Solid-phase Competitive ELISA Research Articles

Foot and Mouth Disease Vaccine Matching and Post-Vaccination Assessment in Abu Dhabi, United Arab Emirates.

Despite the annual vaccination of livestock against foot and mouth disease (FMD) in the United Arab Emirates (UAE), outbreaks of the disease continue to be reported. The effective control of field outbreaks by vaccination requires that the vaccines used are antigenically matched to circulating field FMD viruses. In this study, a vaccine matching analysis was performed using the two-dimensional virus neutralization test (VNT) for three field isolates belonging to the O/ME-SA/PanAsia-2/ANT-10 and O/ME-SA/SA-2018 lineages collected from different FMD outbreaks that occurred within the Abu Dhabi Emirate in 2021 affecting Arabian oryx (Oryx leucoryx), goat, and sheep. In addition, post-vaccination antibodies in sheep and goats were measured using solid-phase competitive ELISA (SPCE) for FMDV serotypes A and O at five months after a single vaccine dose and a further 28 days later after a second dose of the FMD vaccine. An analysis of vaccine matching revealed that five out of the six vaccine strains tested were antigenically matched to the UAE field isolates, with r1-values ranging between 0.32 and 0.75. These results suggest that the vaccine strains (O-3039 and O1 Manisa) included in the FMD vaccine used in the Abu Dhabi Emirate are likely to provide protection against outbreaks caused by the circulating O/ME-SA/PanAsia-2/ANT-10 and O/ME-SA/SA-2018 lineages. All critical residues at site 1 and site 3 of VP1 were conserved in all isolates, although an analysis of the VP1-encoding sequences revealed 14-16 amino acid substitutions compared to the sequence of the O1 Manisa vaccine strain. This study also reports on the results of post-vaccination monitoring where the immunization coverage rates against FMDV serotypes A and O were 47% and 69% five months after the first dose of the FMD vaccine, and they were increased to 81 and 88%, respectively, 28 days after the second dose of the vaccine. These results reinforce the importance of using a second booster dose to maximize the impact of vaccination. In conclusion, the vaccine strains currently used in Abu Dhabi are antigenically matched to circulating field isolates from two serotype O clades (O/ME-SA/PanAsia-2/ANT-10 sublineage and O/ME-SA/SA-2018 lineage). The bi-annual vaccination schedule for FMD in the Abu Dhabi Emirate has the potential to establish a sufficient herd immunity, especially when complemented by additional biosecurity measures for comprehensive FMD control. These findings are pivotal for the successful implementation of the region's vaccination-based FMD control policy, showing that high vaccination coverage and the wide-spread use of booster doses in susceptible herds is required to achieve a high level of FMDV-specific antibodies in vaccinated animals.

Seroprevalence, Serotyping, and Associated Risk Factors of Foot and Mouth Diseases in Bovine in Western Amhara Regional State, North Western Ethiopia

Foot and mouth disease (FMD) is the most contagious disease of animals. The disease affects domestic cloven-hoofed animals, including cattle, swine, sheep, and goats, deer, and is characterized by fever, lameness, and vesicular lesions on the tongue, feet, snout, and teats. The study aimed to determine seroprevalence of FMD (Foot and Mouth Disease), to identifay type of serotypes and to know associated risk factors. A total of 389 sera samples were collected from cattle in four districts of the north western Amhara region and subjected to a 3ABC enzyme-linked immune-sorbent assay. The overall seroprevalence of FMDV was 5.66% (22/389); (95%; CI: 3.34% to 7.98%). The 22 positive samples were subjected to solid phase competitive ELISA to identify specific serotypes. The occurrence of FMD was higher in Adet (OR= 12.8), greater in the semi-intensive than extensive production systems (OR=10.4) and highly occurred in the cross breed than local breed cattle (OR=3.56). Serotypes identified in the four districts were type O, SAT2, and A. This study revealed that FMD is a prevalent and endemic disease. Thus, awareness creation to the stakeholders on the control and prevention of a disease is required. Further epidemiological investigation and vaccine trials should be conducted.

Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

The Immunogenicity of a Foot-and-Mouth Disease Virus Serotype O Vaccine in Commercial and Subsistence Cattle Herds in Zambia.

The recent introduction of foot-and-mouth disease (FMD) virus serotype O (O/EA-2 topotype) in Southern Africa has changed the epidemiology of the disease and vaccine requirements of the region. Commercial and subsistence cattle herds in Zambia were vaccinated with an FMD virus serotype O Manisa vaccine according to a double- or single-dose vaccination schedule. Heterologous antibody responses induced by this vaccine against a representative O/EA-2 virus from Zambia were determined. Virus neutralisation tests (VNTs) showed double-dosed cattle had a mean reciprocal log virus neutralisation titre of 2.02 (standard error [SE] = 0.16, n = 9) for commercial herds and 1.65 (SE = 0.17, n = 5) for subsistence herds 56 days after the first vaccination (dpv). Significantly lower mean titres were observed for single-dosed commercial herds (0.90, SE = 0.08, n = 9) and subsistence herds (1.15, SE = 0.18, n = 3) 56 dpv. A comparison of these results and those generated by solid-phase competitive ELISA (SPCE) tests showed a statistically significant positive correlation by Cohen's kappa coefficient. Therefore, SPCE might be used in assessing the immunogenicity of vaccines in place of VNT. Furthermore, for this vaccine and field strain, a vaccination regime employing a two-dose primary course and revaccination after 4-6 months is likely to be appropriate.

Monitoring of mycotoxin content in compound feeds for productive animals

The article presents the results of monitoring mycotoxins in compound feeds for productive animals. Mycotoxins are secondary metabolites of microscopic fungi with pronounced toxic properties. Since mold fungi spores are ubiquitous, and intensive crop cultivation technologies and environmental pollution disrupt the ecological balance, the problem of mycotoxicosis is global. The purpose of the scientific work was to monitor and analyze the content of mycotoxins in compound feeds for productive animals sold on the territory of St. Petersburg and the Leningrad region. The research was carried out on the basis of the Department of biological safety of feed and veterinary drugs of the North-Western Testing Laboratory of the FGBI "VNIIZH". 40 samples of complete compound feeds for productive animals sold on the territory of St. Petersburg and the Leningrad region were used as research material. The studies were carried out in stages. At the first stage, the total toxicity of feed was determined according to GOST 31674-2012 "Feed, compound feed, feed raw materials. At the next stage, a mycological study was carried out of those samples that, according to the results of biotesting, had toxic properties. Then, to determine the exact cause of the toxicity of the samples, enzyme immunoassay was performed using indirect solid-phase competitive ELISA. Thus, analyzing the data obtained, it can be concluded that four samples of compound feeds out of 40 were contaminated with mycotoxins: deoxynivalenol and zearalenone simultaneously. This is due to the fact that the producer for these mycotoxins is one type of microscopic fungi - Fusarium graminearum, which turned out to be affected by these samples of compound feeds. It should be noted that these mycotoxins are characterized by low toxicity and a high degree of accumulation in the body, which contributes to the occurrence of chronic poisoning, therefore, the need for further monitoring of the welfare of the feed base for farm animals and poultry in veterinary and sanitary terms increases.

Evaluating serological tests for foot-and-mouth disease while accounting for different serotypes and uncertain vaccination status

