Dose and Size-Dependent Antiviral Effects of Silver Nanoparticles on Feline Calicivirus, a Human Norovirus Surrogate.

OBJECTIVES: Silver nanoparticles (AgNPs) as antibacterial agents are incorporated in many consumer products, while the use as antiviral agents is an ongoing area of research. We evaluated the antiviral properties of AgNPs of variable sizes (10, 75, and 110 nm) and doses (25, 50, and 100 µg/mL) at different contact time points against feline calicivirus (FCV), a surrogate for norovirus. MATERIALS AND METHODS: Antiviral effects of the AgNPs were determined by comparing the infectivity of FCV, the appearance of cytopathic effects (CPEs), and the integrity of the viral capsid protein in viral suspension treated with AgNPs with the untreated controls. RESULTS: The 10 nm AgNPs at 50 and 100 µg/mL concentrations inactivated the FCV beyond the limit of detection, resulting in a decrease of up to 6.5 log10 viral titer, prevented development of CPEs, and reduction in the western blot band signal of the viral capsid protein. No significant antiviral effect was observed for the 75 and 110 nm AgNPs. Conclusions and Applications: These results demonstrate that the antiviral effects of AgNPs are both size and dose dependent, thus potential applications of AgNPs as antiviral agents to prevent contamination of foodborne viruses need to consider size and dose effects.

Molecular Epidemiology of Novel Pathogen "Brachyspira hampsonii" Reveals Relationships between Diverse Genetic Groups, Regions, Host Species, and Other Pathogenic and Commensal Brachyspira Species.

Outbreaks of bloody diarrhea in swine herds in the late 2000s signaled the reemergence of an economically significant disease, swine dysentery, in the United States. Investigations confirmed the emergence of a novel spirochete in swine, provisionally designated "Brachyspira hampsonii," with two genetically distinct clades. Although it has since been detected in swine and migratory birds in Europe and North America, little is known about its genetic diversity or its relationships with other Brachyspira species. This study characterizes B. hampsonii using a newly developed multilocus sequence typing (MLST) approach and elucidates the diversity, distribution, population structure, and genetic relationships of this pathogen from diverse epidemiological sources globally. Genetic characterization of 81 B. hampsonii isolates, originating from six countries, with our newly established MLST scheme identified a total of 20 sequence types (STs) belonging to three clonal complexes (CCs). B. hampsonii showed a heterogeneous population structure with evidence of microevolution locally in swine production systems, while its clustering patterns showed associations with its epidemiological origins (country, swine production system, and host species). The close genetic relatedness of B. hampsonii isolates from different countries and host species highlights the importance of strict biosecurity control measures. A comparative analysis of 430 isolates representing seven Brachyspira species (pathogens and commensals) from 19 countries and 10 host species depicted clustering by microbial species. It revealed the close genetic relatedness of B. hampsonii with commensal Brachyspira species and also provided support for the two clades of B. hampsonii to be considered a single species.

Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus.

Newly emergent turkey arthritis reoviruses (TARVs) have been isolated from cases of lameness in male turkeys over 10 weeks of age. In a previous study, experimental inoculation of TARV in one-week-old turkey poults produced lymphocytic tenosynovitis at four weeks post inoculation but without causing clinical lameness. This study was undertaken to determine if TARV infection at an early age can lead to clinical lameness in birds as they age. One-week-old male turkeys were inoculated orally with a TARV (strain TARV-O'Neil) and monitored for the development of gait defects until 16 weeks of age. At 4, 8, 12 and 16 weeks of age, a subset of birds was euthanized followed by the collection of gastrocnemius tendon, digital flexor tendon, and intestines for virus detection by rRT-PCR and for histologic inflammation scoring. Clinical lameness was first displayed in TARV-infected turkeys at 8 weeks of age and ruptured gastrocnemius tendons with progressive lameness were also seen at 12-16 weeks of age. The virus was detected in gastrocnemius tendon of 4- 8- and 12-week-old turkeys but not in 16-week-old turkeys. Histologic inflammation scores of tendons at each of the four time points were significantly higher in the virus-inoculated group than in the control group (p < 0.01). Lesions began as lymphocytic tenosynovitis with mild synoviocyte hyperplasia at four weeks of age and progressed to fibrosis as the birds aged. These results demonstrate the potential of TARV to infect young turkeys and to produce subclinical tenosynovitis that becomes clinically demonstrable as the turkeys age.

Efficacy of Five Commonly Used Disinfectants Against Turkey Arthritis Reovirus.

