Inter-laboratory comparison of 2 ELISA kits used for foot-and-mouth disease virus nonstructural protein serology.

Serologic assays used to detect antibodies to nonstructural proteins (NSPs) of foot-and-mouth disease virus (FMDV) are used for disease surveillance in endemic countries, and are essential to providing evidence for freedom of the disease with or without vaccination and to recover the free status of a country after an outbreak. In a 5-site inter-laboratory study, we compared the performance of 2 commercial NSP ELISA kits (ID Screen FMD NSP ELISA single day [short] and overnight protocols, ID.Vet; PrioCHECK FMDV NS antibody ELISA, Thermo Fisher Scientific). The overall concordance between the PrioCHECK and ID Screen test was 93.8% (95% CI: 92.0-95.2%) and 94.8% (95% CI: 93.1-96.1%) for the overnight and short ID Screen incubation protocols, respectively. Our results indicate that the assays (including the 2 different formats of the ID Screen test) can be used interchangeably in post-outbreak serosurveillance.

A Virus-Like Particle-Based Solid-Phase Competition Enzyme-Linked Immunosorbent Assay for Antibody Detection of Foot-and-Mouth Disease Virus Serotype O.

Foot-and-mouth disease (FMD) is caused by FMD virus (FMDV) is a highly contagious disease of ruminants, which is primarily controlled by vaccination. The monitoring of antisera after vaccination is currently depending on liquid-phase blocking ELISA (LPBE). Recently, bacterium-original FMD virus-like particle (VLP) showed the potential as vaccine candidates. In this study, to minimize the risk of live virus involvement, the Escherichia coli original VLP of FMDV serotype O were used as the immunogen for monoclonal antibodies (Mabs) production and the capture antigen in the development of a solid-phase competition ELISA (SPCE). The samples with a percentage inhibition of >50% were considered positive in the SPCE assay. The concordance rate of the Mab-based SPCE compared with the LPBE for clinical serum samples test was 93.4%, and with a high agreement (kappa = 0.892) with LPBE in antibody duration monitoring. Results indicated that the VLP-based SPCE had high specificity and sensitivity, which provides an alternative method for postimmunization antibody evaluation of FMDV serotype O.

Development and validation of a solid-phase competition ELISA based on virus-like particles of foot-and-mouth disease virus serotype A for antibody detection.

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious epidemic disease, which is controlled primarily by prophylactic vaccination and serological monitoring after vaccination. Here, we have developed a solid-phase competition ELISA (SPCE) method based on virus-like particles (VLPs) of FMDV serotype A. The use of VLPs in the SPCE assay as a replacement for inactivated FMDV provides a high level of biosafety. The SPCE showed high concordance rates when compared with the virus neutralization test and liquid-phase blocking ELISA for testing clinical serum samples and successive serological monitoring (kappa = 0.925). Thus, this SPCE is an alternative method for post-immunization detection of antibodies against FMDV serotype A, with high specificity and sensitivity.

Enhanced efficacy of a multi-epitope vaccine for type A and O foot­and-mouth disease virus by fusing multiple epitopes with Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA), a novel TLR4 agonist.

Foot-and-mouth disease (FMD) is an acute, severe, and highly contagious disease that affects cloven-hoofed animals and can lead to serious economic losses and social effects. Therefore, a safe and effective subunit vaccine is required to prevent and control FMD. Dendritic cells (DCs) are a type of professional antigen presenting cell (APC). Immature DCs are typically stimulated by various adjuvants via immune receptors (e.g., toll-like receptor 4 [TLR4]), which activate DCs to induce their maturation. TLR4 has been well-established to induce both innate and adaptive immune responses to various external microbial or internal damage-related molecular patterns. In this study, the multi-epitope immunogen, HAO, of foot-and-mouth disease virus (FMDV) serotypes A and O was fused with the recombinant protein, heparin-binding hemagglutinin (HBHA), a novel TLR4 agonist, to obtain a new recombinant fusion protein, termed HAO-HBHA. HAO-HBHA was found to be highly efficient at activating murine DCs by the TLR4 pathway, both in vitro and in vivo. HAO-HBHA elicited strong specific humoral immune responses detected with an ELISA and virus neutralizing antibody test (VNT). HAO-HBHA also elevated the cellular immune responses, as indicated by intracellular cytokine (e.g., IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-12p70) expression in Th1 and Th2 cells. As a TLR4 agonist, HBHA has significant advantages for enhancing the immune efficacy of a FMDV serotype A and O bivalent multi-epitope vaccine. These findings provide a novel strategy for the development of a safe and effective multi-epitope vaccine candidate against FMDV and further extends the application of TLR agonist-based vaccine platforms.