Controlling foot-and-mouth disease (FMD) by vaccination requires adequate population coverage and high vaccine efficacy under field conditions. To assure veterinary services that animals have acquired sufficient immunity, strategic post-vaccination surveys can be conducted to monitor the coverage and performance of the vaccine. Correct interpretation of these serological data and an ability to derive exact prevalence estimates of antibody responses requires an awareness of the performance of serological tests. Here, we used Bayesian latent class analysis to evaluate the diagnostic sensitivity and specificity of four tests. A non-structural protein (NSP) ELISA determines vaccine independent antibodies from environmental exposure to FMD virus (FMDV), and three assays measuring total antibodies derived from vaccine antigen or environmental exposure to two serotypes (A, O): the virus neutralisation test (VNT), a solid phase competitive ELISA (SPCE), and a liquid phase blocking ELISA (LPBE). Sera (n=461) were collected by a strategic post-vaccination monitoring survey in two provinces of Southern Lao People's Democratic Republic (PDR) after a vaccination campaign in early 2017. Not all samples were tested by every assay and each serotype: VNT tested for serotype A and O, whereas SPCE and LPBE tested for serotype O, and only NSP-negative samples were tested by VNT, with 90 of them not tested (missing by study design). These data challenges required informed priors (based on expert opinion) for mitigating possible lack of model identifiability. The vaccination status of each animal, its environmental exposure to FMDV, and the indicator of successful vaccination were treated as latent (unobserved) variables. Posterior median for sensitivity and specificity of all tests were in the range of 92-99 %, except for the sensitivity of NSP (∼66%) and the specificity of LPBE (∼71 %). There was strong evidence that SPCE outperformed LPBE. In addition, the proportion of animals recorded as having been vaccinated that showed a serological immune response was estimated to be in the range of 67-86 %. The Bayesian latent class modelling framework can easily and appropriately impute missing data. It is important to use field study data as diagnostic tests are likely to perform differently on field survey samples compared to samples obtained under controlled conditions.

Preliminary validation of a single-spot version of a solid-phase competition ELISA for the detection of southern African territories foot-and-mouth disease serotype exposure in goats

The objective of this study was to perform a preliminary validation of a solid-phase competition ELISA (SPCE) in goats exposed to foot-and-mouth disease virus (FMDV) southern African territories (SAT) serotypes through vaccination or experimental infection. Thirty-nine goats were vaccinated with a FMDV vaccine and 37 subsequently challenged with a SAT1 virus serotype. Blood was collected every 7 days until termination at 14 days post-challenge. Single-spot SAT1 virus serotype SPCE (ss-SPCE) was performed in duplicate at two time points and a half-titration version was performed after a variable time of long-term storage. Coefficient of variation (CV) was calculated and accuracy of the ss-SPCE was estimated relative to a half-titration SPCE log10 titer of 1.6 using mixed-effect logistic regression. Additionally, sensitivity and specificity were estimated based on serological results 14-days post-challenge and at study enrolment, respectively. Three hundred and forty-two serum samples were tested in duplicate on two non-consecutive days. The median (interquartile range (IQR)) CV for the ss-SPCE for SAT1 was 2.1% (0.5, 14.3%) and 2.5% (0.6, 12.8%) for the two testing days, respectively. Median (IQR) inter-assay (different day) CV was 10.6% (2.5, 42.5%). Specificity and sensitivity of the ss-SPCE relative to the log10 titer using a 70% percentage inhibition positive threshold were 83.4% (95% confidence interval, 77.7–87.9) and 95.8% (90.7–98.2), respectively. Specificity was estimated as 100% (92.6, 100) and sensitivity as 97.3% (87.4, 99.9) when only considering serum tested at the beginning and end of the study, respectively. The SAT1 ss-SPCE is repeatable and accurate for determining FMDV serological status in goats.

Determination of foot-and-mouth disease serotypes from naturally infected cattle by solid phase competitive ELISA (SPCE) techniques

Objective: Foot and mouth disease (FMD) is a highly infectious and economically important disease affecting cloven-hoofed domestic and wild animals. Early diagnosis and serotyping of the agent are very important to effectively design and implement the control approach. This study was conducted on serum samples collected from Amhara, Tigray, Oromia and Addis Ababa between October 2018 to February 2020. The animals were kept under a semi-intensive to an extensive system of rearing. Serum samples with low OD values (positive) using competition NSP-ELISA were subjected to serotyping ELISA. Results: In the present study, three serotypes were identified from 186 NSP ELISA positive sera of which 156 serotype O, 40 serotypes A and 28 serotype SAT2. In this analysis, multiple serotype infection was observed which is why the number of serotypes was beyond the samples analyzed. Among 23 samples from Addis Ababa 10, 3 and 5 were O, A and SAT2 serotypes respectively, while in samples from the Oromia region 12 were O and 3 were SAT2 serotypes. From the Amhara region, 99 samples analyzed were found to be serotype O and SAT2 in 7 of the serum samples. From the Tigray region, 30 samples were seen to have Serotype O infection, whereas 13 of them were SAT2. The proportion of serotypes identified based on the production system practices was also found that semi-intensive production takes the largest share in all three serotypes followed by extensive production. Generally, early determination of the serotype from past infection helps to aware of the epidemiology as well as the infection immunity of the herd/individual animals.