Since late 2009, an unusual problem of reovirus-related lameness has been seen in market-age tom turkeys in the upper Midwest area of the United States. In this study, we determined the efficacy of five commonly used disinfectants (Virocid, Keno X5, Synergize, One Stroke, and Tek Trol) against turkey arthritis reoviruses (TARVs). For comparison, turkey enteric reovirus (TERV) and chicken arthritis reovirus (CARV) were also included. At their recommended concentrations, all five disinfectants were found to be effective virucidals, inactivating 99.99% of all viruses within 10 min. However, oxidizing agents and quaternary ammonium compounds + aldehyde types of disinfectants were more effective, killing the viruses in a shorter time (2-5 min) than the other types of disinfectants. These results indicate that these disinfectants can be an effective tool in the control of these viruses.

Pathogenicity of newly emergent turkey arthritis reoviruses in chickens.

Turkey arthritis reoviruses (TARVs) were isolated recently from gastrocnemius and digital flexor tendons of lame turkeys with swollen joints and tenosynovitis. These TARVs were genetically different from chicken arthritis reoviruses (CARVs) and produced gastrocnemius tenosynovitis when inoculated into turkey poults. The purpose of this study was to determine the pathogenicity of TARVs in chickens. One-week-old, specific-pathogen-free chicks were inoculated with either a TARV (TARV-MN2 or TARV-O'Neil) or CARV via oral, intratracheal, or footpad routes. At 2 and 3 weeks post inoculation (PI), a subset of chicks from each group was euthanized followed by collection of tissues for real-time RT-PCR (rRT-PCR), virus isolation, and histopathology. Chickens inoculated with CARV via intratracheal and footpad routes developed gastrocnemius lymphocytic tenosynovitis at 2 and 3 weeks PI. Both TARV-MN2 and TARV-O'Neil induced gastrocnemius lymphocytic tenosynovitis in chicks inoculated only via the footpad route at 2 and 3 weeks PI. Although there was no evidence of clinical lameness, the virus was present in leg tendons, internal organs, and intestines of all TARV-inoculated chicks regardless of route of inoculation, as indicated by rRT-PCR and virus isolation. These results indicate that TARVs do not produce gastrocnemius tenosynovitis in chicks by 3 weeks PI when administered via the most probable natural route (e.g., oral and intratracheal). Further studies are needed to determine the long term effects these viruses might play in inducing lameness in chickens.

Survival of airborne MS2 bacteriophage generated from human saliva, artificial saliva, and cell culture medium.

Laboratory studies of virus aerosols have been criticized for generating airborne viruses from artificial nebulizer suspensions (e.g., cell culture media), which do not mimic the natural release of viruses (e.g., from human saliva). The objectives of this study were to determine the effect of human saliva on the infectivity and survival of airborne virus and to compare it with those of artificial saliva and cell culture medium. A stock of MS2 bacteriophage was diluted in one of three nebulizer suspensions, aerosolized, size selected (100 to 450 nm) using a differential mobility analyzer, and collected onto gelatin filters. Uranine was used as a particle tracer. The resulting particle size distribution was measured using a scanning mobility particle sizer. The amounts of infectious virus, total virus, and fluorescence in the collected samples were determined by infectivity assays, quantitative reverse transcription-PCR (RT-PCR), and spectrofluorometry, respectively. For all nebulizer suspensions, the virus content generally followed a particle volume distribution rather than a number distribution. The survival of airborne MS2 was independent of particle size but was strongly affected by the type of nebulizer suspension. Human saliva was found to be much less protective than cell culture medium (i.e., 3% tryptic soy broth) and artificial saliva. These results indicate the need for caution when extrapolating laboratory results, which often use artificial nebulizer suspensions. To better assess the risk of airborne transmission of viral diseases in real-life situations, the use of natural suspensions such as saliva or respiratory mucus is recommended.

The role of avian reoviruses in turkey tenosynovitis/arthritis.

