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.
This work presents an AC electrokinetic impedance sensing method that is capable of detecting specific interactions between macromolecules such as antigen-antibody binding. Serum samples were added ...to the surface of interdigitated electrodes that had been coated with bacterial antigens. After applying an AC signal of 100 mV at a specific frequency continuously, the electrodes' impedance change was recorded and used to determine the occurrence and level of antibody binding to the antigen. Our theoretical analysis indicated that with this AC signal, the target macromolecules will experience a sufficiently strong attraction force towards the electrode surface for acceleration of the binding process. Using this method, 11 human tuberculosis and 10 bovine tuberculosis serum samples were tested. The results were consistent with those obtained by a conventional ELISA method. The limit of detection of the impedance sensing method was estimated to be better than 10 ng mL
−1
. In summary, we demonstrate that AC electrokinetic impedance sensing can be used for rapid and sensitive detection of specific antibodies in serum samples. This method may form a basis for development of a point of care diagnostic device for human and bovine tuberculosis.
This work presents an AC electrokinetic impedance sensing method that is capable of detecting specific interactions between macromolecules such as antigen-antibody binding.
A full nucleoprotein gene sequencing of 68 isolates collected from passive rabies surveillance system in Georgia between 2015 and 2016 identified two distinct dog rabies phylogroups, GEO_V1 and ...GEO_V2, which both belonged to the cosmopolitan dog clade. GEO_V1 was found throughout the country and was further divided into four sub-phylogroups that overlapped geographically; GEO_V2 was found in the southeast region and was closely related to dog rabies in Azerbaijan. A sequence analysis of the full N gene, partial nucleoprotein gene of N-terminal and C-terminal, and the amplicon sequences of pan-lyssavirus RT-qPCR LN34 showed that all four sequencing approaches provided clear genetic typing results of canine rabies and could further differentiate GEO_V1 and GEO_V2. The phylogenetic analysis results vary and were affected by the length of the sequences used. Amplicon sequencing of the LN34 assay positive samples provided a rapid and cost-effective method for rabies genetic typing, which is important for improving rabies surveillance and canine rabies eradication globally.
Rabies surveillance in the United States during 2014 Monroe, Benjamin P; Yager, Pamela; Blanton, Jesse ...
Journal of the American Veterinary Medical Association,
2016-Apr-01, Letnik:
248, Številka:
7
Journal Article
Serologic assays developed for SARS-CoV-2 detect different antibody subtypes and are based on different target antigens. Comparison of the performance of a SARS-CoV-2 Spike-Protein ELISA and the ...nucleocapsid-based Abbott Architect.sup.TM SARS-CoV-2 IgG assay indicated that the assays had high concordance, with rare paired discordant tests results.
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Environmental surveillance can be used for monitoring enteric disease in a population by detecting pathogens, shed by infected people, in sewage. Detection of pathogens depends on ...many factors: infection rates and shedding in the population, pathogen fate in the sewerage network, and also sampling sites, sample size, and assay sensitivity. This complexity makes the design of sampling strategies challenging, which creates a need for mathematical modeling to guide decision making.
In the present study, a model was developed to simulate pathogen shedding, pathogen transport and fate in the sewerage network, sewage sampling, and detection of the pathogen. The simulation study used Salmonella enterica serovar Typhi (S. Typhi) as the target pathogen and two wards in Kolkata, India as the study area. Five different sampling strategies were evaluated for their sensitivity of detecting S. Typhi, by sampling unit: sewage pumping station, shared toilet, adjacent multiple shared toilets (primary sampling unit), pumping station + shared toilets, pumping station + primary sampling units. Sampling strategies were studied in eight scenarios with different geographic clustering of risk, pathogen loss (decay, leakage), and sensitivity of detection assays. A novel adaptive sampling site allocation method was designed, that updates the locations of sampling sites based on their performance. We then demonstrated how the simulation model can be used to predict the performance of environmental surveillance and how it is improved by optimizing the allocation of sampling sites.
The results are summarized as a decision tree to guide the sampling strategy based on disease incidence, geographic distribution of risk, pathogen loss, and the sensitivity of the detection assay. The adaptive sampling site allocation method consistently outperformed alternatives with fixed site locations in most scenarios. In some cases, the optimum allocation method increased the median sensitivity from 45% to 90% within 20 updates.