Establishment and validation of a liquid-phase blocking ELISA for the detection of antibodies elicited by the foot-and-mouth disease virus A/ASIA/Sea-97 lineage

ABSTRACT It has been reported that a commercial solid-phase competition ELISA (SPCE) commonly used in year-round serum surveillance in South Korea may not be sufficient for use in the sero-monitoring of protective antibody responses following vaccination. Here, we established a liquid-phase blocking ELISA (LPBE) method based on the purified 146S antigen of the foot-and-mouth disease virus (FMDV) A/Yeoncheon/SKR/2017 strain, and its analytical performance was assessed using a wide range of pig sera. The established LPBE performed well regardless of whether antisera were elicited by a homologous (A/Yeoncheon/SKR/2017) or heterologous virus (A/Pocheon/SKR/2010) classified in the A/ASIA/Sea-97 lineage. The LPBE performance was also assessed using sera obtained from pigs in the field that underwent routine vaccination, demonstrating that the assay exhibited higher consistency with the virus neutralization test (standard reference test) than the commercial SPCE. This suggested that the LPBE performed well for antisera induced by the FMDV serotype A vaccine strains used in South Korea, namely, A22/IRQ/24/64, A/Argentina/2001 + A24/Cruzeiro and A/Zabaikalsky/2013. Overall, the established LPBE is considered to be a convenient serological assay for use in postvaccination sero-monitoring.

The importance of smoking cessation during pregnancy and its association with perinatal outcomes

Abstract In utero exposure to tobacco smoke is associated with an increased risk of multiple adverse perinatal outcomes. Smoking cessation during pregnancy has been related to the improvement of these outcomes, but often relies on self-reporting, impairing an accurate assessment of smoking cessation impact on newborńs health. Building on data obtained in the frame of the NEOGENE project, this work aimed to estimate the association between smoking cessation among pregnant women, confirmed by maternal urinary cotinine concentrations, and perinatal outcomes. The study population included 595 pregnant women who sought prenatal care in a public hospital in Porto (Portugal), from April 2017 to July 2018. Data on tobacco consumption and cessation was obtained in a face-to-face interview, during the hospital stay. Perinatal outcomes, namely birth weight (BW), length (BL) and head circumference (HC) were retrieved from the hospital medical records. Maternal self-reported tobacco use was validated by urinary cotinine concentrations, using the solid-phase competitive ELISA technique. Maternal active smoking was associated with a significant decrease in BW, BL and HC of 157.66 g (p &amp;lt; 0.001), 0.78 cm (p = 0.001) and 0.39 cm (p = 0.016). Notably, maternal smoking cessation led to a significant increase in BW of approximately 172 g (p = 0.006), when compared to mothers who have not ceased. Increases in BL and HC did not reach significance. This study upholds that tobacco consumption is still an important public health threat in Portugal and that smoking cessation during pregnancy reverses smoking-associated deficits in perinatal outcomes, emphasizing the crucial need for awareness campaigns to promote smoking cessation during pregnancy. Acknowledgments: This work was supported by FCT and FAPESP (FAPESP/19914/2014). AIS, JM and CC also acknowledge FCT for individual funding (SFRH/BD/145101/2019, SFRH/BPD/115112/2016 and SFRH/BPD/96196/2013 grants, respectively). Key messages Maternal active smoking was significantly associated with lower birth weight, length and head circumference. Maternal smoking cessation reverses the smoking-associated deficits in birth weight.

