Development and comparison of loop-mediated isothermal amplification and quantitative polymerase chain reaction assays for the detection of Mycoplasma bovis in milk

Bovine mastitis is an economic burden for dairies worldwide. Mycoplasma species, and especially Mycoplasma bovis, are among the most important causative agents, and rapid, precise, and low-cost methods for Mycoplasma detection are urgently needed. For this purpose, loop-mediated isothermal amplification (LAMP) and quantitative PCR (qPCR) assays were developed and compared. The LAMP assay was designed and primer concentrations optimized to M. bovis oppD, encoding oligopeptide permease D. For qPCR, a Taqman assay (Applied Biosystems, Carlsbad, CA) targeting M. bovis gltX, encoding glutamate transfer RNA ligase, was optimized for primer concentration, annealing temperature, and DNA polymerase. Both assays were similarly sensitive, with a detection limit of approximately 10⁴ to 10⁵ M. bovis cells/mL. Both assays were also successful in confirming M. bovis identity in laboratory culture suspensions and in bovine milk. The LAMP and qPCR assays combined with the MoBio DNA extraction kit (MoBio Laboratories Inc., Carlsbad, CA) resulted in the correct detection of 13 out of 13 M. bovis isolates and 14 out of 16 M. bovis-positive milk samples collected from commercial dairies in California. When combined with the PrepMan Ultra reagent (Applied Biosystems), the qPCR assay resulted in confirming 21 out of 21 M. bovis-positive milk samples. Comparison of the assays to milk containing either Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma californicum, M. alkalescens, or Acholeplasma laidlawii or milk lacking any detectable Mycoplasma species or relatives resulted in 3 out of 17 (LAMP with MoBio), 1 out of 17 (qPCR with MoBio), and 2 out of 36 (qPCR with PrepMan Ultra) false positives. Overall, the qPCR assay was more robust than LAMP and could be used on DNA recovered from milk prepared with the PrepMan Ultra reagent, a method that does not include a DNA purification step. The use of this qPCR method enables M. bovis detection in bovine milk in 40 to 55 min, and therefore provides new opportunities to accelerate and simplify M. bovis detection in unpasteurized milk to reduce the incidence of M. bovis mastitis outbreaks.     

Colonizing capability of Campylobacter jejuni genotypes from low-prevalence avian species in broiler chickens

Genetic variations in Campylobacter jejuni or host factors result in low prevalence rates among nonchicken poultry species. The objective of this study was to determine the colonizing potential, in broiler chickens, of C. jejuni that was recovered from low-prevalence avian species. Twenty-day-old Campylobacter-negative broiler chicks were inoculated by oral gavage with genetically different primary isolates of C. jejuni recovered from squab, duck, or chicken. Serial sampling and microbiologic testing of ceca were used to determine the level of colonization and the prevalence of positive chickens. All isolates were recovered from chickens by 10 days postinoculation. The C. jejuni strains recovered from challenged birds were genetically identical to the inoculated strains. By 10 days postinoculation, treatment groups inoculated with duck or control chicken isolates were 100% positive. The level of colonization by the squab isolate on day 2 postinoculation was significantly less than the duck or chicken isolates and had not colonized all birds by day 10 postinoculation.     

Accurate atmospheric correction of ASTER thermal infrared imagery using the WVS method

The water vapor scaling (WVS) method involves an atmospheric correction algorithm for thermal infrared (TIR) multispectral data, designed mainly for the five TIR spectral bands of the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) on the Terra satellite. First, this method is improved for better applicability to ASTER/TIR imagery. The major improvement is the determination of a water vapor scaling factor on a band-by-band basis, which can reduce most of the errors induced by various factors such as algorithm assumptions. Next, the WVS method is validated by assessing the surface temperature and emissivity retrieved for a global-based simulation model (416 448 conditions), 183 ASTER scenes selected globally, and ASTER scenes from two test sites, Hawaii Island and Tokyo Bay. In situ lake surface temperatures measured in 13 vicarious calibration experiments, Moderate Resolution Imaging Spectroradiometer sea surface temperature products, and a climatic lake temperature are also used in validation. All the results indicate that although the ASTER/TIR standard atmospheric correction algorithm performs less well in humid conditions, the WVS method will provide more accurate retrieval of surface temperature and emissivity in most conditions including notably humid conditions. The expected root mean square error is about 0.6 K in temperature. Since the WVS method will be degraded by errors in gray pixel selection and cloud detection, these processing steps should be applied accurately.     

