Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

Background

We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.

Results

The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively.

Conclusions

These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.     

Evaluation of Nitric Acid Extraction of Elements from Soils and Sediments in Two watersheds in East Tennessee

Determining the total elemental concentrations of soils requires a total dissolution method, in which hydrofluoric acid (HF) is commonly used. However, this method is tedious and risky due to the dangerous reagent HF. This study compared a single acid extraction technique using a nitric acid (HNO₃) extraction method to total dissolution (HF + microwave assisted aqua regia) for evaluating the total elemental concentrations in soils and sediments. The two methods were used to analyze the elemental content of soils and sediments from the Oostanaula Creek and Pond Creek watersheds in East Tennessee. Twenty-one elements (Al, Ba, Ca, Co, Cr, Cu, Fe, K, La, Li, Mg, Mn, Nd, P, S, Si, Sr, Ti, V, Zn, and Zr) were subsequently measured using inductively coupled plasma optical emission spectrometry (ICP-OES). The extraction efficiency for HNO₃ relative to the total was compared and discussed. In general, HNO₃ can extract the elements in absorbed phases and those residing in non-silicate minerals. Six elements (Ba, Co, Fe, Ca, Mn, and P) generated significant correlations between total and HNO₃ in both OC and PC watersheds. Finally, the elements were grouped according to the interrelationships of their total elemental concentrations based on the dendrogram plots, suggesting their geochemical association in soils and sediment forming minerals.     

Exploring drivers of the usage of anticoagulant rodenticides on UK farms

Anticoagulant rodenticides (ARs) are considered inhumane, show increasingly limited efficacy due to acquired resistance, and carry environmental consequences associated with non-target species uptake. In a questionnaire study of 499 UK farms that all deployed chemical rodenticide we found a high mean reliance (79%), on second generation ARs with just over half of the respondents using no other rodent control methods. Additional methods where deployed, alone or in combination, included predation (41%), kill-trap deployment (16%) and shooting (1%). Nearly 40% of all respondents deployed rodenticides year-round. There was no evidence to suggest that “tidy-farm” measures, such as clearing food spills and minimising on-farm rodent harbourage sites aimed at minimising rodent-associated problems, were associated with a lower likelihood of year-round deployment; in fact trends in our analyses suggested the opposite. We therefore encourage operators to fully evaluate the true necessity of rodenticide deployment before AR use.     

Persistence and Profiles of Tetracycline Resistance Genes in Swine Farms and Impact of Operational Practices on Their Occurrence in Farms’ Vicinities

Animal farms are recognized as a major contributor to environmental pollution with antibiotic resistance genes, the persistence at the source and the discharge routes to the environment of which need better understanding. The presence of 16 tetracycline resistance genes (TRG) was assessed in feed, feces, manure lagoons, and the vicinities of three Georgia swine farms varied on antibiotic usage and other operational characteristics. In these farms, TRG profiles of feces and lagoons were composed of the same “persistent” and “transient” genes, with the former consistently observed in farms’ proximities and the latter quickly attenuated in the lagoons. Fourteen TRG incidents, 13 of which occurred in gullies, ponds, and creeks, were detected around farms. Their frequency correlated to the cumulative antibiotic usage (R ² = 0.99), number of lagoons (R ² = 0.73), and precipitation (R ² = 0.83), but not to the size of herd or the amount of manure used for fertilization. Gene-specific amplicons of TRG from the farms’ proximities were similar to those of “persistent” TRG from corresponding farms. Our data suggested the independency of TRG profiles of antibiotic usage, variability in TRG persistence in manure lagoons, correlations between operational practices and TRG incidence in farms’ vicinities, and spills and seepage from lagoons as TRG environmental routes.     

Canine distemper with spinal cord lesions.

A case of distemper in a 6-month-old dog is described. The dog was presented with a history of tetraparesis suggestive of trauma. Neurological examination and clinical pathology findings of lymphopenia and pleocytosis suggested a viral cause. Microscopic findings of a nonsuppurative meningoencephalomyelitis with numerous intranuclear inclusions in the cerebellum, brain stem, and all parts of the spinal cord suggested a diagnosis of distemper.     

On the interpretation of test sensitivity in the two-test two-population problem: Assumptions matter

Bayesian analyses of diagnostic test accuracy often require the assumption of constant test accuracy among populations to ensure model identifiability. In a prior study (Toft, N., Jørgensen, E., Højsgaard, S., 2005. Diagnosing diagnostic tests: evaluating the assumptions underlying the estimation of sensitivity and specificity in the absence of a gold standard. Prev. Vet. Med. 68, 19–33), the sensitivity estimate from a two-test two-population model was shown to be weighted toward the population with the higher prevalence of infection. In the present study, we provided analytical formulae that give insight into the effect of assuming constant sensitivity when this assumption was false. To further investigate the effect of failure of the assumption of constant sensitivity, we also simulated several data sets under the assumption that the first test's sensitivity varied in the two populations. Bayesian conditional independence models that presumed constant sensitivities were implemented in WinBUGS and posterior estimates (mean and 95% probability intervals) were evaluated based on the known true values of the parameters. Findings from the Bayesian analyses of several scenarios indicated that the posterior mean was a good estimate of the weighted mean of the sensitivities in the two populations, when one test was perfectly specific. When neither test was perfectly specific, the Bayesian posterior mean for test 1 sensitivity was either greater than the larger of the two true sensitivities, or smaller than both, and estimates of prevalence and the second test's specificity were incorrect. The implication is that estimates of some parameters will be biased if test sensitivities are not constant across populations. Without a perfectly specific test, and if the assumption of constant sensitivity fails, the only solution we are aware of would involve incorporating prior information on at least two parameters.     

Babesia gibsoni infection among dogs in the southeastern United States

OBJECTIVE: To identify subclinical Babesia gibsoni infection in American Pit Bull Terriers from the southeastern United States and to determine the genetic sequence of parasite DNA isolated from these dogs. DESIGN: Case series. ANIMALS: 33 American Pit Bull Terriers and 87 dogs of various other breeds. PROCEDURE: Blood smears were examined for microscopic evidence of the parasite, and DNA was extracted from blood samples and used in a polymerase chain reaction (PCR) assay designed to amplify the small subunit ribosomal RNA gene sequence of B. gibsoni. Amplification products of the expected size were sequenced, and sequences were compared with published sequences for B. gibsoni isolates. Hematocrit, platelet count, mean platelet volume, WBC count, and eosinophil count were compared between dogs with positive PCR assay results and dogs with negative results. RESULTS: Results of the PCR assay were positive for 18 of the 33 (55%) American Pit Bull Terriers, including all 10 dogs with microscopic evidence of parasitemia. Only 1 of these dogs was clinically ill at the time blood samples were collected. Results of microscopic evaluation of blood smears and of the PCR assay were negative for the 87 other dogs. Hematocrit and platelet count were significantly lower in dogs with positive PCR assay results than in dogs with negative results. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that American Pit Bull Terriers in the southeastern United States may be subclinically infected with B. gibsoni. However, subclinical infection was not identified in dogs of other breeds from the same geographic area.