Antibody-modified gold nanoparticles (AuNPs) are central to many novel and emerging biosensing technologies due to the specificity provided by antibody-antigen interactions and the unique properties ...of nanoparticles. These AuNP-enabled assays have the potential to provide significant improvements in sensitivity and multiplexed analysis compared to conventional immunoassays. However, a major challenge for these AuNP platform technologies is the synthesis of stable antibody-AuNP conjugates that resist aggregation in high salt environments and biological matrices. Moreover, synthetic strategies to form stable conjugates often require different solution conditions, e.g., pH, for each unique antibody. Herein we describe our effort to develop an approach to chemically modify lysine residues on antibodies to facilitate the formation of stable antibody-AuNP conjugates over a wide pH range. In this work, we systematically investigated the immobilization of native and chemically modified antibodies to 60 nm citrate-capped AuNPs as a function of pH and evaluated the stability of the antibody-AuNP conjugate in a saline environment. We have developed a method to chemically modify the lysine residues on an antibody prior to conjugation to the AuNP that results in stable conjugates over a wide pH range (6.0-8.5). Amino acid analysis and zeta potential measurements of native and modified antibodies reveal that the requisite modification correlates with the number of lysine residues, and a reduction in positive charge contribution from protonated lysine is required to form stable, pH-independent conjugates. Furthermore, we demonstrate that the chemically modified antibodies maintain antigen-binding capabilities. We apply this novel conjugation strategy to develop a surface-enhanced Raman spectroscopy (SERS)-based assay for the accurate subtyping of avian influenza viruses.
Snake fungal disease (SFD) is a clinical syndrome associated with dermatitis, myositis, osteomyelitis, and pneumonia in several species of free-ranging snakes in the US. The causative agent has been ...suggested as Ophidiomyces ophiodiicola, but other agents may contribute to the syndrome and the pathogenesis is not understood. To understand the role of O. ophiodiicola in SFD, a cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A simple, rapid, and sensitive immunoassay has been developed based on antigen-mediated aggregation of gold nanoparticles (AuNP) and surface-enhanced Raman spectroscopy (SERS). Central to this ...platform is the extrinsic Raman label (ERL), which consists of a gold nanoparticle modified with a mixed monolayer of a Raman active molecule and an antibody. ERLs are mixed with sample, and antigen induces the aggregation of the ERLs. A membrane filter is then used to isolate and concentrate the ERL aggregates for SERS analysis. Preliminary work to establish proof-of-principle of the platform technology utilized mouse IgG as a model antigen. The effects of membrane pore diameter and AuNP size on the analytical performance of the assay were systematically investigated, and it was determined that a pore diameter of 200nm and AuNP diameter of 80nm provide maximum sensitivity while minimizing signal from blank samples. Optimization of the assay provided a detection limit of 1.9ng/mL, 20-fold better than the detection limit achieved by an ELISA employing the same antibody–antigen system. Furthermore, this assay required only 60min compared to 24h for the ELISA. To validate this assay, mouse serum was directly analyzed to accurately quantify IgG. Collectively, these results demonstrate the potential advantages of this technology over current diagnostic tests for protein biomarkers with respect to time, simplicity, and detection limits. Thus, this approach provides a framework for prospective development of new and more powerful tools that can be designed for point-of-care diagnostic or point-of-need detection.
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•SERS immunoassay reduces assay time and lowers detection limit compared to ELISA.•Antigen mediates assembly of extrinsic Raman labels (ERLs) in suspension.•Filters can be used to selectively capture and concentrate ERL aggregates.•ERLs exhibit high stability and specificity in complex biological environment.