Spatial distribution and risk factors for foot and mouth disease virus in Uganda: Opportunities for strategic surveillance.

Foot-and-mouth disease virus (FMDV) has a substantial impact on cattle populations in Uganda, causing short- and long-term production losses and hampering local and international trade. Although FMDV has persisted in Uganda for at least 60 years, its epidemiology there and in other endemic settings remains poorly understood. Here, we utilized a large-scale cross-sectional study of cattle to elucidate the dynamics of FMDV spread in Uganda. Sera samples (n = 14,439) from 211 herds were analyzed for non-structural protein reactivity, an indication of past FMDV exposure. Serological results were used to determine spatial patterns, and a Bayesian multivariable logistic regression mixed model was used to identify risk factors for FMDV infection. Spatial clustering of the disease was evident, with higher risk demonstrated near international borders. Additionally, high cattle density, low annual rainfall, and pastoralism were associated with increased likelihood of FMD seropositivity. These results provide insights into the complex epidemiology of FMDV in Uganda and will help inform refined control strategies in Uganda and other FMDV-endemic settings.

Co-infection with different serotypes of FMDV in vaccinated cattle in Southern Egypt.

During 2015-2016 period, an outbreak of foot-and-mouth disease virus (FMDV) was observed in cattle in four governorates of the upper of Egypt. The infection was extended to the vaccinated cattle. A total of 54 mouth swabs and serum samples were collected from vaccinated cattle for serological and virological investigation. The typical clinical signs of FMDV infection were observed in all cattle under investigation. All samples were positive for FMDV using molecular methods, while the serological method showed 85% positive of tested samples. Typing of FMDV-positive samples using serotype-specific primers showed that 51.8% of samples were serotype O, 9.2% were serotype A, and 18.5% were SAT 2. Surprisingly, co-infections of serotypes A/SAT 2 (12.9%) and O/SAT 2 (7.4%) were also detected. By geographical location, the 3 serotypes A, O, and SAT2 were detected in all four governorates. The phylogenetic assessment of the detected viruses showed that two distinct groups of FMDV serotype O of East Africa-3 (EA-3) topotype were most closely related to circulating viruses in Sudan, as well as FMDV strains belonging to the topotype VII of serotype SAT 2. The detected SAT 2 strains clustered in separate clades in topotype VII, indicating new incursions. The VP1 signatures and protein sequences of some characterized viruses were analyzed. Multiple mutations were detected in VP1. Therefore, to enhance the control of FMD in Egypt, we recommend establishing an active surveillance system to characterize newly emerging virus strains/serotypes and subsequently updating vaccine strains.

Foot and mouth disease virus serological study of dromedary camels in Oman.

The potential role of camels in the epidemiology of foot and mouth disease in Oman was investigated. Sera from local dromedaries (n = 151) that graze with animals (cattle and small ruminants) positive for foot and mouth disease virus (FMDV) non-structural protein antibody (NSP-Ab) were tested for the detection of FMDV NSP-Ab. The samples were tested using a commercial competitive enzyme-linked immunosorbent assay (cELISA) , a rapid immunochromatographic assay and a solid-phase cELISA for the detection of antibodies specific to FMDV serotype O. The results from all three assays were negative when tested with dromedary sera. This indicates that FMDV was not transmitted to dromedary camels kept with FMDV NSP-Ab-positive ruminants.

Une étude a été entreprise dans le but de déterminer le rôle potentiel joué par les camélidés dans l'épidémiologie de la fièvre aphteuse à Oman. À cette fin, des échantillons ont été prélevés à partir de dromadaires autochtones (n = 151) qui pâturaient sur les mêmes prairies que des bovins et des petits ruminants possédant des anticorps contre la protéine non structurale du virus de la fièvre aphteuse en vue de rechercher la présence de ces anticorps. Les sérums ont été soumis à trois tests sérologiques : une épreuve immuno-enzymatique de compétition (cELISA), un essai immunochromatographique rapide et une cELISA en phase solide pour la détection spécifique d'anticorps dirigés contre le sérotype O du virus de la fièvre aphteuse. Les sérums des dromadaires ont tous donné des résultats négatifs aux trois tests. Ces résultats indiquent qu'il n'y a pas eu transmission du virus de la fièvre aphteuse entre les ruminants possédant des anticorps contre la protéine non structurale du virus et les dromadaires.

