Ashutosh Wadhwa

Bead-based microfluidic immunoassay for diagnosis of Johne's disease.

Microfluidics technology offers a platform for development of point-of-care diagnostic devices for various infectious diseases. In this study, we examined whether serodiagnosis of Johnes disease (JD) can be conducted in a bead-based microfluidic assay system. Magnetic micro-beads were coated with antigens of the causative agent of JD, Mycobacterium avium subsp. paratuberculosis. The antigen-coated beads were incubated with serum samples of JD-positive or negative serum samples and then with a fluorescently-labeled secondary antibody (SAB). To confirm binding of serum antibodies to the antigen, the beads were subjected to flow cytometric analysis. Different conditions (dilutions of serum and SAB, types of SAB, and types of magnetic beads) were optimized for a large degree of differentiation between the JD-negative and JD-positive samples. Using the optimized conditions, we tested a well-classified set of 155 serum samples from JD-negative and JD-positive cattle by using the bead-based flow cytometric assay. Of 105 JD-positive samples, 63 samples (60%) showed higher antibody binding levels than a cut-off value determined by using antibody binding levels of JD-negative samples. In contrast, only 43-49 JD-positive samples showed higher antibody binding levels than the cut-off value when the samples were tested using commercially-available immunoassays. Microfluidic assays were performed by magnetically immobilizing a number of beads within a microchannel of a glass microchip and detecting antibody on the collected beads using laser-induced fluorescence. Antigen-coated magnetic beads treated with the bovine serum sample and fluorescently-labeled SAB were loaded into a microchannel to measure the fluorescence (reflecting level of antibody binding) on the beads in the microfluidic system. When the results of five bovine serum samples with the microfluidic system were compared to those analyzed with the flow cytometer, a high level of correlation (linear regression, r(2)=0.994) was observed. In a further experiment, we magnetically immobilized antigen-coated beads in a microchannel, reacted the beads with serum and SAB in the channel, and detected antibody binding to the beads in the microfluidic system. A strong antibody binding in JD-positive serum was detected, whereas there was only negligible binding in negative control experiments. Our data suggest that the bead-based microfluidic system may form a basis for development of an on-site serodiagnosis of JD.

Rabies surveillance in the United States during 2015

OBJECTIVE To describe rabies and rabies-related events occurring during 2015 in the United States. DESIGN Observational study based on passive surveillance data. ANIMALS All animals submitted for rabies testing in the United States during 2015. PROCEDURES State and territorial public health programs provided data on animals submitted for rabies testing in 2015. Data were analyzed temporally and geographically to assess trends in domestic and sylvatic animal rabies cases. RESULTS During 2015, 50 states and Puerto Rico reported 5,508 rabid animals to the CDC, representing an 8.7% decrease from the 6,033 rabid animals reported in 2014. Of the 5,508 cases of animal rabies, 5,088 (92.4%) involved wildlife. Relative contributions by the major animal groups were as follows: 1,704 (30.9%) bats, 1,619 (29.4%) raccoons, 1,365 (24.8%) skunks, 325 (5.9%) foxes, 244 (4.4%) cats, 85 (1.5%) cattle, and 67 (1.2%) dogs. There was a 4.1% decrease in the number of samples submitted for testing in 2015, compared with the number submitted in 2014. Three human rabies deaths were reported in 2015, compared with only 1 in 2014. A 65-year-old man in Massachusetts was bitten by a rabid dog while abroad. A 77-year-old woman in Wyoming had contact with a bat. A 54-year-old man in Puerto Rico was bitten by a mongoose. The only connection among these 3 cases was that none received postexposure prophylaxis. CONCLUSIONS AND CLINICAL RELEVANCE Laboratory testing of animals suspected to be rabid remains a critical public health function and continues to be a cost-effective method to directly influence human rabies postexposure prophylaxis recommendations. (J Am Vet Med Assoc 2017;250:1117-1130).