Urinary cotinine assessment of maternal smoking and environmental tobacco smoke exposure status and its associations with perinatal outcomes: a cross-sectional birth study

Tobacco consumption and environmental tobacco smoke (ETS) exposure remains an important public health concern. Pregnant women require particular attention as active and passive smoking during pregnancy are associated with multiple adverse perinatal outcomes. This study aimed to biochemically validate self-reported smoking and ETS exposure status among pregnant women, to more precisely ascertain its association with adverse perinatal outcomes. Data refers to 595 pregnant women who sought prenatal care in a public hospital in Porto, Portugal. A standard questionnaire on smoking and ETS-related variables was completed. Urinary cotinine (UC) concentrations were assessed by solid-phase competitive ELISA, in maternal urine samples collected on the day of delivery. The results showed that the optimal UC cut-off value to distinguish smokers from non-smokers and within non-smokers those who were exposed to ETS from those non-exposed in the third trimester of pregnancy was 74.1 ng/mL (sensitivity and specificity of 96.7% and 98.0%, respectively) and 1.6 ng/mL (sensitivity of 66.2% and specificity of 75.7%, respectively). The agreement between maternal self-reported and UC-based smoking status was very good (κ=0.919, p<0.001), but much lower for ETS exposure (κ=0.386, p<0.001). Maternal active smoking in the third trimester of pregnancy was associated with a significant decrease in birth weight, length and head circumference of 157.66 g (95% CI: −245.81, −69.52; p<0.001), 0.78 cm (95% CI: −1.22, −0.34; p=0.001) and 0.39 cm (95% CI: −0.70, −0.07; p=0.016), respectively. Maternal ETS exposure in the third trimester of pregnancy was associated with a non-significant increase in birth weight of 38.37 g (95% CI: −28.91, 105.64; p=0.263). Furthermore, maternal smoking cessation was associated with the increase of approximately 172 g in birth weight (95% CI: 50.00, 293.19). As such, there is an urgent need for increased public health awareness campaigns to encourage smoking cessation during pregnancy, in order to improve perinatal outcomes.

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.

Seroprevalence of Foot and Mouth Disease Virus Infection in Some Wildlife and Cattle in Bauchi State, Nigeria.

Foot and mouth disease (FMD) is an important transboundary viral disease of both domestic and wild cloven-hoofed animals characterized by high morbidity with devastating consequence on the livestock worldwide. Despite the endemic nature of FMD in Nigeria, little is known about the epidemiology of the disease at the wildlife-livestock interface level. To address this gap, blood samples were collected between 2013 and 2015 from some wildlife and cattle, respectively, within and around the Yankari Game Reserve and Sumu Wildlife Park in Bauchi State, Nigeria. Wild animals were immobilized using a combination of etorphine hydrochloride (M99® Krüger-Med South Africa) at 0.5–2 mg/kg and azaperone (Stresnil®, Janssen Pharmaceuticals (Pty.) Ltd., South Africa) at 0.1 mg/kg using a Dan-Inject® rifle (Dan-Inject APS, Sellerup Skovvej, Denmark) fitted with a 3 ml dart syringe and for reversal, naltrexone (Trexonil® Kruger-Med South Africa) at 1.5 mg IM was used, and cattle were restrained by the owners for blood collection. Harvested sera from blood were screened for presence of antibodies against the foot and mouth disease virus (FMDV) using the PrioCHECK® 3ABC NSP ELISA kit, and positive samples were serotyped using solid-phase competitive ELISA, (IZSLER Brescia, Italy). Out of the 353 sera collected from cattle and wildlife 197 (65.7%) and 13 (24.5%) (P < 0.05), respectively, tested positive for antibodies to the highly conserved nonstructural 3ABC protein of FMDV by the FMDV-NS blocking ELISA. Classification of cattle into breed and sex showed that detectable antibodies to FMDV were higher (P < 0.05) in White Fulani 157 (72.8%) than in Red Bororo 23 (39.7%) and Sokoto Gudali 17 (33.3%) breeds of cattle, whereas in females, detectable FMDV antibodies were higher (P < 0.05) 150 (72.8%) than in males 47 (50.0%). In the wildlife species, antibodies to FMDV were detected in the waterbucks 2 (28.6%), elephant 1 (25.0%), wildebeests 4 (33.3%), and elands 6 (25.0%). Four serotypes of FMDV: O, A, SAT 1, and SAT 2 were detected from the 3ABC positive reactors in waterbucks, elephants, wildebeests, and elands. The results showed presence of antibodies to FMDV in some wildlife and cattle and suggested that wildlife could equally play an important role in the overall epidemiology of FMD in Nigeria. FMD surveillance system, control, and prevention program should be intensified in the study area.