Los autores describen un estudio encaminado a estudiar la posible función del dromedario en la epidemiología de la fiebre aftosa en Omán. Tras extraer suero de dromedarios locales (n = 151) que pastaban junto con animales (ganado vacuno y pequeños rumiantes) positivos para el anticuerpo contra la proteína no estructural del virus de la fiebre aftosa, se sometieron las muestras de suero a técnicas de detección de ese anticuerpo, empleando para ello: un ensayo inmunoenzimático (ELISA) de competición comercial; una prueba rápida de inmunocromatografía; y un ELISA de competición en fase sólida para la detección de anticuerpos específicos contra el serotipo O del virus. Las tres técnicas arrojaron resultado negativo ante los sueros de dromedario, hecho indicativo de que los rumiantes con anticuerpos contra la proteína no estructural del virus de la fiebre aftosa no habían transmitido el virus a los dromedarios con los que convivían.

Evaluation and optimization of a conventional SPCE for FMD post-vaccination monitoring.

BACKGROUND: Foot-and-mouth disease (FMD) can be controlled by either stamping out or vaccination, a choice which depends on both the economic importance of the livestock sector as well as the disease status. In FMD-free countries with vaccination, such as Korea, vaccination programs should guarantee prevention against transmission of FMD. Monitoring of vaccination programs is also essential for ensuring sufficient coverage that will limit the transmission of FMDV. There are several methods to screen FMD virus (FMDV) structural protein (SP) antibodies including SPCE (Solid-phase competitive ELISA), LPBE (Liquid-phase blocking ELISA), and VNT (Virus neutralization test). Among these, SPCE is widely used for serological monitoring since VNT-the gold standard method-has certain practical limitations, such as high costs in terms of time and labor. However, whether SPCE can ensure the vaccination status of individual animals and whole farms is unclear. In this study, SPCE, LPBE and VNT were compared with respect to correlation with each other and sensitivity at commercial pig farms. RESULTS: The positive results obtained by PrioCHECK SPCE differed from those obtained by LPBE and VNT. The sensitivity of SPCE relative to those of the other tests was fairly low. The raw data of SPCE were most highly correlated with those of VNT with XJ strain, while their positivity and negativity were most highly correlated with LPBE. The results of ROC analysis proposed new cut-off for PrioCHECK SPCE higher than the previous 50% inhibition. CONCLUSIONS: The high false positive rate of PrioCHECK SPCE suggested that high seropositivity by SPCE may not guarantee a true vaccination coverage. Adjusting the cut-off percentage (%) inhibition value for SPCE is needed to address this problem, and it is highly recommended that routine FMDV serological monitoring programs using PrioCHECK SPCE should be combined with alternative methods such as LPBE or VNT.

Visual and equipment-free reverse transcription recombinase polymerase amplification method for rapid detection of foot-and-mouth disease virus.

BACKGROUND: Foot-and-mouth disease (FMD), which is caused by foot-and-mouth disease virus (FMDV), is a highly contagious tansboundary disease of cloven-hoofed animals and causes devastating economic damages. Accurate, rapid and simple detection of FMDV is critical to containing an FMD outbreak. Recombinase polymerase amplification (RPA) has been explored for detection of diverse pathogens because of its accuracy, rapidness and simplicity. A visible and equipment-free reverse-transcription recombinase polymerase amplification assay combined with lateral flow strip (LFS RT-RPA) was developed to detect the FMDV using primers and LF probe specific for the 3D gene. RESULTS: The FMDV LFS RT-RPA assay was performed successfully in a closed fist using body heat for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. The assay could detect FMDV serotypes O, A and Asia1, and there were no cross-reactions with vesicular stomatitis virus (VSV), encephalomyocarditis virus (EMCV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2) and pseudorabies virus (PRV). The analytical sensitivity was 1.0 × 102 copies in vitro transcribed FMDV RNA per reaction, which was the same as a real-time RT-PCR. For the 55 samples, FMDV RNA positive rate was 45.5% (25/55) by LFS RT-RPA and 52.7% (29/55) by real-time RT-PCR. For the LFS RT-RPA assay, the positive and negative predicative values were 100% and 80%, respectively. CONCLUSIONS: The performance of the LFS RT-RPA assay was comparable to real-time RT-PCR, while the LFS RT-RPA assay was much faster and easier to be performed. The developed FMDV LFS RT-RPA assay provides an attractive and promising tool for rapid and reliable detection of FMDV in under-equipped laboratory and at point-of-need facility, which is of great significance in FMD control in low resource settings.