Modeling for cost analysis of Johne's Disease control based on evelisa testing

Use of enzyme-linked immunosorbent assay (ELISA) is recommended for control of Johnes disease (JD) in the cattle industry. A recent report showed that prevalence of JD in dairy farms could be reduced by applying an ELISA-based control strategy, even though the sensitivity of the current ELISA has been reported to be lower than 30%. We previously developed a more sensitive ELISA test (EVELISA; Ethanol Vortex ELISA) for diagnosis of JD and, in this report, aimed to evaluate cost-effectiveness of the EVELISA in JD control compared to that of a current ELISA test. For simulation of population dynamics, we developed a deterministic, discrete-time mathematical model incorporating contact structure, possibility of adult infection and the concept of order of events. In our model, the number of animals infected with the causative agent of JD, Mycobacterium avium subsp. paratuberculosis (MAP), increases in a 10-year simulation if no JD control measure is applied. When test results of ELISA or EVELISA are used for JD control, the increase in MAP-infected animals is less significant. According to our model, EVELISA-based control measures increase the annual per capita revenue of US dairy farms when compared to no JD control and ELISA-based JD control, respectively.

Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer ( Cervus elaphus ) with bovine tuberculosis

BackgroundBovine tuberculosis (bTB) in wildlife species poses a threat to domestic livestock in many situations. Control programs for bTB in livestock depend on testing and slaughtering the positive animals; however, the currently available diagnostic tests often have poor specificity. In our previous study, we developed a specific and sensitive enzyme linked immunosorbent assay (ELISA) for another mycobacterial disease – Johne’s disease, using surface antigens of Mycobacterium avium ssp. paratuberculosis (MAP) extracted by briefly agitating the bacilli in 80% ethanol solution. The ELISA test was named ethanol vortex ELISA (EVELISA). The objective of this study is to examine whether EVELISA technique could be used to specifically detect anti-Mycobacterium bovis (M. bovis) antibodies in the serum of M. bovis-infected farmed red deer (Cervus elaphus). We tested a total of 45 red deer serum samples, divided in 3 groups – uninfected animals (n = 15), experimentally infected with M. bovis (n = 15) and experimentally infected with MAP (n = 15).ResultsThe presence of anti-M. bovis antibodies was tested using an ethanol extract of M. bovis. Without absorption of anti-MAP cross reactive antibodies, it was found that 13 out of the 15 MAP-infected animals showed high antibody binding. Using heat killed MAP as an absorbent of cross reactive antibodies, anti-M. bovis antibodies were detected in 86.7% of M. bovis-infected animals with minor false positive results caused by MAP infection.ConclusionsThe results from this study suggest that EVELISA may form a basis for a sensitive and specific test for the diagnosis of bTB in farmed red deer.

Optimization of Serum EVELISA for Milk Testing of Johne's Disease

Johnes disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most economically important diseases of dairy cattle. Control of JD could be achieved by good herd management practices, and diagnosis; however, this approach has been hampered by the low sensitivity of currently available enzyme-linked immunosorbent assay (ELISA) tests. In our previous study, we developed a sensitive serum ELISA test, ethanol-vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti-MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD-free herds. We evaluated five environmental mycobacteria as absorbents of cross-reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal polymerase chain reaction test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean±SD=0.355±0.455) was significantly higher than that in the JD-negative milk samples (mean±SD=0.071±0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.

Evaluation of ethanol vortex ELISA for detection of bovine tuberculosis in cattle and deer

BackgroundThe use of serological assays for diagnosis of bovine tuberculosis (TB) has been intensively studied and use of specific antigens have aided in improving the diagnostic accuracy of the assays. In the present study, we report an in-house enzyme linked immunosorbent assay (ELISA), developed by using ethanol extract of Mycobacterium bovis (M. bovis). The assay, named (ethanol vortex ELISA [EVELISA]), was evaluated for detection of anti- M. bovis antibodies in the sera of cattle and white-tailed deer.MethodsBy using the EVELISA, we tested sera obtained from two species of animals; cattle (n = 62 [uninfected, n = 40; naturally infected, n = 22]) and white-tailed deer (n = 41 [uninfected, n = 25; naturally infected, n = 7; experimentally infected, n = 9]). To detect species specific molecules, components in the ethanol extract were analyzed by thin layer chromatography and western blotting.ResultsAmong the tested animals, 77.2% of infected cattle and 87.5% of infected deer tested positive for anti- M. bovis antibody. There were only minor false positive reactions (7.5% in cattle and 0% in deer) in uninfected animals. M. bovis -specific lipids and protein (MPB83) in the ethanol extract were detected by thin layer chromatography and western blotting, respectively.ConclusionThe results warrant further evaluation and validation of EVELISA for bovine TB diagnosis of traditional and alternative livestock as well as for free-ranging animal species.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.