Production of a bovine IL-12p40 probe and application using in situ hybridization on ruminant fixed tissues.

Pro-inflammatory cytokines (particularly IL-12) are important for initiating protective T helper 1 (Th1)-type immune responses and hence vital for combating intracellular infections and tumours. In situ hybridization (ISH) provides a powerful diagnostic tool allowing the identification and localization of cells producing these mediators in fixed tissues. The objective of this work was to produce a bovine IL-12p40 probe that allows detection of IL-12p40 mRNA in fixed tissues from different ruminant species. The RNA probe sequence is 447bp in length and from a region with high cross-species-sequence homology (>97.3% homology) to the ovine, cervine, caprine and bubaline IL-12p40 genes. ISH was carried out on paraformaldehyde fixed tissues collected from cattle, sheep and goats. The probe was efficient in identifying IL-12p40 expressing cells in fixed tissues from all these species. In conclusion, the IL-12p40 probe was efficient in identifying and localizing cells that express IL-12p40, and provides a good immuno-diagnostic technique to characterize immune responses in fixed tissues.

The role of nuclear technologies in the diagnosis and control of livestock diseases--a review.

Nuclear and nuclear-related technologies have played an important role in animal health, particularly in relation to disease diagnosis and characterization of pathogenic organisms. This review focuses primarily on how and where nuclear technologies, both non-isotopic and isotopic methods, have made their impact in the past and where it might be expected they could have an impact in the future. The review outlines the extensive use of radiation attenuation in attempts to create vaccines for a multiplicity of pathogenic organisms and how the technology is being re-examined in the light of recent advances in irradiation techniques and cryopreservation/lyophilization that might obviate some of the problems of maintenance of viable, attenuate vaccines and their transport and use in the field. This approach could be used for a number of parasitic diseases where vaccination has been problematic and where investigations into the development of molecular vaccines have still failed to deliver satisfactory candidates for generating protective immune responses. Irradiation of antigens or serum samples also has its uses in diagnosis, especially when the samples need to be transported across international boundaries, or when handling the pathogens in question when carrying out a test presents serious health hazards to laboratory personnel. The present-day extensive use of enzyme immunoassays and molecular methods (e.g., polymerase chain reaction) for diagnosis and characterization of animal pathogens has its origins in the use of isotope-labeled antigens and antibodies. These isotopic techniques that included the use of 75Se, 32P, 125I, and 35S isotopes enabled a level of sensitivity and specificity that was hitherto unrealized, and it is prescient to remind ourselves of just how successful these technologies were, in spite of their infrequent use nowadays. Finally, the review looks at the potential for stable isotope analysis for a variety of applications--in the tracking of animal migrations, where the migrant are potential carriers of transboundary animal diseases, and where it would be useful to determine the origins of the carrier, e.g., Highly Pathogenic Avian Influenza and its dissemination by wild water fowl. Other applications could be in monitoring sequestered microbial culture (e.g., rinderpest virus) where in the case of accidental or deliberate release of infective culture it would be possible to identify the laboratory from which the isolate originated.

U-SPECT-II: An Ultra-High-Resolution Device for Molecular Small-Animal Imaging.

UNLABELLED: We present a new rodent SPECT system (U-SPECT-II) that enables molecular imaging of murine organs down to resolutions of less than half a millimeter and high-resolution total-body imaging. METHODS: The U-SPECT-II is based on a triangular stationary detector set-up, an XYZ stage that moves the animal during scanning, and interchangeable cylindric collimators (each containing 75 pinhole apertures) for both mouse and rat imaging. A novel graphical user interface incorporating preselection of the field of view with the aid of optical images of the animal focuses the pinholes to the area of interest, thereby maximizing sensitivity for the task at hand. Images are obtained from list-mode data using statistical reconstruction that takes system blurring into account to increase resolution. RESULTS: For (99m)Tc, resolutions determined with capillary phantoms were smaller than 0.35 and 0.45 mm using the mouse collimator with 0.35- and 0.6-mm pinholes, respectively, and less than 0.8 mm using the rat collimator with 1.0-mm pinholes. Peak geometric sensitivity is 0.07% and 0.18% for the mouse collimator with 0.35- and 0.6-mm pinholes, respectively, and 0.09% for the rat collimator. Resolution with (111)In, compared with that with (99m)Tc, was barely degraded, and resolution with (125)I was degraded by about 10%, with some additional distortion. In vivo, kidney, tumor, and bone images illustrated that U-SPECT-II could be used for novel applications in the study of dynamic biologic systems and radiopharmaceuticals at the suborgan level. CONCLUSION: Images and movies obtained with U-SPECT-II provide high-resolution radiomolecule visualization in rodents. Discrimination of molecule concentrations between adjacent volumes of about 0.04 microL in mice and 0.5 microL in rats with U-SPECT-II is readily possible.

