Rapid detection and quantification of the marine toxic algae, Alexandrium minutum, using a super-paramagnetic immunochromatographic strip test

The dinoflagellates of Alexandrium genus are known to be producers of paralytic shellfish toxins that regularly impact the shellfish aquaculture industry and fisheries. Accurate detection of Alexandrium including A. minutum is crucial for environmental monitoring and sanitary issues. In this study, we firstly developed a quantitative lateral flow immunoassay (LFIA) using super-paramagnetic nanobeads for A. minutum whole cells. This dipstick assay relies on two distinct monoclonal antibodies used in a sandwich format and directed against surface antigens of this organism. No sample preparation is required. Either frozen or live cells can be detected and quantified. The specificity and sensitivity are assessed by using phytoplankton culture and field samples spiked with a known amount of cultured A. minutum cells. This LFIA is shown to be highly specific for A. minutum and able to detect reproducibly 105 cells/L within 30 min. The test is applied to environmental samples already characterized by light microscopy counting. No significant difference is observed between the cell densities obtained by these two methods. This handy super-paramagnetic lateral flow immnunoassay biosensor can greatly assist water quality monitoring programs as well as ecological research.

Acute phase proteins and biomarkers for health in chickens

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.

Avaliação citotóxica de Amoxilina e Clavulanato de Potássio em mexilhões Perna perna

Dissertação (Mestrado em Tecnologia Nuclear)

Usefulness of the Hepatocyte Growth Factor as a Predictor of Mortality in Patients Hospitalized With Acute Heart Failure Regardless of Ejection Fraction

Hepatocyte growth factor (HGF) plays a role in the improvement of cardiac function and remodeling. Their serum levels are strongly related with mortality in chronic systolic heart failure (HF). The aim of this study was to study prognostic value of HGF in acute HF, interaction with ejection fraction, renal function, and natriuretic peptides. We included 373 patients (age 76 ± 10 years, left ventricular ejection fraction [LVEF] 46 ± 14%, 48% men) consecutively admitted for acute HF. Blood samples were obtained at admission. All patients were followed up until death or close of study (>1 year, median 371 days). HGF concentrations were determined using a commercial enzyme-linked immunosorbent assay (human HGF immunoassay). The predictive power of HGF was estimated by Cox regression with calculation of Harrell C-statistic. HGF had a median of 1,942 pg/ml (interquartile rank 1,354). According to HGF quartiles, mortality rates (per 1,000 patients/year) were 98, 183, 375, and 393, respectively (p <0.001). In Cox regression analysis, HGF (hazard ratio1SD = 1.5, 95% confidence interval 1.1 to 2.1, p = 0.002) and N-terminal pro b-type natriuretic peptide (NT-proBNP; hazard ratio1SD = 1.8, 95% confidence interval 1.2 to 2.6, p = 0.002) were independent predictors of mortality. Interaction between HGF and LVEF, origin, and renal function was nonsignificant. The addition of HGF improved the predictive ability of the models (C-statistic 0.768 vs 0.741, p = 0.016). HGF showed a complementary value over NT-proBNP (p = 0.001): mortality rate was 490 with both above the median versus 72 with both below. In conclusion, in patients with acute HF, serum HGF concentrations are elevated and identify patients at higher risk of mortality, regardless of LVEF, ischemic origin, or renal function. HGF had independent and additive information over NT-proBNP.

