Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey.

Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one-step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine-thiocyanate-based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column-based purification coupled with one-step RT-PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri-phasic extraction method (Tri-reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT-PCR was as low as 0.505 to 2.84 TCID(50) for VSV-IND and VSV-NJ, respectively. The multiplex RT-PCR consistently detected VSV-IND and NJ RNA in as little as 0.1-1.0 fg of total RNA from spiked BHK-21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT-PCR assay was capable of detecting both types of VSV in a one-step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID(50) (IND), 0.0946 TCID(50) (NJ), and 0.057 fg (IND and NJ) per 2 µl PCR sample, which is significantly more sensitive than reported previously (0.28-2.8 TCID50/1 µl). So the present study improved the sensitivity of previously reported multiplex RT-PCR for the detection and differentiation of VSV-IND and VSV-NJ in a single assay.

Effect of time and temperature on PrPCWD immunoreactivity as evidenced by Western blot.

The protease-resistant infectious prion protein, PrPres, that causes transmissible spongiform encephalopathies, is remarkably resistant to conventional physical and chemical sterilization methods, including heat. It was hypothesized that thermal-dependent PrPres degradation has been underestimated, and the effect of prolonged incubation at 37 degrees C, 55 degrees C, and 80 degrees C on PrPres detection was examined using brain homogenates from chronic wasting disease-affected elk and mule deer (PrPCWD). Immunoblotting demonstrated progressive loss of PrPCWD immunoreactivity with time in all incubated samples as temperature increased, and PrPCWD was virtually undetectable after 90 days of incubation at 55 degrees C and 80 degrees C. These results indicate that decontamination methods and tissue disposal systems maintaining elevated temperatures for long periods of time could interfere with immunodetection, and the reliability of assays for PrPres detection could be compromised when applied to tissues exposed to heat with time. Although these results may suggest that such prolonged heat treatment could destroy prions, the observed loss of immunoreactivity does not necessarily correlate with a concurrent loss of infectivity. Bioassay is needed to determine if samples that have been incubated under these conditions retain infectivity.

Determining the persistence of Mycobacterium bovis bacille Calmette-Guerin Danish in select tissues of orally vaccinated feral swine (Sus scrofa ssp.).

Mycobacterium bovis bacille Calmette-Guerin (BCG) is being considered for vaccination of feral swine (Sus scrofa ssp.). Since BCG is a live bacterium, evaluation of its safety and persistence in tissues is important. Fifteen feral swine received approximately 4.5 × 10(6) colony forming units of BCG Danish via oral bait. Four animals received bait without BCG. At 1, 3, 6, and 9 months post-vaccination, four vaccinates were euthanized. Non-vaccinates were euthanized at 9 months. Clinical signs were not noted in vaccinated pigs at any time. Tissues from all 20 pigs were culture-negative for mycobacteria. Based on our data, BCG is safe and appears not to persist in feral swine tissues after one month post-oral vaccination. However, further work must be performed at higher doses, and on a larger number of animals representing the target population, and further evaluation of persistence in tissues within the first month post-vaccination is needed.

Inhibiting avian influenza virus shedding using a novel RNAi antiviral vector technology: proof of concept in an avian cell model.

Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.

A single-tube multiplex reverse transcription-polymerase chain reaction for detection and differentiation of vesicular stomatitis Indiana 1 and New Jersey viruses in insects.

A multiplex single-tube reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10-30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.

Time-dependent changes in plasma concentrations of 3-methylindole and blood concentrations of 3-methyleneindolenine-adduct in feedlot cattle.

OBJECTIVE: To describe time-dependent changes in plasma concentrations of 3-methylindole (3MI) and blood concentrations of 3-methyleneindolenine (3MEIN)-adduct in feedlot cattle. ANIMALS: 64 yearling steers. PROCEDURES: Steers were assigned to 2 groups (32 steers/group). During the first 8 weeks, blood samples were collected from group 1 before the morning ration was fed, whereas samples from group 2 were collected 2 to 3 hours after the ration was fed. Blood samples were collected from all steers approximately 4 times/wk for 3 weeks and 3 times/wk for the subsequent 5 weeks. Samples were collected at the same time for all steers for an additional 10 weeks. Plasma samples were analyzed for 3MI concentrations. Blood samples collected from cattle in group 2 during the first 8 weeks were analyzed for 3MEIN-adduct concentrations. RESULTS: Mean blood concentration of 3MEIN-adduct increased to a maximum value on day 33 (0.80 U/microg of protein) and then decreased to a minimum on day 54 0.40 U/microg of protein). Plasma 3MI concentrations initially decreased and remained low until after day 54. Group-1 cattle had lower plasma 3MI concentrations, compared with concentrations for group-2 cattle. Blood 3MEIN-adduct concentrations and plasma 3MI concentrations were not associated with deleterious effects on weight gains. CONCLUSIONS AND CLINICAL RELEVANCE: Blood 3MEIN-adduct concentrations peaked during the period of greatest risk for development of bovine respiratory disease complex. Conversely, plasma 3MI concentrations decreased during the same period. Animal-to-animal variation in metabolic capacity to convert 3MI to 3MEIN may be of more importance than differences in plasma 3MI concentration.