Turkey arthritis reovirus (TARV) has been isolated from the gastrocnemius tendons and tibiotarsal joint fluid of lame male turkeys >12 weeks old in the Midwest. Two experiments were conducted to compare the pathogenicity in turkeys of three TARVs (TARV-MN2, TARV-MN4 and TARV-O'Neil), one turkey enteric reovirus (TERV strain MN1) and one chicken arthritis reovirus (CARV strain MN1). Two hundred microlitres of virus were inoculated by the oral, intratracheal, or footpad route into 6-day-old poults placed in isolator units. Poults were necropsied at 1 and 4 weeks post infection in Experiment 1, and at 2 and 4 weeks post infection in Experiment 2. Reovirus was detected by reverse transcription-polymerase chain reaction and virus isolation in tendons of TARV-inoculated poults at 1, 2 and 4 weeks post infection. TARV-O'Neil and TARV-MN2 were detected in tendons of sentinal birds at 1 and 4 weeks and 1 week p.i., respectively. In general, TARVs produced lymphocytic tenosynovitis of the gastrocnemius and digital flexor tendon sheaths without inflammation of the tendons proper. In Experiment 1, poults inoculated with TARV-MN2 and TARV-O'Neil had significantly higher gastrocnemius tendon inflammation scores, as determined by histology, than those inoculated with TERV-MN1 or CARV-MN1. In Experiment 2, poults inoculated with TARV-MN2 and TARV-O'Neil had significantly higher gastrocnemius tendon inflammation scores than those inoculated with TARV-MN4 and virus-free medium (negative control group). Koch's postulates was fulfilled when TARV-MN2 and TARV-O'Neil were re-isolated from tendons of poults that had originally been challenged with either of these viruses. Results of these experiments indicate that TARVs have a unique ability to induce gastrocnemius tenosynovitis in turkeys and that administration of TARV-O'Neil through the oral or intratracheal route is a reproducible model to study pathogenesis of TARV infection.

One-step real-time reverse transcription-PCR for the detection of turkey reoviruses.

During late 2010 and early 2011, an unusual problem of lameness and swollen hock joints in commercial turkeys was reported in the upper Midwest, which continues to this day. The disease caused substantial economic losses to turkey producers. Reovirus was isolated from tendons and joint fluids of lame turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory. This study was undertaken to develop a TaqMan real-time reverse transcription-PCR (rRT-PCR) assay for the early detection of turkey reoviruses (both enteric and lameness strains). A primer probe set was designed from the conserved region of the S4 segment of the turkey reovirus genome. The newly developed rRT-PCR was specific for the detection of turkey reoviruses. The detection limit of this assay was 10 genome copies per reaction. For the TARV-MN4 strain of turkey arthritis reovirus, one 50% tissue culture infectious dose was equivalent to 11.6 +/- 0.2 genome copies. The highest coefficient of variation for intraexperimental and interexperimental variability was 0.08 and 0.06, respectively, indicating the reproducibility of the assay. This new test should be useful for the detection of turkey enteric and arthritis reoviruses.

Responses of intestinal virome to silver nanoparticles: safety assessment by classical virology, whole-genome sequencing and bioinformatics approaches.

BACKGROUND: Effects of silver nanoparticles (AgNP) on the intestinal virome/phage community are mostly unknown. The working hypothesis of this study was that the exposure of pharmaceutical/nanomedicine and other consumer-use material containing silver ions and nanoparticles to the gastrointestinal tract may result in disturbance of the beneficial gut viruses/phages. METHODS: This study assesses the impact of AgNP on the survival of individual bacteriophages using classical virology cultivation and electron microscopic techniques. Moreover, how the ingested AgNP may affect the intestinal virus/phages was investigated by conducting whole-genome sequencing (WGS). RESULTS: The viral cultivation methods showed minimal effect on selected viruses during short-term exposure (24 h) to 10 nm AgNP. However, long-term exposure (7 days) resulted in significant reduction in the viral/phage population. Data obtained from WGS were filtered and compared with a nonredundant viral database composed of the complete viral genomes from NCBI using KRAKEN (confidence scoring threshold of 0.5). To compare the relative differential changes, the sequence counts in each treatment group were normalized to account for differences in DNA sequencing library sizes. Bioinformatics techniques were developed to visualize the virome comparative changes in a phylogenic tree graph. The computed data revealed that AgNP had an impact on several intestinal bacteriophages that prey on bacterial genus Enterobacteria, Yersinia and Staphylococcus as host species. Moreover, there was an independent effect of nanoparticles and released ions. CONCLUSION: Overall, this study reveals that the small-size AgNP could lead to perturbations of the gut microbial ecosystem, leading to the inactivation of resident phages that play an important role in influencing gastrointestinal health.

Molecular characterization of quail bronchitis virus isolated from bobwhite quail in Minnesota.