PREVALENCE AND PREDICTORS OF VITAMIN A DEFICIENCY AMONG INFANTS IN WESTERN KENYA USING A CROSS-SECTIONAL ANALYSIS

Vitamin A (VA) deficiency (VAD) is a major nutritional public health problem among children under-5-years-old in the developing world including Kenya. A community-based cross-sectional survey among 1,630 children (aged 6-23 mos) was undertaken in Western Kenya. A questionnaire was administered to collect demographic, socio-economic and dietary intake information. Prevalence of low retinol-binding protein (RBP) concentrations was assessed using Dried Blood Spot (DBS) methodology. Analysis of RBP was carried out using rapid enzyme immunoassay (EIA) and C-reactive protein (CRP) was carried out using enzyme linked immunosorbent assay (ELISA) to estimate VA and sub-clinical inflammation statuses, respectively. Values were adjusted for influence of inflammation using CRP (CRP >5 mg/L) and population prevalence of VAD (RBP <0.825 μmol/L, biologically equivalent to 0.70 μmol/L retinol) estimated. Anthropometric data gave three indices: stunting, wasting and underweight—all of which took age and sex into consideration. Mean (geometric± SD) concentration of RBP was adequate (1.56±0.79μmol/L) but the inflammation-adjusted mean (±SE) prevalence of VAD was high (20.1±1.1%) in this population. The level of CRP was within normal range (1.06±4.95 mg/L) whilst 18.4±0.9% of the children had subclinical inflammation (CRP>5 mg/L). Intake of VA capsule (VAC) by a child was a predictor of VAD with children who have not taken VA during the past 1 year prior to the survey having a 30% increased risk of VAD (OR (CI): 1.3 (1.1-1.7); p=0.025. Additionally, age of the child was a predictor with older children (18-23 mos) having a 30 % increased risk of VAD (OR (CI): 1.3 (1.1-1.9); p=0.035); the caretaker’s knowledge on VA and nutrition was also a predictor of VAD with children whose caretaker’s had poor knowledge having a 40 % increased risk of VAD (OR (CI): 1.4 (1.0-1.9); p=0.027. A child’s district of residence was also a significant predictor of VAD. Prevalence of VAD in this sample of infants was high. Predictors of VAD included child intake of VAC in the last 1 year before the survey, older children, children whose caretakers had poor VA and nutritional knowledge and a child’s district of residence. There is a need to improve knowledge on nutrition and VA of caretakers; undertake a targeted VAC distribution, particularly in children older than 1 year and above and use a sustainable food-based intervention in the areas with severe VAD.

Primary dengue virus infections induce differential cytokine production in Mexican patients

Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF) and dengue haemorrhagic fever (DHF) from dengue virus (DENV) 1 (n = 116) or 2 (n = 55) were compared. DF and DHF were defined according to the patient’s clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcriptionpolymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL)-12p70, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

Characteristics of Clostridium difficile infection in patients with discordant diagnostic test results

Background: Clinical features of Clostridium difficile infection (CDI) cases diagnosed by detection of polymerase chain reaction (PCR), with negative toxin enzyme immunoassay results (EIA) have not been fully elucidated. The purpose of this study was to determine the magnitude of CDI patients who had negative EIA toxin determinations but positive PCR tests, and their differences in clinical presentation. Methods: We performed a retrospective study comparing the clinical features of CDI cases detected by EIA (toxins A + B) with cases detected by PCR (toxin negative, PCR positive) over a 16-month period. Only patients with an initial Clostridium difficile infection episode that fulfilled a standardized definition were included. Results: During the study period, 107 episodes of CDI were detected. Seventy-four patients (69%) had positive glutamate dehydrogenase (GDH) antigen and EIA determinations (EIA positive patients). Thirty-three patients (31%) had GDH positive, negative toxin EIA and positive PCR determination (PCR positive patients). PCR positive patients were younger, 57 (27) years (mean [SD]), than EIA positive patients, 71 (16) years, (p < 0.001). Fewer PCR positive patients were receiving proton pump inhibitors (21 patients, 64%) than EIA positive patients (61 patients, 82%, p = 0.034). The clinical presentation was similar in both groups. In the multivariate analysis, lower age was identified as the only independent variable associated with PCR positive patients. Conclusions: One third of Clostridium difficile infection patients present negative toxin EIA and PCR positive tests. Performing PCR determination after the negative EIA test is more relevant in younger patients.