Involvement of the renin-angiotensin system in the progression of severe hand-foot-and-mouth disease.

BACKGROUND: Hand-foot-and-mouth disease (HFMD) is generally considered as a mild exanthematous disease to infants and young children worldwide. HFMD cases are usually mild and self-limiting but for few cases leads to complicated severe clinical outcomes, and even death. Previous studies have indicated that serum Ang II levels in patients with H7N9 infection were related to the severity of infection. However, the mechanisms underlying the pathogenesis of severe HFMD remain unclear. This study was undertaken to clarify the role of the renin-angiotensin system (RAS) in the progression of severe HFMD. METHODS: In the present study, 162 children including HFMD patients and healthy controls were recruited. The data was analyzed by time-series fashion. Concentrations of angiotensin II (Ang II) and noradrenaline (NA) in serum of patients were measured with ELISA. We established a mouse model for enterovirus 71 (EV71) infection and determined concentrations of Ang II, NA in tissue lysates at 3, 5 and 7 days post infection (dpi). RESULTS: The concentrations of Ang II and NA in serum of the HFMD patients with mild or severe symptoms were significantly higher than that in healthy controls. Additionally, the concentrations of Ang II and NA in serum of severe cases were significantly higher than those mild cases and the increased concentrations of Ang II and NA showed the same time trend during the progression of HFMD in the severe cases. Furthermore, the concentrations of Ang II and NA in target organs of EV71-infected mice including brains, skeletal muscle, and lungs were increased with the progression of EV71 infection in mice. Histopathological alterations were observed in the brains, skeletal muscle and lungs of EV71-infected mice. CONCLUSION: Our study suggested that activation of the RAS is implicated in the pathogenesis of severe HFMD.

Host serum microRNA profiling during the early stage of foot-and-mouth disease virus infection.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals, with many outbreaks in the developing world. MicroRNAs (miRNAs) are non-coding RNAs that regulate antiviral defence by post-transcriptional regulation of gene expression. In this study, the host miRNA response following FMDV infection was investigated in cattle, a natural host for FMDV. A significant alteration in serum miRNA expression was detected at early stages of infection. Compared to prior to infection, on day 2 postinfection (PI), 119 miRNAs were upregulated, of which 39 were significantly upregulated (P < 0.05). Gene target prediction and pathway enrichment analysis suggested that upregulated miRNAs target innate immune signalling pathways, suggesting a homeostasis effect, possibly to limit inappropriate immune responses. Further, for the significantly upregulated miRNAs, nine miRNA recognition elements were identified in the genome sequence of FMDV serotype O, which was used for infection. The antiviral effect of four of these miRNAs was confirmed in a cell culture system. These data demonstrate that changes in miRNA expression occur during early pathogenesis, and the identification of possible miRNA targets genes could help in elucidating molecular events involved in virus-host interaction and thus could be useful in developing therapeutic strategies.

Enhanced sensitivity in detection of antiviral antibody responses using biotinylation of foot-and-mouth disease virus (FMDV) capsids.

Analysis of the immune response to infection of livestock by foot-and-mouth disease virus (FMDV) is most often reported as the serum antibody response to the virus. While measurement of neutralizing antibody has been sensitive and specific, measurements of the quality of the antibody response are less robust. Determining the immunoglobulin (Ig) isotype of the serum antibody response provides a deeper understanding of the biology of the response and more sensitive methods for these assays will facilitate analyses of B cell mediated immunity. We tested the hypothesis that using the virus as the molecular probe could be achieved by adding tags to the surface of the FMDV capsid, and that would enhance sensitivity in assays for anti-FMDV antibody responses. The use of a FLAG-tagged virus in these assays failed to yield improvement whereas chemically biotinylating the virus capsid resulted in significant enhancement of the signal. Here we describe methods using biotinylated virus for measuring anti-viral antibody in serum and antibody secreting cells (ASCs) in blood that are sensitive and specific. Finally, we describe using the biotinylated virus in flow cytometry where such assays should greatly enhance the analysis of anti-virus antibody producing B cells, allowing the investigator to focus on only the FMDV specific B cells when analyzing the development of the B cell response to either infection or vaccination.