Seroprevalence of foot-and-mouth disease virus in cattle herds raised in Maasai Mara ecosystem in Kenya

A cross-sectional study was carried out to determine foot-and-mouth disease (FMD) seroprevalence and identify risk factors of exposure among cattle herds raised in three zones with different types of land use and progressively distant from the Maasai Mara National Reserve (MMNR) boundary. We selected five villages purposively; two in zone 1 (area < 20 km from the MMNR), another two in zone 2 (area between 20−40 km away from the MMNR) and one in zone 3 (area >40 km away from the MMNR). A total of 1170 cattle sera were collected from 390 herds in all the zones and tested for antibodies against the non-structural proteins (NSPs) of FMD virus (FMDV) using two 3ABC-based Enzyme-Linked Immunosorbent Assay ELISA kits. All sera samples were also screened for serotype-specific antibodies using Solid Phase Competitive ELISA (SPCE) kits (IZSLER, Italy). We targeted FMDV serotypes A, O, South African Territory [SAT] 1 and SAT 2, known to be endemic in East Africa including Kenya. Data on putative risk factors for FMD seropositivity in cattle were collected using a questionnaire. The overall apparent animal-level FMD seroprevalence based on the parallel comparison of the two anti-NSPs ELISA kits was 83.8 % (95 % CI; 81.8–85.9), and differed significantly across zones. Zone 1 had a higher seroprevalence than zones 2 and 3 (χ2 = 116.1, df = 2, p < 0.001). In decreasing order, the overall seroprevalences of FMDV serotypes A, SAT 2, O and SAT 1 were 26.3 % (95 % CI; 23.5–29.2), 21.4 % (95 % CI; 18.8–24.0), 21.2 % (95 % CI; 18.7–23.9) and 13.1 % (95 % CI; 11.1–15.3), respectively. The distribution of these serotypes differed significantly between zones (p < 0.05) except for SAT 2 serotype (χ2 = 0.90, df = 2, p = 0.639). Both serotypes A and O were more prevalent in zones 1 and 2 than zone 3 while serotype SAT 1, was higher in zone 3 compared to other zones. The results of multivariable analyses identified animal sex (i.e., female), raising of cattle in zones 1 and 2 (areas < 40 km away from the MMNR); mixing of cattle from multiple herds at watering points, and pastoral husbandry practices, as significant predictors of animal-level FMD seropositivity. This study established that FMD seroprevalence declined with distance from the MMNR.

Development of Monoclonal Antibody Specific to Foot-and-Mouth Disease Virus Type A for Serodiagnosis.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease affecting cloven-hoofed livestock worldwide. FMD virus (FMDV) type A is one of the most common causes of FMD outbreaks among the seven FMDV serotypes, and its serological diagnosis is therefore important to confirm FMDV type A infection and to determine FMD vaccine efficacy. Here, we generated monoclonal antibodies (mAbs) specific to FMDV type A via hybridoma systems using an inactivated FMDV type A (A22/Iraq/1964) and found 4 monoclones (#29, #106, #108, and #109) with high binding reactivity to FMDV type A among 594 primary clones. In particular, the #106 mAb had a higher binding reactivity to the inactivated FMDV type A than the other mAbs and a commercial mAb. Moreover, the #106 mAb showed no cross-reactivity to inactivated FMDV type South African territories 1, 2, and 3, and low reactivity to inactivated FMDV type O (O1 Manisa). Importantly, the solid-phase competitive ELISA (SPCE) using horseradish peroxidase (HRP)-conjugated #106 mAb detected FMDV type A-specific Abs in sera from FMD type A-vaccinated cattle more effectively than a commercial SPCE. These results suggest that the newly developed FMDV type A-specific mAb might be useful for diagnostic approaches for detecting Abs against FMDV type A.