Salivary cortisol level and uncooperative behavior in pediatric dental practice

Aim: To investigate the relation between uncooperative behavior and salivary cortisol level in children who underwent preventive dental care. Methods: The sample was composed by 10 children of both sexes aged 40 to 52 months, presenting uncooperative behavior during dental preventive treatments. The saliva collection was performed using a cotton wheel and an Eppendorf tube (Sarstedt Salivete®) in 3 different moments: a) at home, on a day without dental treatment and at the same time on the day of the sessions treatment; b) 30 min after the end of the session, when there was manifestation of uncooperative behavior; c) 30 min after the end of the session, when there was a cooperative behavior of the child. A sample of saliva was centrifuged for 5 minutes at 2400 rpm, 1 of mL of saliva was pipetted in an Eppendorf tube and stored in a freezer at -20 ° C. For the determination of the levels of salivary cortisol was used an Active® kit for cortisol enzyme immunoassay (EIA) DSL-10-67100, composed of specific rabbit antibody anti-cortisol. Data were analyzed statistically for the uncooperative behavior issued in the beginning and at the end of sessions, using the paired t test (p<0.05) and for cortisol levels in saliva samples at home, after the beginning and at the end of sessions, using repeated-measures ANOVA and Tukey’s test (p<0.05). Results: During expression of uncooperative behavior in preventive dental care sessions the salivary cortisol level was significantly higher (0.65 ± 0.25 μg/dL) compared with expression of collaborative behavior (0.24 ± 0.10 μg/dL). Conclusions: It is possible to conclude that, even under preventive intervention, the stress must be controlled in order to reduce dental anxiety and fear.

Circuit resistance training improved endothelial dysfunction in obese aged women

Introduction: It is widely accepted that obesity is associated with endothelial dysfunction. In a recent paper, we have also found circuit resistance training may reduce visceral fat in obese aged women. Accordingly, the current study was conducted to ascertain the effects of circuit resistance training on markers of endothelial dysfunction in this population group. Methods: In the present interventional study, a total of 48 obese aged women were recruited from the community. Twenty-four of them were randomly assigned to perform a 12-week resistance circuit training programme, 3-days per week. This training was circularly performed in 6 stations: arm curl, leg extension, seated row, leg curl, triceps extension and leg press. The Jamar handgrip electronic dynamometer was used to assess maximal handgrip strength of the dominant hand. Lastly, serum samples were analysed using an immunoassay (ELISA) for endothelin-1, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Results: When compared to baseline, resistance training significantly reduced serum levels of endothelin-1 (2.28 ± 0.7 vs. 1.98 ± 1.1 pg/ml; p = 0.019; d = 0.67) and ICAM-1 (290 ± 69 vs. 255 ± 76 ng/ml; p = 0.004; d = 0.92) in the experimental group. No significant changes in any of the tested outcomes were found in the control group. Conclusion: A short-term circuit resistance program improved endothelial dysfunction in aged obese women. Further studies on this topic are still required to consolidate this approach in clinical application.

Anti-müllerian hormone serum concentrations of women with germline BRCA1 or BRCA2 mutations

