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.     

Detection of Assemblage A, Giardia duodenalis and Eimeria spp. in alpacas on two Maryland farms

Sixty-one fecal samples were collected from adult alpacas and crias (ages 10 weeks to 10 years) on two farms in central Maryland. The farms raised both suri (silky-haired) and huacaya (crimpy-haired) breeds. Females and crias were housed together on pasture, whereas older/breeding males were maintained on separate pastures. Samples were subjected to a density gradient centrifugation protocol to concentrate parasites and remove fecal debris and were examined by immuno-fluorescent and differential interference contrast microscopy. Oocysts of Eimeria spp. were noted in 14 fecal samples, 6 on MD-1 and 8 on MD-2. Based on oocyst morphometrics two species of Eimeria were present: E. punoensis (19.2 microm x 16.5 microm) and E. alpacae (23.7 microm x 19.5 microm). Five animals shed exclusively E. punoensis, seven shed exclusively E. alpacae, and two had mixed infections. The Eimeria infections were not associated with obvious clinical signs. To determine the presence of Cryptosporidium and Giardia species and genotypes, DNA was extracted from feces and subjected to PCR utilizing specific primers for the ssu-rRNA gene for both parasites. All PCR positive samples were further analyzed by DNA sequencing to identify the species or genotypes that were present. Assemblage A, G. duodenalis was detected in fecal samples from two alpacas on MD-1 and in one alpaca on MD-2. Assemblage E, G. duodenalis and Cryptosporidium spp. were not detected on either farm. Although the prevalence on these two farms was low, alpacas can harbor zoonotic G. duodenalis, and this should be borne in mind by persons interacting with the animals.     

Prevalence of species and genotypes of Cryptosporidium found in 1-2-year-old dairy cattle in the eastern United States

The prevalence of Cryptosporidium species in 1-2-year-old heifers was determined for 571 animals on 14 dairy farms in seven states on the East Coast of the United States. A fecal specimen collected directly from each heifer was processed to concentrate oocysts that were then examined by polymerase chain reaction (PCR). For every PCR-positive specimen the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified by PCR from heifers on 13 of 14 farms. On all except four farms groups of heifers were housed in a barn or in large covered pens. Others were pastured. From many of the same farms an earlier study reported that 41% of 393 pre-weaned calves and 26.2% of 447 post-weaned calves were infected. In the present study, 11.9% of 571 heifers were infected with Cryptosporidium, 0.7% with Cryptosporidium parvum, the zoonotic species. Of 68 PCR-positive specimens characterized by gene sequencing 1, 4, 10, 24, and 29 calves were infected with Cryptosporidium suis, Cryptosporidium parvum, Cryptosporidium deer-like genotype, Cryptosporidium bovis, and Cryptosporidium andersoni, respectively. These findings demonstrate a lower prevalence of infection in 1-2-year-old dairy cattle than in younger cattle as well as a change in the diversity of species present. Consequently, the risk of humans acquiring infection with C. parvum from exposure to feces from yearling and older cattle appears much lower than from exposure to pre-weaned calves.     

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.     

Quantification of naloxone binding sites in brains from suckled beef cows during postpartum anestrus and resumption of estrous cycles

Thirty beef cows, approximately 3 yr of age, were randomly assigned to be slaughtered on d 7, 14, 28, 42 or 56 postpartum. Each cow suckled one calf until slaughter. Data from cows slaughtered on d 42 and 56 were pooled and further classified as anestrous or cyclic based on the presence of a corpus luteum and elevated serum concentrations of progesterone at slaughter. Specific binding of [3H]naloxone (3H-NAL) to homogenates of tissue from hypothalamus (HYP), preoptic area (POA) and basal forebrain (BF) was assessed using multiple-point Scatchard analyses. Nonspecific binding was estimated in the presence of 10(-6) M naloxone. Separation of bound from free 3H-NAL was achieved by centrifugation at 20,000 X g. Concentration (fmol/mg original tissue wet wt) of 3H-NAL binding sites in POA tissue was higher (P less than .05) on d 28 postpartum in anestrous cows than in cyclic cows on d 42 + 56 postpartum (2.58 +/- .32 vs 1.58 +/- .10). When all anestrous cows were compared with cyclic cows, concentrations of 3H-NAL binding sites in POA tissues and in BF tissue were higher (P less than .05) in anestrous cows (anestrous POA, 2.12 +/- .17, cyclic POA, 1.58 +/- .10; anestrous BF, 2.94 +/- .41, cyclic BF, 2.19 +/- .16). Compared across brain regions for all cows, the concentration of specific binding sites for 3H-NAL was greater (P less than .01) in BF (2.5 +/- .2) than in POA (1.9 +/- .1) and greater (P less than .01) in POA than in HYP (1.5 +/- .1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of in vitro corticosterone on chicken T‐ and B‐lymphocyte proliferation

1. The effects of corticosterone and the time of its addition to cultures, on concanavalin A (Con‐A) and pokeweed mitogen (PWM)‐induced lymphocyte proliferation were studied.

2. Peripheral blood lymphocytes (PBL) were isolated from Cornell K‐strain Single Comb White Leghorn immature male chickens and cultured with various concentrations of Con‐A or PWM. Corticosterone, in different concentrations, was added to the cultures either 2 h before or 2 h after the addition of the respective mitogen.

3. Addition of corticosterone 2 h before the mitogens caused a significant suppression of lymphocyte proliferation in response to both Con‐A and PWM stimulation. Also addition of corticosterone 2 h after the mitogens caused significant suppression of proliferation in response to both mitogens; however, the degree of suppression was not as great.

4. The results indicate that after early activation events are initiated by the mitogens, lymphocytes are less sensitive to the effects of corticosterone. Because less suppression was seen in the cultures preincubated with PWM than those with Con‐A, it is likely that there are different sensitivities to corticosterone in the cell populations that respond to these mitogens.     

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.     

Specific binding of naloxone to ovine brain tissue: comparison of brain regions and endocrine states

Binding of [3H]naloxone ([3H]NAL) to brain membranes was quantified by Scatchard analysis using two methods of separating bound from free [3H]NAL. In the centrifugation method, membranes that were soluble at 1,000 x g, but sedimented at 20,000 x g, were incubated with [3H]NAL. For filtration, all membranes that sedimented at 20,000 x g were incubated and filtered through glass filter fibers. Nonspecific binding was estimated using greater than 500-fold excess of unlabeled naloxone (10(-6) M). Specific binding of [3H]NAL was used to generate linear multiple-point Scatchard plots, which indicated a single class of high-affinity sites. In Exp. 1, 10 ovariectomized (OVX) ewes were injected with estradiol-17 beta alone or in combination with progesterone. Compared with OVX controls, these hormonal treatments did not affect binding of [3H]NAL (centrifugation method) to combined hypothalamus (HYP) + preoptic (POA) tissues. In cyclic ewes (Exp. 2, filtration method), affinity constants (2.4 +/- .2 x 10(8) M-1) did not differ among HYP, POA and basal forebrain (BF) tissues, but BF had more sites (39 +/- 3 fmol/mg) than either HYP (14 +/- 1) or POA (17 +/- 1). Binding affinity and concentration of sites within each brain area (HYP, POA, BF) did not differ between d 8 and d 16 (preovulatory but after luteolysis) in normally cycling ewes. Overall, neural tissue dissected from BF had a greater concentration of binding sites than HYP or POA. Exogenous and endogenous fluctuations in ovarian steroids did not affect binding of [3H]NAL to these tissues.