Seroprevalence and molecular characterization of foot-and-mouth disease virus in Chad.

This study aimed at determining the seroprevalence of foot‐and‐mouth disease (FMD) in domestic ruminants and at characterizing the virus strains circulating in four areas of Chad (East Batha, West Batha, Wadi Fira and West Ennedi). The study was carried out between October and November 2016. A total of 1,520 sera samples (928 cattle, 216 goats, 254 sheep and 122 dromedaries) were collected randomly for FMD serological analyses. Nine epithelial tissue samples were also collected from cattle showing clinical signs, for FMDV isolation and characterization. Serological results showed an overall NSP seroprevalence of 40% (375/928) in cattle in our sample (95% CrI [19–63]). However, seroprevalences of 84% (27/32), 78% (35/45) and 84% (21/25) were estimated in cattle over 5 years of age in East Batha, West Batha and Wadi Fira, respectively. In cattle under 1 year of age, 67% (18/27) seroprevalence was estimated in Wadi Fira, 64% (14/22) in East Batha and 59% (13/22) in West Batha. It was found that the high seroprevalences have been obtained in areas where pastures are shared by several different herds but also in farms where two to three species (bovine, caprine and ovine) are raised together. ELISA PrioCHECK®FMDV types O and A and in‐house solid phase competition ELISA serotyping results showed that the four O, A, SAT1 and SAT2 serotypes have circulated in Chad in 2016. However, the type SAT2 dominated with an overall seroprevalence of 43% (29/67) and was present in the four areas investigated. The phylogenetic analyses of the VP1 coding sequence allowed determining the serotype SAT2 topotype VII, close to viral strains found in Cameroon in 2015 with a similarity of 98.60%.

Serological and molecular epidemiology of foot-and-mouth disease viruses in agro-pastoralist livestock herds in the kachia grazing reserve, Nigeria.

The Kachia Grazing Reserve (KGR) is located in Kaduna state in north-western Nigeria and consists of 6 contiguous blocks housing 744 defined households (HH), all engaged in livestock keeping. It is considered as a homogenous epidemiological unit and a defined study area. In 2012, all cattle and sheep of 40 selected HH were sampled to determine sero-prevalence of antibodies to foot-and-mouth disease virus (FMDV) and of FMDV. The overall sero-prevalence of antibodies to the non-structural 3ABC protein (NSP-3ABC ELISA) was 28.9% (380/1,315) (30.6% cattle; 16.3% sheep), and in 4.5% (62/1,380) (5% cattle; 0.6% sheep) of the examined sera FMD viral RNA could be detected by real-time RT-PCR (rRT-PCR). Additionally, in 2012 and 2014 serum, epithelium and probang samples were collected from cattle in reported FMD outbreaks and the causative FMDVs were molecularly characterized. Approximately half (28/59) of the outbreak sera reacted positive in NSP-3ABC ELISA, and 88% (52/59) of the outbreak sera contained detectable viral RNA. Overall, antibodies against five FMDV serotypes (O, A, SAT1, SAT2 and SAT3) were detected by solid phase competitive ELISA with combinations of two or more serotypes being common. Of the 21 FMDVs that could be isolated 19 were sequenced and 18 were confirmed as SAT2 (lineage VII) while one was characterized as serotype O (EA-3 topotype). Phylogenetic analysis revealed a close relationship between Nigerian FMDV strains and strains in this region and even with strains in North-Africa. Our findings indicate that FMD constitutes an endemic health problem to cattle rearing in the agro-pastoralist community in the KGR and that the KGR is not a closed epidemiological unit. Insight into the local FMDV epidemiology and in the circulating FMDV serotypes/strains is of support to the relevant authorities in Nigeria when considering the need for an FMD control policy to improve animal production in grazing reserves.