Chemiluminescence Immunoassay for the Detection of Antibodies against the 2C and 3ABC Nonstructural Proteins Induced by Infecting Pigs with Foot-and-Mouth Disease Virus.

The potential diagnostic value of chemiluminescence immunoassays (CLIAs) has been accepted in recent years, although their use for foot-and-mouth disease (FMD) diagnostics has not been reported. Full-length 3ABC and 2C proteins were expressed in bacteria and purified by affinity chromatography to develop a rapid and accurate approach to distinguish pigs infected with foot-and-mouth disease virus (FMDV) from vaccinated pigs. The recombinant proteins were then used as antigens to develop two CLIAs for the detection of antibodies against nonstructural viral proteins. The diagnostic performance of the two assays was compared by analyzing serum from pigs (naive pigs, n = 63; vaccinated, uninfected pigs, n = 532; naive, infected pigs, n = 117) with a known infection status. The 3ABC-2C CLIA had a higher accuracy rate, with a diagnostic sensitivity of 100% and a diagnostic specificity of 96.5%, than the 3ABC CLIA, which had a diagnostic sensitivity of 95.7% and a diagnostic specificity of 96.0%. The results of the 3ABC-2C CLIA also had a high rate of concordance with those of two commercial FMDV enzyme-linked immunosorbent assay (ELISA) kits used to assess serum collected from 962 pigs in the field (96.2% and 97.8%, respectively). The 3ABC-2C CLIA detected infection in serum samples from infected pigs earlier than the commercial ELISA kits. In addition, the 3ABC-2C CLIA produced results within 15 min. On the basis of these findings, the 3ABC-2C CLIA could serve as the foundation for the development of penside FMD diagnostics and offers an alternative method to detect FMDV infections.

Foot-and-Mouth Disease Seroprevalence in Cattle in Eritrea.

Information about seroprevalence of foot-and-mouth disease (FMD) and virus serotypes in Eritrea is unavailable, but is very important as it may guide the choice of intervention measures including vaccination to be implemented. We carried out a cross-sectional study from February to June 2011 in Eritrea with a two-stage cluster design, sampling cattle in 155 villages with the objective of determining the seroprevalence of FMD in four administrative regions of the country. We analysed cattle sera (n = 2429) for FMD virus antibodies using the non-structural ELISA (NS ELISA) and virus neutralization test (VNT). The overall seroprevalence was 26% and 30% for the NS ELISA and VNT, respectively. FMD virus serotypes O (14%) and A (11%) were the most prevalent. Gash Barka showed the highest (39%) seroprevalence both in NS ELISA and VNT compared to the other three administrative regions. Strategic FMD virus vaccination with type O and A (matching circulating strains) in combination of zoo-sanitary measures would be the best control option for Eritrea which could be started in areas where the disease is less endemic.

Prevalence and risk factors for foot and mouth disease infection in cattle in Israel.

Foot and mouth disease (FMD) is a highly contagious viral disease with major economic consequences. In Israel, FMD epidemics recur almost every year and mostly affect cattle. The highest number of outbreaks occurs among beef cattle farms, followed by feedlot farms and dairy farms. We performed several cross-sectional serological studies in Israel during 2006-2014, aimed to reveal if the virus is endemic among cattle and to determine the sero-prevalence of antibodies directed against non-structural proteins (NSP) of FMD virus. Additionally we aimed to determine the risk factors for such sero-positivity. A risk based sampling was performed and the presence of anti-NSP antibodies was estimated using the PrioCHECK(®) ELISA kit. Beef cattle showed the highest sero-prevalence (13.2%, CI95%=10.8-15.8%). Higher FMD sero-prevalence in beef cattle sampled in 2014 was associated with previous FMD outbreaks in the farm and with age (adult cows versus calves (p<0.05)). Sero-prevalence in feedlot calves was significantly lower with only one sero-positive calf out of 256 (0.4%, CI95%=0-2.2%). Sero-prevalence among dairy cattle was 2.7% (CI95%=2-3.6%) with location of up to 3km from FMD outbreaks in multiple farms and location of up to 5km from the nearest border standing out as significant (p<0.05) risk factors for sero-positivity. The extremely low sero-prevalence of FMD in feedlot cattle and the significant association of infection in beef cattle with previous outbreaks suggest absence of virus circulation between these two populations during the study period, although previous data show that during outbreaks such transmission can occur. Low sero-prevalence in dairy cattle located in areas adjacent to previous FMD outbreaks may be attributed to intense routine vaccination and stringent control measures that were applied during outbreaks such as emergency vaccination and strict quarantine. Early detection of FMD outbreaks among grazing beef herds as well as the implementation of control measures among these farms are therefore the methods of choice to prevent future outbreaks in Israel.