Quantum dots for in vivo small-animal imaging.

Nanotechnology is poised to transform research, prevention, and treatment of cancer through the development of novel diagnostic imaging methods and targeted therapies. In particular, the use of nanoparticles for imaging has gained considerable momentum in recent years. This review focuses on the growing contribution of quantum dots (QDs) for in vivo imaging in small-animal models. Fluorescent QDs, which are small nanocrystals (1-10 nm) made of inorganic semiconductor materials, possess several unique optical properties best suited for in vivo imaging. Because of quantum confinement effects, the emission color of QDs can be precisely tuned by size from the ultraviolet to the near-infrared. QDs are extremely bright and photostable. They are also characterized by a wide absorption band and a narrow emission band, which makes them ideal for multiplexing. Finally, the large surface area of QDs permits the assembly of various contrast agents to design multimodality imaging probes. To date, biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Here we consider these novel breakthroughs in light of their potential clinical applications and discuss how QDs might offer a suitable platform to unite disparate imaging modalities and provide information along a continuum of length scales.

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe.

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.

Merging molecular and anatomical information: a feasibility study on rodents using microPET and MRI.

OBJECTIVE: The use of the micro positron emission tomography (microPET) technique provides a powerful means for molecular imaging on small animals, while its inferior spatial resolution offers insufficient anatomical information which impedes the interpretations of the scans. To improve this limitation, it often relies on a clinical magnetic resonance imaging (MRI) for providing anatomical details. In this study, we designed and developed a new image co-registration platform which contains a stereotactic frame and external fiducial markers for microPET and MRI studies. The image co-registration accuracies were also validated by this new platform using various imaging protocols for microPET and MRI. METHODS: The microPET images were reconstructed by filtered back-projection (FBP) and ordered subset expectation maximization (OSEM) methods. Two MRI pulse sequences, two-dimensional T1-weighted fast spin-echo (FSE) and three-dimensional spoiled gradient recalled (SPGR), were employed in the studies. Two MRI scanning protocols were proposed for small animal imaging: the whole-body high-speed mode and the partial high-resolution mode. RESULTS: Reconstructed images from two different modalities were integrated by point-to-point registration via the external fiducials. Four inter-modality matched co-registration pairs (FBP-FSE, FBP-SPGR, OSEM-FSE, OSEM-SPGR) were obtained for both the high speed and high resolution modes. Co-registration accuracy was given as the average fiducial registration error (FRE) between the centroids of six markers from the registered images. The overall systemic FREs were about 0.50 mm. CONCLUSIONS: From the inter-modality FRE comparison, MRI imaging with FSE performed better than that with SPGR sequence, due to its higher signal-to-noise ratio and less magnetic susceptibility effects. In the microPET perspective, the OSEM was superior to the FBP, as a result of fewer image artifacts.

Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification.

A new method for the molecular detection of the fish pathogens, infectious haematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV), is described. By employing molecular padlock probe (MPP) technology combined with rolling circle amplification (RCA) and hyperbranching (Hbr), it is possible to detect RNA target sequence from these viruses at levels comparable with those detected by the polymerase chain reaction (PCR), but without prior reverse transcription. The use of MPP technology combined with RCA and Hbr for the detection of IHNV and ISAV in fish exhibited selectivity comparable with that of PCR while potentially reducing the time and cost required for analysis. The method described was used to detect as few as 10(4) DNA oligonucleotide targets and was sequence-specific at the single base level. Viral RNA could be detected directly, either alone or in the presence of non-viral RNA from fish tissue. This technology is applicable for detecting a variety of microbes, in addition to IHNV and ISAV, and is ideal for further integration into a biosensor platform for on-site diagnosis of pathogen infection in fish.

Correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with infectious salmon anemia virus.

We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U5575-1, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P < 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R = 0.81, 0.70, and 0.78, respectively; P < 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.

A submillimeter resolution fluorescence molecular imaging system for small animal imaging.

Most current imaging systems developed for tomographic investigations of intact tissues using diffuse photons suffer from a limited number of sources and detectors. In this paper we describe the construction and evaluation of a large dataset, low noise tomographic system for fluorescence imaging in small animals. The system consists of a parallel plate-imaging chamber and a lens coupled CCD camera, which enables conventional planar imaging as well as fluorescence tomography. The planar imaging data are used to guide the acquisition of a Fluorescence Molecular Tomography (FMT) dataset containing more than 106 measurements, and to superimpose anatomical features with tomographic results for improved visual representation. Experimental measurements exhibited good agreement with the diffusion theory models used to predict light propagation within the chamber. Tests of the instrument's capacity to quantitatively reconstruct fluorochrome distributions in three dimensions showed less than 5% errors between actual fluorochrome concentrations and FMT findings, and suggested a detection threshold of approximately 100 femptomoles for small localized objects. Experiments to assess the instrument's spatial resolution demonstrated the ability of the system to resolve objects placed at clear distances of less than 1 mm. This is a significant resolution increase over previously developed systems for animal imaging, and is primarily due to the large dataset employed and the use of inversion methods. Finally, the in vivo imaging capacity is showcased. It is expected that the large dataset collected can enable superior imaging of molecular probes in vivo and improve quantification of fluorescence signatures.

A comparison of enzymatic and molecular approaches to characterize the cellulolytic microbial ecosystems of the rumen and the cecum.

We used RNA probes and enzyme activities to compare the cellulolytic microbial ecosystems of the rumen and the cecum. Four rumen- and cecum-cannulated wethers were fed a diet of barley plus hay (60:40). Digesta samples were collected 1 h before feeding and 3, 6, and 9 h after feeding for measurements on microbial populations, and 1 h before feeding and 3 and 6 h after feeding for digestion measurements, pH, and VFA. Polysaccharidase and glycosidase specific activities of solid-adherent microorganisms were measured respectively by the amount of reducing sugars released from xylan or avicel or p-nitrophenol from the p-nitrophenol derivatives of xylose and glucose. The distribution and amounts of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) were determined by dot-blot hybridization using specific 16SrRNA-targeting probes. Enzyme activities were higher in the rumen than in the cecum and before feeding than at 3 h after feeding. The sum of the three cellulolytic bacterial species represented, on average, 4.5% of the total bacterial RNA in the two compartments and did not vary with sampling time. The cellulolytic bacterial community structure was different in the two compartments, with F. succinogenes as the main species in the rumen and R. flavefaciens in the cecum. The lower cellulolytic activity in the cecum than in the rumen could not be ascribed to any difference in the structure of the cellulolytic bacterial community between these two compartments, and other hypotheses related to digestion are proposed.

Cereal supplementation modified the fibrolytic activity but not the structure of the cellulolytic bacterial community associated with rumen solid digesta.

4 ruminally cannulated cows were fed a forage diet (93% hay + 7% straw) and a mixed diet (33 % hay + 7% straw + 40% barley) in a 2 x 2 crossover experimental design. In sacco degradation of forage, fibrolytic activities (polysaccharidases and glycosidases) of the solid-associated bacteria (SAB), and distribution of the 3 main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens) were determined for both diets. Barley supplementation decreased the hay degradation rate and mainly the polysaccharidase activities of the SAB (30% on average). The sum of rRNA of the 3 cellulolytic bacterial species represented on average 17% of the total bacterial signal and R. albus was the dominant cellulolytic bacterial species of the 3 studied. Barley supplementation did not modify the proportion of the 3 cellulolytic bacteria attached to plant particles. The negative effect of barley on the ruminal hay degradation rate is due to a decrease in fibrolytic activity of the SAB, and not to a modification of the balance of the three cellulolytic bacterial species examined.

