, this quantitative slpELISA provides an adequate method for monitoring the effectiveness of vaccination campaigns and is also suitable for the assessment of seroconversion of naive animals during early stages of infection.