Application and Comparison of Laboratory Parameters for Forecasting Severe Hand-Foot-Mouth Disease Using Logistic Regression, Discriminant Analysis and Decision Tree.

BACKGROUND: In recent years, the prevalence of hand-foot-mouth disease (HFMD) in China and some other countries has caused worldwide concern. Mild cases tend to recover within a week, while severe cases may progress rapidly and tend to have bad outcome. Since there is no vaccine for HFMD and anti-inflammatory treatment is not ideal. In this study, we aimed to establish a valid forecasting model for severe HFMD using common laboratory parameters. METHODS: Retrospectively, 77 severe HFMD cases from Zhengzhou Children's hospital in the peaking period between years 2013 to 2015 were collected, with 77 mild HFMD cases in the same area. The study recorded common laboratory parameters to assist in establishment of the severe HFMD model. After screening the important variables using Mann-Whitney U test, the study also matched the logistic regression (LR), discriminant analysis (DA), and decision tree (DT) to make a comparison. RESULTS: Compared with that of the mild group, serum levels of WBC, PLT, PCT, MCV, MCH, LCR, SCR, LCC, GLO, CK-MB, K, S100, and B in the severe group were higher (p < 0.05), while MCR, EOR, BASOR, SCC, MCC, EO, BASO, NA, CL, T, Th, and Th/Ts were lower (p < 0.05). Five indicators including MCR, LCC, Th, CK-MB, and CL were screened out by LR and the same for DA, and five variables including EO, LCC, CL, GLO, and MCC screened out by DT. The area under the curve (AUC) of LR, DA, and DT was 0.805, 0.779 and 0.864, respectively. CONCLUSIONS: The findings were that common laboratory indexes were effectively used to distinguish the mild HFMD cases and severe HFMD cases by LR, DA, and DT, and DT had the best classification effect with an AUC of 0.864.

Immunoprotective mechanisms in swine within the "grey zone" in antibody response after immunization with foot-and-mouth disease vaccine.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals caused by the FMD virus (FMDV). Vaccination represents one approach for limiting the effects of FMD. The level of protection in vaccinated animals after challenge with foot and mouth disease virus (FMDV) is closely related to the antibody titer, which can be classified into three zones: a "white zone", a "grey zone", and a "black zone". The aim of the present study was to clarify the immunoprotective mechanisms operating in the grey zone, in which vaccinated animals have intermediate antibody titers, making it difficult to predict the level of protection. Thirty-three pigs were used to analyze the distribution of lymphocyte subpopulations in whole blood and the expression levels of 40 cytokines before vaccination and challenge. The antibody titer in pigs in the grey zone ranged from 1:6-1:45. Cytotoxic T lymphocyte subpopulations, expression levels of Th1 cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-12, IL-15, IL-18, and monocyte interferon gamma inducing factor (MIG), and of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1α, transforming growth factor-α (TGF-α), and TWEAK R varied between protected and unprotected animals. The results of this study suggest that the cellular immune response is the key factor responsible for immunoprotection in vaccinated animals with antibody titers within the grey zone.

Effect of storage conditions on subpopulations of peripheral blood T lymphocytes isolated from naïve cattle and cattle infected with foot-and-mouth disease virus.