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

BackgroundFoot-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.ResultsThe 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.ConclusionsThe 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.

MAb-based competitive ELISA for the detection of antibodies against influenza D virus.

Influenza D virus (IDV) was first reported in 2011 in swine in Oklahoma and consequently found in cattle, sheep, and goats across North America and Eurasia. Cattle have been proposed as the natural reservoir. In this study, we developed and validated a MAb-based competitive ELISA for the detection of antibodies against IDV. Thirty-one hybridomas specific to IDV were generated using Balb/C mice immunised with purified IDV/Swine/Italy/199724-3/2015. The specificity of MAbs was determined by comparing their reactivity with the homologous and other influenza A viruses along with additional bovine and swine viruses. A solid-phase competitive ELISA (IDV-cELISA) was set up using the partially purified antigen coated to the plate and incubation of two serum dilutions (1/10 and 1/20) followed by addition of a peroxidase-conjugated MAb as competitor, which had shown wide intratype cross-reactivity and positivity in HI. To evaluate the diagnostic performances of IDV-cELISA, we used 884 sera (414 negative and 470 positive) from different species. ROC analyses were performed to enable the selection of best cut-off value and estimation of diagnostic specificity and sensitivity. The agreement between IDV-cELISA and HI test was assessed by Cohen's kappa value (κ). The κ analysis showed an almost perfect agreement (κ=0.93; 95%CI -0.899 to 0.961) between HI test and IDV-cELISA. ROC analysis showed that IDV-cELISA was accurate with an area under the curve (AUC)=0.999, 95% CI 0.993-1.000). A cut-off value of 65% was selected with Se and Sp values of 99.35 (95% CI 98.1-99.9) and 98.75 (95% CI 97.1-99.6). These results proved excellent diagnostic performances of IDV-cELISA, which compared to HI presented major advantages, such as suitability for automation, low dependence on individual skills, spectrophotometric reading, and easy interpretation of the results. This assay can be exploited to detect anti-IDV antibodies in different animal species.

Two novel blood-based biomarker candidates measuring degradation of tau are associated with dementia: A prospective study

BackgroundTruncated tau appears to be specifically related to disease pathology and recent studies have shown the presence and elevation of several truncated tau species in Cerebrospinal fluid (CSF) of subjects with Alzheimer’s disease (AD); however, the relevance of truncated Tau measurements in blood is still being studied.ObjectiveThe aim of the current study was to assess the longitudinal associations between baseline levels of two novel blood biomarker candidates measuring truncated tau, Tau-A and Tau-C, and the risk of incident dementia and AD in elderly women.MethodsUsing solid phase competitive ELISA, two tau fragments were detected in serum of 5,309 women from the Prospective Epidemiological Risk Factor study. The study was an observational, prospective study of Danish postmenopausal women. Subjects were followed with registry-linkage for up to 15 years (median follow-up time 13.7 years). Cox regression was used to assess the utility of the biomarker candidates in relation to dementia and AD.ResultsHigh levels of Tau-A and Tau-C (above the median) in blood were associated with lower risk of dementia and AD (Tau-A: Dementia HR[95% CI] = 0.85[0.70–1.04]; AD 0.71[0.52–0.98] and Tau-C: Dementia 0.84[0.70–1.00]; AD 0.78[0.60–1.03]). Tau-C gave a very modest increase in the AUC in a 5-year prediction horizon as compared to a reference model with age and education, while a combination of the two did not improve their predictive capacity.ConclusionsMeasurement of tau in serum is feasible. The serological tau turnover profile may be related to the diagnosis and development of dementia and AD. The exact processing and profile in serum in relation to cognitive disorders remains to be further assessed to provide simple non-invasive tests to identify subjects with progressive cognitive disorders.