STUDY QUESTION Do women with BRCA1 or BRCA2 mutations have reduced ovarian reserve, as measured by circulating anti-Müllerian hormone (AMH) concentration?SUMMARY ANSWER Women with a germline mutation in BRCA1 have reduced ovarian reserve as measured by AMH.WHAT IS KNOWN ALREADY The DNA repair enzymes encoded by BRCA1 and BRCA2 are implicated in reproductive aging. Circulating AMH is a biomarker of ovarian reserve and hence reproductive lifespan.STUDY DESIGN, SIZE, DURATION This was a cross-sectional study of AMH concentrations of 693 women at the time of enrolment into the Kathleen Cuningham Foundation Consortium for research in the Familial Breast Cancer (kConFab) cohort study (recruitment from 19 August 1997 until 18 September 2012). AMH was measured on stored plasma samples between November 2014 and January 2015 using an electrochemiluminescence immunoassay platform.PARTICIPANTS/MATERIALS, SETTING, METHODS Eligible women were from families segregating BRCA1 or BRCA2 mutations and had known mutation status. Participants were aged 25–45 years, had no personal history of cancer, retained both ovaries and were not pregnant or breastfeeding at the time of plasma storage. Circulating AMH was measured for 172 carriers and 216 non-carriers from families carrying BRCA1 mutations, and 147 carriers and 158 non-carriers from families carrying BRCA2 mutations. Associations between plasma AMH concentration and carrier status were tested by linear regression, adjusted for age at plasma storage, oral contraceptive use, body mass index and cigarette smoking.MAIN RESULTS AND THE ROLE OF CHANCE Mean AMH concentration was negatively associated with age (P < 0.001). Mutation carriers were younger at blood draw than non-carriers (P ≤ 0.031). BRCA1 mutation carriers had, on average, 25% (95% CI: 5%–41%, P = 0.02) lower AMH concentrations than non-carriers and were more likely to have AMH concentrations in the lowest quartile for age (OR 1.84, 95% CI: 1.11–303, P = 0.02). There was no evidence of an association between AMH concentration and BRCA2 mutation status (P = 0.94).LIMITATIONS, REASONS FOR CAUTION AMH does not directly measure the primordial follicle pool. The clinical implications of the lower AMH concentrations seen in BRCA1 mutation carriers cannot be assessed by this study design.WIDER IMPLICATIONS OF THE FINDINGS Women with a germline mutation in BRCA1 may have reduced ovarian reserve. This is consistent with other smaller studies in the literature and has potential implications for fertility and reproductive lifespan.STUDY FUNDING/COMPETING INTEREST(S) kConFab is supported by a grant from the Australian National Breast Cancer Foundation, and previously by the National Health and Medical Research Council (NHMRC), the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania and South Australia, and the Cancer Foundation of Western Australia. K.A.P. is an Australian National Breast Cancer Foundation Practitioner Fellow. J.L.H. is a NHMRC Senior Principal Research Fellow. M.H. is a NHMRC Practitioner Fellow. R.A.A. reports personal fees from Roche Diagnostics & Beckman Coulter outside the submitted work and C.S. reports other earnings from Melbourne IVF outside the submitted work. The remaining authors have nothing to declare and no conflicts of interest.

One size does not fit all : monitoring faecal glucocorticoid metabolites in marsupials

Marsupial research, conservation, and management can benefit greatly from knowledge about glucocorticoid (GC) secretion patterns because GCs influence numerous aspects of physiology and play a crucial role in regulating an animal's response to stressors. Faecal glucocorticoid metabolites (FGM) offer a non-invasive tool for tracking changes in GCs over time. To date, there are relatively few validated assays for marsupials compared with other taxa, and those that have been published generally test only one assay. However, different assays can yield very different signals of adrenal activity. The goal of this study was to compare the performance of five different enzyme immunoassays (EIAs) for monitoring adrenocortical activity via FGM in 13 marsupial species. We monitored FGM response to two types of events: biological stressors (e.g., transport, novel environment) and pharmacological stimulation (ACTH injection). For each individual animal and assay, FGM peaks were identified using the iterative baseline approach. Performance of the EIAs for each species was evaluated by determining (1) the percent of individuals with a detectable peak 0.125-4.5days post-event, and (2) the biological sensitivity of the assay as measured by strength of the post-event response relative to baseline variability (Z-score). Assays were defined as successful if they detected a peak in at least 50% of the individuals and the mean species response had a Z⩾2. By this criterion, at least one assay was successful in 10 of the 13 species, but the best-performing assay varied among species, even those species that were closely related. Furthermore, the ability to confidently assess assay performance was influenced by the experimental protocols used. We discuss the implications of our findings for biological validation studies.

Effects of Increased Levels of Prenatal Mesotocin on Postnatal Individual Recognition and Stress Responsiveness in Northern bobwhite quail (Colinus Virginianus)

Oxytocin (OT) plays a key role in the mediation of social and stress behaviors across many species; however, the mechanism is still unclear. The present study investigated the influence of prenatal levels of mesotocin (MT; avian homologue of OT) on postnatal social and stress behavior in Northern bobwhite quail. Experiment one determined endogenous levels of MT during prenatal development using an enzyme-linked immunoassay kit. Experiment two examined the influence of increased MT during prenatal development on chicks' individual recognition ability and stress response to a novel environment. Experiment one showed MT levels increased significantly throughout embryonic development. Experiment two showed significant differences in stress behavior for chicks with increased MT during prenatal development; however, no significant differences were found for social behavior. This study suggests MT serves different functions depending on the stage of embryonic development and that increasing MT levels affects postnatal stress behavior, but not social behavior.

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.