Direct transmission of avian influenza viruses to mammals has become an increasingly investigated topic during the past decade; however, isolates that have been primarily investigated are typically ...ones originating from human or poultry outbreaks. Currently there is minimal comparative information on the behavior of the innumerable viruses that exist in the natural wild bird host. We have previously demonstrated the capacity of numerous North American avian influenza viruses isolated from wild birds to infect and induce lesions in the respiratory tract of mice. In this study, two isolates from shorebirds that were previously examined in mice (H1N9 and H6N1 subtypes) are further examined through experimental inoculations in the ferret with analysis of viral shedding, histopathology, and antigen localization via immunohistochemistry to elucidate pathogenicity and transmission of these viruses. Using sequence analysis and glycan binding analysis, we show that these avian viruses have the typical avian influenza binding pattern, with affinity for cell glycoproteins/glycolipids having terminal sialic acid (SA) residues with α 2,3 linkage Neu5Ac(α2,3)Gal. Despite the lack of α2,6 linked SA binding, these AIVs productively infected both the upper and lower respiratory tract of ferrets, resulting in nasal viral shedding and pulmonary lesions with minimal morbidity. Moreover, we show that one of the viruses is able to transmit to ferrets via direct contact, despite its binding affinity for α 2,3 linked SA residues. These results demonstrate that avian influenza viruses, which are endemic in aquatic birds, can potentially infect humans and other mammals without adaptation. Finally this work highlights the need for additional study of the wild bird subset of influenza viruses in regard to surveillance, transmission, and potential for reassortment, as they have zoonotic potential.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A rapid one-step screening method to evaluate the specificity of antibody-antigen binding was developed using antibody-conjugated gold nanoparticles (Ab-AuNPs) and dynamic light scattering (DLS). ...Influenza A virus was used as a model antigen to develop this platform and antigen-specific antibodies were attached to the surface of AuNPs. Introduction of virus to the nanoparticle solution resulted in aggregation of the AuNP probes provided that the antibody had an affinity for the virus. A fundamental concern of the work was to ensure aggregates only formed in the presence of the antigen. Therefore, optimal conditions for synthesizing and maintaining the stability of the Ab-AuNP conjugates were investigated by varying pH and antibody concentration, and a protocol for preparing stable Ab-AuNP conjugates is presented. Thereafter the AuNP probes were exploited in a DLS assay to screen the binding specificity of four antibodies to two different isolates of influenza virus (subtype H1N1). The DLS data for antibody binding were in concordance with the results obtained with a conventional ELISA, thereby validating the DLS platform. Importantly, the DLS assay was completed in 30 minutes relative to 24 hours
via
ELISA.
Antigen-mediated aggregation of antibody-gold nanoparticle conjugates is detected with dynamic light scattering to evaluate antibody-antigen binding.
Abstract The direct transmission of highly pathogenic avian influenza (HPAI) viruses to humans in Eurasia and subsequent disease has sparked research efforts leading to better understanding of HPAI ...virus transmission and pathogenicity in mammals. There has been minimal focus on examining the capacity of circulating low pathogenic wild bird avian influenza viruses to infect mammals. We have utilized a mouse model for influenza virus infection to examine 28 North American wild bird avian influenza virus isolates that include the hemagglutinin subtypes H2, H3, H4, H6, H7, and H11. We demonstrate that many wild bird avian influenza viruses of several different hemagglutinin types replicate in this mouse model without adaptation and induce histopathologic lesions similar to other influenza virus infections but cause minimal morbidity. These findings demonstrate the potential of wild avian influenza viruses to directly infect mice without prior adaptation and support their potential role in emergence of pandemic influenza.
The purpose of this report is to discuss the use of topical 1% 5‐fluorouracil as a sole therapy for canine corneal squamous cell carcinoma (SCC). A 12‐year‐old castrated male pug was evaluated for a ...well‐demarcated, central, 3 mm in diameter, pale pink, raised, right corneal mass. An incisional biopsy was obtained using a #64 beaver blade after topical anesthesia and without sedation. A definitive diagnosis of corneal SCC was obtained after histopathologic evaluation of the biopsy. Topical 1% 5‐fluorouracil ointment was applied to the right eye four times daily for 2 weeks followed by no treatment for 2 weeks, then treatment again twice daily for 2 weeks. The cornea remained free of recurrence 10 months after cessation of treatment. In dogs affected with corneal SCC, topical 1% 5‐fluorouracil monotherapy may be a viable and cost‐effective treatment option with minimal side effects. This chemotherapy agent may also have an effect on corneal pigmentation. Chronic cyclosporine therapy did not contribute to the pathogenesis of corneal SCC in the case described.
All viruses strategically alter the antiviral immune response to their benefit. The vaccinia virus (VACV) K1 protein has multiple immunomodulatory effects in tissue culture models of infection, ...including NF-κB antagonism. However, the effect of K1 during animal infection is poorly understood. We determined that a
-less vaccinia virus (vΔK1L) was less pathogenic than wild-type VACV in intranasal and intradermal models of infection. Decreased pathogenicity was correlated with diminished virus replication in intranasally infected mice. However, in intradermally inoculated ears, vΔK1L replicated to levels nearly identical to those of VACV, implying that the decreased immune response to vΔK1L infection, not virus replication, dictated lesion size. Several lines of evidence support this theory. First, vΔK1L induced slightly less edema than vK1L, as revealed by histopathology and noninvasive quantitative ultrasound technology (QUS). Second, infiltrating immune cell populations were decreased in vΔK1L-infected ears. Third, cytokine and chemokine gene expression was decreased in vΔK1L-infected ears. While these results identified the biological basis for smaller lesions, they remained puzzling; because K1 antagonizes NF-κB
, antiviral gene expression was expected to be higher during vΔK1L infection. Despite these diminished innate immune responses, vΔK1L vaccination induced a protective VACV-specific CD8
T cell response and protected against a lethal VACV challenge. Thus, vΔK1L is the first vaccinia virus construct reported that caused a muted innate immune gene expression profile and decreased immune cell infiltration in an intradermal model of infection yet still elicited protective immunity.
