We investigated the efficacy of oral and parenteral Mycobacterium bovis bacille Calmette-Guerin Danish strain 1331 (BCG) in its ability to protect white-tailed deer (Odocoileus virginianus) against disease caused by M. bovis infection. Twenty-two white-tailed deer were divided into four groups. One group (n=5) received 10(9) colony-forming units (cfu) BCG via a lipid-formulated oral bait; one group (n=5) received 10(9) cfu BCG in culture directly to the oropharynx, one group (n=6) was vaccinated with 10(6) cfu BCG subcutaneously, and one group served as a control and received culture media directly to the oropharynx (n=6). All animals were challenged 3 mo after vaccination. Five months postchallenge the animals were examined for lesions. Results indicate that both oral forms of BCG and parenterally administered BCG offered significant protection against M. bovis challenge as compared to controls. This study suggests that oral BCG vaccination may be a feasible means of controlling bovine tuberculosis in wild white-tailed deer populations.
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.
Abstract. The objective of this study was to evaluate the use of a one-tube nested polymerase chain reaction (OTN PCR) with 5 concentration and lytic treatments for the detection of Mycobacterium bovis in experimentally inoculated milk samples (spiked samples). OTN PCR and the following treatments were tested in inoculated samples: 1) centrifugation; 2) C 18 -carboxypropylbetaine ϩ capture resin 1 ϩ Proteinase K (CB18-CH-PK); 3) centrifugation ϩ capture resin 1 ϩ Proteinase K; 4) centrifugation ϩ capture resin 2 ϩ Proteinase K; and 5) centrifugation ϩ immunomagnetic separation (IMS). The OTN PCR and the 5 treatments were evaluated in 2 different sets of spiked milk samples. One set consisted of 10-fold serial dilutions of a phenol-killed M. bovis in milk to final concentrations ranging from 5 to 50,000 cells/ml of milk. The other set of samples consisted of 2.5 serial dilutions of milk spiked with M. bovis to final concentrations ranging from 20.5 to 5,000 cells/ml of milk. Each treatment was repeated 5 times at each cell concentration. CB18-CH-PK and IMS were significantly more sensitive than other treatments. The lowest detection limit for these techniques was 20-50 cells/ ml of spiked milk. The specificity of OTN PCR in this study was high as demonstrated by the lack of DNA amplification products when M. bovis cells were not present in the samples. [The OTN PCR used in conjunction with CB18-CH-PK or IMS could be effectively used as a diagnostic and/or screening test for the detection of M. bovis in milk from herds with bovine tuberculosis.] Bovine tuberculosis (TB) is a chronic zoonotic disease caused by Mycobacterium bovis. Bovine TB generates financial losses to the livestock industry and can be trasmitted to humans. Screening and diagnosis of TB in cattle is still problematic. Since 1917, the tuberculin test has been used for bovine TB screening in live cattle in the USA. 6,8 The sensitivity of the various tuberculin tests ranges between 68.6% and 91.2% and the specificity between 75.5% and 98.8%. 6 These values suggest that the efficacy of any intradermal tuberculin test in control and/or eradication programs is limited. Direct diagnostic methods based on detection of the agent have been used to confirm TB in tissues collected at postmortem examinations. Mycobacterial isolation is highly specific but has poor sensitivity and is time consuming and labor intensive. Isolation of M. bovis via traditional culture methods may require 4-8 weeks. 17 The use of the polymerase chain reaction (PCR) in samples collected by noninvasive procedures (i.e., samples of milk, colostrum, and respiratory secretions) appears to be an acceptable alternative to my-
Abstract.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.Vesicular stomatitis virus (VSV) causes vesicular lesions on the epithelia of the tongue and mouth, as well as coronary bands of hooves of cattle, pigs, and horses. 9 Humans, rodents, and numerous other mammals and fowl can also be infected. 12,16 Vesicular stomatitis virus has been isolated on many occasions in several species of insects, including mosquitoes, during viral epidemics and in forested endemic foci. 2,21-23 At least 2 groups of insects, sand flies (Lutzomyia spp) and black flies (Diptera: Simuliidae), have been shown to carry the virus in nature and to replicate and transmit VSV to susceptible hosts after laboratory infection. 2,13,20 In addition, it has been shown that black flies can act as vectors to disseminate VSV by cofeeding on a nonviremic host. 14 However, the exact role played by insects in VSV's natural cycle has not been definitely proven. Vesicular stomatitis (VS) is of considerable economic importance as quarantines must be enforced until diagnosis is complete because its clinical symptoms are indistinguishable from those of foot and mouth disease (FMD).
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.
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