From 2008 to 2012, 4 separate cases of quail bronchitis virus infection were seen in bobwhite quail (Colinus virginianus) raised in Minnesota. The quail chicks ranged in age from 5 d to 8 wk and suffered from respiratory distress and elevated mortality. On necropsy, gross lesions consisted of mucus in trachea, congested lungs, caseous air sacculitis, accumulation of chalky white urates on internal organs, necrotic foci in liver, and enlarged spleen. Histologic examination revealed fibrinoheterophilic rhinitis, heterophilic bronchitis, heterophilic tracheitis, and interstitial pneumonia in addition to deciliation, desquamation, and necrosis of bronchial respiratory epithelium. Karyomegaly with basophilic intranuclear inclusions was also seen in affected epithelium. Severe epicarditis, pericarditis, myocarditis, multifocal necrotizing hepatitis, and splenitis were additional pathological findings. Quail bronchitis virus (QBV) was isolated from all four samples when inoculated in specific-pathogen-free (SPF) embryonated chicken eggs. The virus was confirmed by electron microscopy and polymerase chain reaction using fowl adenovirus (FAdV) hexon gene-specific primers. Nucleotide sequences of the four isolates showed 99.0% identity with CELO strain of fowl adenovirus A. Nine nucleotide substitutions were observed; 3 of these were nonsynonymous (A281G, C314T and G565C), leading to changes in deduced amino acid sequences (S94G, T105M and A189P, respectively). Based on partial sequence of the hexon gene, QBV isolates of this study clustered closely with fowl adenovirus A and were different from FAdV groups B through E and from adenoviruses of goose, duck, turkey, and pigeon. Further studies are indicated to determine the impact of nonsynonymous substitutions on host specific pathogenicity of these viruses.

Genotypes, Antibiotic Resistance, and ST-8 Genetic Clone in Campylobacter Isolates from Sheep and Goats in Grenada.

Rectal swabs from 155 sheep and 252 goats from Grenada were evaluated to determine the prevalence of Campylobacter spp., antibiotic resistance, and multilocus sequence types. Fifteen Campylobacter isolates were obtained (14 C. jejuni and 1 C. coli). The prevalence (3.7%) did not differ significantly between sheep (4.5%) and goats (3.2%). Among the seven antimicrobials tested, resistance was only detected for tetracycline (30.8%) and metronidazole (38.5%). Campylobacter isolates showed no significant difference between sheep and goats for type of antimicrobial resistance or percent of resistant isolates. Twelve of the isolates were successfully genotyped consisting of four recognized clonal complexes and three novel sequence types. Importantly, one isolate from one goat was identified as the C. jejuni sequence type-8, a zoonotic and tetracycline-resistant clone reported to be a highly virulent clone associated with ovine abortion in the USA. Although most samples were from comingled sheep and goat production units, there were no shared sequence types between these two host species. None of the sequence types identified in this study have previously been reported in poultry in Grenada, suggesting sheep- and goat-specific Campylobacter clones in Grenada. This is the first report of genotyping of Campylobacter isolates from sheep and goats in the Eastern Caribbean.

Phylogenetic diversity and dietary association of rumen Treponema revealed using group-specific 16S rRNA gene-based analysis.

Treponema spp. are a commonly detected bacterial group in the rumen that are involved in the degradation of soluble fibers. In this study, a ruminal Treponema group-specific PCR primer targeting the 16S rRNA gene was designed and used to assess the phylogenetic diversity and diet association of this group in sheep rumen. Total DNA was extracted from rumen digesta of three sheep fed a diet based on alfalfa/orchardgrass hay or concentrate. The real-time PCR quantification indicated that the relative abundance of the Treponema group in the total rumen bacteria was as high as 1.05%, while the known species Treponema bryantii accounted for only 0.02%. Fingerprints of the Treponema community determined by 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE) analysis tended to differ among the diets. Principal component analysis of the DGGE profiles distinguished those Treponema associated with either the hay or the concentrate diets. Analysis of a Treponema 16S rRNA gene clone library showed phylogenetically distinct operational taxonomic units for a specific dietary condition, and significant (P=0.001) differences in community composition were observed among clone libraries constructed from each dietary regimen. The majority of clones (75.4%) had <97% sequence similarity with known Treponema. These results suggest the predominance of uncultured Treponema that appear to have distinct members related to the digestion of either hay or concentrate diet.

Genetic diversity and diet specificity of ruminal Prevotella revealed by 16S rRNA gene-based analysis.

16S rRNA gene-based analysis of rumen Prevotella was carried out to estimate the diversity and diet specificity of bacteria belonging to this genus. Total DNA was extracted from the rumen digesta of three sheep fed two diets with different hay-to-concentrate ratios (10 : 1 and 1 : 2). Real-time PCR quantification of Prevotella revealed that the relative abundance of this genus in the total rumen bacteria was up to 19.7%, while the representative species Prevotella bryantii and Prevotella ruminicola accounted for only 0.6% and 3.8%, respectively. Denaturing gradient gel electrophoresis analysis for Prevotella revealed shifts in the community composition with the diet. Analysis of 16S rRNA gene clone libraries showed significant differences (P=0.001) between clones detected from the sheep on the diets with different hay-to-concentrate ratios. The majority (87.8%) of Prevotella clones had <97% sequence similarity with known rumen Prevotella. These data suggest that uncultured Prevotella is more abundant than known Prevotella and that members of this genus appear to have specific metabolic niches.