The vaccinia virus (VACV) K1 protein inhibits NF-κB activation among its other antagonistic functions. A virus lacking K1 (vΔK1L) was predicted to be less pathogenic because it would trigger a more robust antiviral immune response than VACV. Indeed, vΔK1L was less pathogenic in intradermally infected mouse ear pinnae. However, vΔK1L infection unexpectedly elicited dramatically reduced infiltration of innate immune cells into ears. This was likely due to decreased expression of cytokine and chemokine genes in vΔK1L-infected ears. As such, our finding contradicted observations from cell culture systems. Interestingly, vΔK1L conferred protective immunity against lethal VACV challenge. This suggests that the muted immune response triggered during vΔK1L infection remained sufficient to mount an effective protective response. Our results highlight the complexity and unpredictable nature of virus-host interactions, a relationship that must be understood to better comprehend virus pathogenesis or to manipulate viruses for use as vaccines.
Bovine viral diarrhea virus (BVDV) has a great economic impact on the United States cattle industry. The Academy of Veterinary Consultants, the American Association of Bovine Practitioners, and the ...National Cattlemen's Beef Association have called for the goal of BVDV control and eventual eradication in the USA. One of the key factors in such efforts will be the detection of BVDV infections, particularly targeting persistently infected animals. To assess current BVDV detection methods in the USA, 26 veterinary diagnostic laboratories in 23 states were surveyed. Survey questions related to the types of tests currently offered, the number of tests performed, the reasons for test requests, the type of samples used, whether sample pooling was performed, and whether follow-up testing or information regarding bovine viral diarrhea (BVD) management was provided after positive tests. There was no clear consensus on an individual BVDV testing method, the pooling of samples or the retesting of positive animals. Ear-notch antigen capture enzyme-linked immunosorbent assay (ACE) was the test most frequently performed based on the absolute number of tests. However, when the data were adjusted to reflect individual laboratory choices, the number of ACE and immunohistochemistry tests performed on ear notches was nearly equal. Only 55% of diagnostic laboratories provided BVD management information to producers or veterinarians who submitted positive samples. There was no significant difference in the number of positive tests in laboratories that received the majority of their samples for screening purposes versus laboratories that received the majority of their samples because BVDV was suspected based on clinical signs in a herd.
Snake fungal disease (SFD; Ophidiomyces ophiodiicola) is posing a significant threat to several free-ranging populations of pitvipers. Triazole antifungals have been proposed for the treatment of ...mycoses in reptiles; however, data are lacking about their safety and efficacy in snakes with SFD. Study 1 investigated in vitro susceptibility, and identified that plasma concentrations >250 ng/ml (voriconazole) and >1,000 ng/ml (itraconazole) may be effective in vivo for SFD. In Study 2, the pharmacokinetics after a single subcutaneous voriconazole injection were assessed in apparently healthy free-ranging cottonmouths (Agkistrodon piscivorus). Based on pilot-study results, four snakes were administered a single injection of voriconazole (5 mg/kg). One pilot snake and three full-study snakes died within 12 hr of voriconazole administration. All surviving snakes maintained plasma concentrations >250 ng/ml for 12–24 hr. In Study 3, two Eastern massasaugas (Sistrurus catenatus) and a timber rattlesnake (Crotalus horridus horridus) diagnosed with SFD were treated with voriconazole delivered by subcutaneous osmotic pumps. The timber rattlesnake (12.1–17.5 mg/kg/hr) reached therapeutic concentrations, whereas the massasaugas (1.02–1.6 mg/kg/hr) did not. In Study 4, the pharmacokinetics of a single 10-mg/kg per-cloaca dose of itraconazole (Sporanox®) was evaluated in seven apparently healthy free-ranging cottonmouths. Similarly, the plasma and tissue concentrations did not meet therapeutic concentrations based on in vitro data. The data presented in this report serve as an initial step toward understanding the pharmacokinetics, efficacy, and safety of triazole antifungals in pitviper species with SFD. Further study is needed to determine the appropriate dose and route of administration of triazole antifungals in pitviper species.
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