Phages, such as those infecting
spp., have been proven to be reliable indicators of human fecal contamination in microbial source tracking (MST) studies, and the efficacy of these MST markers found ...to vary geographically. This study reports the application and evaluation of candidate MST methods (phages infecting previously isolated
strain GB-124, newly isolated
strains (K10, K29, and K33) and recently isolated
strain ASH-08), along with non-source specific somatic coliphages (SOMCPH infecting strain WG-5) and indicator bacteria (
) for identifying fecal contamination pathways in Kolkata, India. Source specificity of the phage-based methods was first tested using 60 known non-human fecal samples from common animals, before being evaluated with 56 known human samples (municipal sewage) collected during both the rainy and dry season. SOMCPH were present in 40-90% of samples from different animal species and in 100% of sewage samples. Phages infecting
strain GB-124 were not detected from the majority (95%) of animal samples (except in three porcine samples) and were present in 93 and 71% of the sewage samples in the rainy and dry season (Mean = 1.42 and 1.83 log
PFU/100mL, respectively), though at lower levels than SOMCPH (Mean = 3.27 and 3.02 log
PFU/100mL, respectively). Phages infecting strain ASH-08 were detected in 89 and 96% of the sewage samples in the rainy and dry season, respectively, but were also present in all animal samples tested (except goats). Strains K10, K29, and K30 were not found to be useful MST markers due to low levels of phages and/or co-presence in non-human sources. GB-124 and SOMCPH were subsequently deployed within two low-income neighborhoods to determine the levels and origin of fecal contamination in 110 environmental samples.
, SOMCPH, and phages of GB-124 were detected in 68, 42, and 28% of the samples, respectively. Analyses of 166 wastewater samples from shared community toilets and 21 samples from sewage pumping stations from the same districts showed that SOMCPH were present in 100% and GB-124 phages in 31% of shared toilet samples (Median = 5.59 and <1 log
PFU/100 mL, respectively), and both SOMCPH and GB-124 phages were detected in 95% of pumping station samples (Median = 5.82 and 4.04 log
PFU/100 mL, respectively). Our findings suggest that GB-124 and SOMCPH have utility as low-cost fecal indicator tools which can facilitate environmental surveillance of enteric organisms, elucidate human and non-human fecal exposure pathways, and inform interventions to mitigate exposure to fecal contamination in the residential environment of Kolkata, India.
Orf (contagious ecthyma) is an exanthematic disease caused by a parapoxvirus and occurs primarily in sheep and goats with zoonotic implications. In the present investigation, an orf outbreak in the ...Muzzaffarnagari sheep flock at the Central Institute for Research on Goats (CIRG), Makhdoom, Mathura, Uttar Pradesh, India, was investigated. Primary goat testes cell culture was used for isolation of the orf virus (ORFV) for the first time. The identity of the virus was confirmed by amplification and sequence analysis of the major envelope glycoprotein (B2L) gene and named ORFV/sheep/India/2012/CIRG. On phylogenetic analysis of B2L protein gene, it clustered with the ORFV strains from China suggesting distinct ORFV strains are circulating in India. On comparison of nucleotide and deduced amino acid sequence analysis (n = 63), a unique 126S residue was observed in ORFV/sheep/India/2012/CIRG. On further sequence analysis (B2L) of different ORFV strains (n = 63), some conserved amino acid residues were identified as host-specific (sheep, human, camel, takin, and musk ox) and have been summarized.
Antigens extracted using ethanol (EtOH) and incorporated in the EtOH vortex ELISA (EVELISA) test have previously shown high specificity and sensitivity for detecting
subspecies
(
) and
infections in ...cattle. The objective of this study is to define the components present in the EtOH extract. We show that this extract is composed of lipid, carbohydrate, and proteins on the surface of the bacilli, and that EtOH removes the outer layer structure of
which comprise these elements. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a
genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits, but this protein is not strongly immunogenic in cattle with Johne's disease. A panel of monoclonal antibodies was used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that a well-known antigen, termed MPB83, is present in
EtOH extracts and a fatty acid desaturase (MAP_2698c) is present in
EtOH extracts, while lipoarabinomannan was common to both. The lipid and carbohydrate components of the extract were analyzed using thin layer chromatography and lectin binding, respectively. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle.
Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease ...(paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)–amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH2 (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.
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