BACKGROUND: Immunophenotyping of blood lymphocytes by flow cytometry is important in infectious disease research. In animal experiments and other longitudinal studies, the processing, prompt staining, and analysis of fresh samples is a logistical challenge and daily assay variation can confound data interpretation. OBJECTIVE: This study examined the feasibility of cryopreservation and deferred analysis of bovine peripheral blood T lymphocytes from normal or infected animals. METHODS: Peripheral blood mononuclear cells were collected from 4 naïve Holstein steers and 4 steers infected with foot-and-mouth-disease virus serotype Asia1. Identical aliquots were labeled and analyzed immediately, labeled for deferred analysis, or stored at -70°C or over liquid nitrogen for up to 3 weeks before labeling. RESULTS: Freezing of unlabeled cells induced statistically significant changes in phenotypic recognition. In infected animals, the γδ T-cell population increased by 28% and CD8(+) αßT cells by 32%, while total CD3(+) cells decreased by 16%, and CD4(+) αßT cells decreased by 12%. Subsequent storage of frozen cells for the duration of the study, however, had no significant effect. There was less than 20% relative change in subpopulation sizes, and storage at -70°C or over liquid nitrogen was equivalent. CONCLUSIONS: Depending on the objectives and practical limitations of a study, deferred labeling of peripheral blood lymphocytes can be a viable option. Although frozen storage of lymphocytes can introduce some artifactual distortion of relative cell populations, frozen cells can be maintained in storage until all samples in a longitudinal study can be analyzed in batch under standardized conditions and without introducing further bias.

Assessing the potential spread and maintenance of foot-and-mouth disease virus infection in wild ungulates: general principles and application to a specific scenario in Thrace.

Foot-and-mouth disease (FMD), due to infection with serotype O virus, occurred in wild boar and within eleven outbreaks in domestic livestock in the south-east of Bulgaria, Thrace region, in 2011. Hence, the issue of the potential for the spread and maintenance of FMD virus (FMDV) infection in a population of wild ungulates became important. This assessment focused on the spread and maintenance of FMDV infection within a hypothetical wild boar and deer population in an environment, which is characterized by a climate transitional between Mediterranean and continental and variable wildlife population densities. The assessment was based on three aspects: (i) a systematic review of the literature focusing on experimental infection studies to identify the parameters describing the duration of FMDV infection in deer and wild boar, as well as observational studies assessing the occurrence of FMDV infection in wild deer and wild boar populations, (ii) prevalence survey data of wild boar and deer in Bulgaria and Turkey and (iii) an epidemiological model, simulating the host-to-host spread of FMDV infections. It is concluded, based on all three aspects, that the wildlife population in Thrace, and so wildlife populations in similar ecological settings, are probably not able to maintain FMD in the long term in the absence of FMDV infection in the domestic host population. However, limited spread of FMDV infection in time and space in the wildlife populations can occur. If there is a continued cross-over of FMDV between domestic and wildlife populations or a higher population density, virus circulation may be prolonged.

Detection of Foot-and-mouth Disease Virus RNA and Capsid Protein in Lymphoid Tissues of Convalescent Pigs Does Not Indicate Existence of a Carrier State.

A systematic study was performed to investigate the potential of pigs to establish and maintain persistent foot-and-mouth disease virus (FMDV) infection. Infectious virus could not be recovered from sera, oral, nasal or oropharyngeal fluids obtained after resolution of clinical infection with any of five FMDV strains within serotypes A, O and Asia-1. Furthermore, there was no isolation of live virus from tissue samples harvested at 28-100 days post-infection from convalescent pigs recovered from clinical or subclinical FMD. Despite lack of detection of infectious FMDV, there was a high prevalence of FMDV RNA detection in lymph nodes draining lesion sites harvested at 35 days post-infection, with the most frequent detection recorded in popliteal lymph nodes (positive detection in 88% of samples obtained from non-vaccinated pigs). Likewise, at 35 dpi, FMDV capsid antigen was localized within follicles of draining lymph nodes, but without concurrent detection of FMDV non-structural protein. There was a marked decline in the detection of FMDV RNA and antigen in tissue samples by 60 dpi, and no antigen or viral RNA could be detected in samples obtained at 100 dpi. The data presented herein provide the most extensive investigation of FMDV persistence in pigs. The overall conclusion is that domestic pigs are unlikely to be competent long-term carriers of infectious FMDV; however, transient persistence of FMDV protein and RNA in lymphoid tissues is common following clinical or subclinical infection.