Validation of ASTER/TIR standard atmospheric correction using water surfaces

The standard atmospheric correction algorithm for the five thermal infrared (TIR) bands of the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) is based on radiative transfer calculation using the MODTRAN code. Atmospheric profiles input to MODTRAN are extracted from either the Global Data Assimilation System (GDAS) product or the Naval Research Laboratory (NRL) climatology model. The present study provides validation results of this algorithm. First, in situ lake surface temperatures measured in 13 vicarious calibration (VC) experiments were compared with surface temperatures retrieved from ASTER data. As the results, the mean bias was 0.8 and 1.8 K for GDAS and NRL, respectively. The NRL model performed worse than GDAS for four experiments at Salton Sea, CA, probably because the model was not suitable for this site, which has typically higher surface temperature and humidity than other VC sites. Next, the algorithm was validated based on the max-min difference (MMD) of water surface emissivity retrieved from each of 163 scenes acquired globally. As a result, the algorithm error increased quadratically with the precipitable water vapor (PWV) content of the atmosphere, and the expected MMD error was 0.049 and 0.067 for GDAS and NRL, respectively, with a PWV of 3 cm, where 0.05 on MMD is roughly corresponding to -0.8 or +2.3 K on the retrieved surface temperature error. The algorithm performance degraded markedly when the surface temperature exceeded about 25/spl deg/C, particularly for NRL. Consequently, GDAS performs better than NRL as expected, while both will perform less well for humid conditions.     

Quinine Actinometry as a Method for Calibrating Ultraviolet Radiation Intensity in Light-Stability Testing of Pharmaceuticals

A collaborative study was carried out by seven different laboratories to evaluate quinine actinometry as a universal, standardized method for calibrating UV radiation intensity from light sources used in light-stability testing of pharmaceutical products. Near UV fluorescent lamps, white fluorescent lamps, metal halide lamps and xenon arc lamps were employed as light sources. The increase in absorbance at 400 nm of aqueous quinine solutions was found to be proportional to the integrated UV energy emitted from the light sources. The linearity observed between absorbance and integrated UV energy indictates that quinine actinometry can be used to measure the intensity of UV radiation at wavelengths around 330 nm. The slopes of regression curves of absorbance vs integrated UV energy varied among the lamps used due to differing spectral distributions. Light degradation of nifedipine, a model photosensitive drug, was studied based on quinine actinometry.     

ASTER preflight and inflight calibration and the validation ofLevel 2 products

Describes the preflight and inflight calibration approaches used for the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER). The system is a multispectral, high-spatial resolution sensor on the Earth Observing System's EOS-AM1 platform. Preflight calibration of ASTER uses well-characterized sources to provide calibration and preflight round-robin exercises to understand biases between the calibration sources of ASTER and other EOS sensors. These round-robins rely on well-characterized, ultra-stable radiometers. An experiment field in Yokohama, Japan, showed that the output from the source used for the visible and near-infrared (VNIR) subsystem of ASTER may be underestimated by 1.5%, but this is still within the 4% specification for the absolute, radiometric calibration of these bands. Inflight calibration will rely on vicarious techniques and onboard blackbodies and lamps. Vicarious techniques include ground-reference methods using desert and water sites. A recent joint field campaign gives confidence that these methods currently provide absolute calibration to better than 5%, and indications are that uncertainties less than the required 4% should be achievable at launch. The EOS-AM1 platform will also provide a spacecraft maneuver that will allow ASTER to see the Moon, allowing further characterization of the sensor. A method for combining the results of these independent calibration results is presented. The paper also describes the plans for validating the Level 2 data products from ASTER. These plans rely heavily upon field campaigns using methods similar to those used for the ground-reference, vicarious calibration methods     

Diversity of flaA genotypes among Campylobacter jejuni isolated from six niche-market poultry species at farm and processing

PCR-restriction fragment length polymorphism of the flagellin (flaA) gene in Campylobacter jejuni was used to determine the relationships of isolates collected at the farm and throughout processing for six niche-market poultry species. This study focused on two specialty chicken products, poussin and free range, and four other specialty products, squab, duck, guinea fowl, and quail. Cloacal and carcass samples were collected from three flocks from each of the six niche species. Three processing plants in California participated in a 2-year investigation. A total of 773 isolates from farm, posttransport, and the processing plants were genotyped, yielding a total of 72 distinct flaA profiles for the six commodities. Genetic diversity of C. jejuni at the farm was greatest for ducks with up to 12 distinct flaA types in two flocks and least for squab 1 flaA type between two farms. For two of the guinea fowl flocks, one free-range flock, two squab flocks, and all three poussin flocks, the flaA types recovered at the prepackage station matched those from the farm. Cross-contamination of poultry carcasses was supported by the observation of flaA types during processing that were not present at the farm level. New C. jejuni strains were detected after transport in ducks, guinea fowl, and free-range chickens. Postpicker, postevisceration, and prewash sampling points in the processing plant yield novel isolates. Duck and free-range chickens were the only species for which strains recovered within the processing plant were also found on the final product. Isolates recovered from squab had 56 to 93% similarity based on the flaA types defined by PCR-restriction fragment length polymorphism profiles. The 26 duck isolates had genetic similarities that ranged from 20 to 90%. Guinea fowl and free-range chickens each had 40 to 65% similarity between isolates. Poussin isolates were 33 to 55% similar to each other, and quail isolates were 46 to 100% similar. Our results continue to emphasize the need to clean processing equipment and posttransport crates in order to decrease cross contamination between flocks. This study also determined that several strains of C. jejuni had unique flaA types that could only be recovered in their host species.