Comparison of two molecular techniques for the detection of avian reoviruses in formalin-fixed, paraffin-embedded chicken tissues.

Reverse transcription (RT) in situ polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques were used to detect the sigma c-encoded gene of avian reovirus (ARV) in chicken tissue sections. The advantage of using in situ methods is to make more rapid and accurate diagnosis of ARV infections. The sensitivity of these two techniques were compared. Of the two techniques, the RT in situ PCR test was found to be more sensitive than ISH and provided the rapid, sensitive, and specific detection of ARV infections.

Detection of rabbit haemorrhagic disease virus (RHDV) by in situ hybridisation with a digoxigenin labelled RNA probe.

An in-situ hybridisation (ISH) technique for the detection of rabbit haemorrhagic disease virus (RHDV) was developed. Thirteen seronegative adult rabbits were infected oro-nasally using the BS89 RHDV strain. Liver and spleen samples were collected from 4 h post infection (p.i.) and repeated every 4 h till 44 h p.i. Each sample was tested immunohistochemically, by sandwich ELISA and by ISH. A 2.482-kb RNA probe, matching the genomic fragment coding for the VP60 structural protein of RHDV, was arranged. Two RNA probes (sense and antisense) were transcribed in vitro and UTP-digoxigenin-labelled. The antisense probe clearly detected positivity in the cytoplasm of the hepatocytes at 8 h p.i. Labelled hepatocytes were scattered throughout the sections until 24 h p.i. followed by a more diffuse perilobular positive reaction. A much weaker signal of similar distribution was detected up to 24 h p.i. using the sense RNA probe. All spleen samples tested negative for both probes. Liver samples were positive at 32 h p.i. using both ELISA and the immunoperoxidase test. Spleen samples were positive using only the ELISA at 32 h p.i. This study showed that RHDV replication occurred almost immediately after inoculation and that the liver appears to be the main site of replication.

The prevalence of Fasciola hepatica in its snail intermediate host determined by DNA probe assay.

Accurate snail intermediate host infection prevalence data have the potential to be extremely useful in determining seasonal transmission dynamics of Fasciola hepatica. Because the microscopic techniques currently used lack the sensitivity and specificity necessary to obtain meaningful infection prevalence data, we developed a highly accurate and efficient DNA probe assay. The assay has a sensitivity of 100%, a specificity of > 99%, easily detects a single miracidia and does not cross-hybridize with DNA of Fascioloides magna, Paramphistomum liorchis or Heterobilharzia americana, trematodes that share the same intermediate host and enzootic range as Fasciola hepatica. Using this assay, we determined the prevalence of F. hepatica in its snail intermediate host, Fossaria cubensis, during the second year of a 2-year study on the epizootiology of Fasciola hepatica in Florida. The overall infection prevalence of snails assayed in this study (n = 5246) was 1.5% and ranged from 0.1% to 3.1% for individual cattle ranches. Additionally, infection prevalence differed significantly for successive size groupings of snails, varying from 0% for 1-mm snails to 18.5% for 9- and 10-mm snails. The accuracy and efficiency of the DNA probe assay reported here for determining snail infection prevalence offers an inexpensive alternative to tracer animal studies for determining the epizootiology of F. hepatica.

Development and evaluation of a non-isotopically labeled DNA probe for the diagnosis of infectious laryngotracheitis.

A digoxigenin-labeled cloned infectious laryngotracheitis virus (ILTV) DNA fragment was evaluated as a nonradioactive alternative probe in the diagnosis of infectious laryngotracheitis. The dot-blot hybridization protocol was optimized and was capable of detecting 40 pg of purified ILTV DNA and as few as 50 ILTV-infected chicken embryo liver cells. The utility of this approach for diagnostic use was evaluated through four ILTV inoculation trials using a mild field isolate, a virulent challenge strain, a tissue-culture-origin vaccine, and an egg-origin vaccine. Birds were examined for clinical signs of ILT, and conjunctival and pharyngeal swabs from inoculated and sentinel birds were tested for ILTV by the digoxigenin-labeled probe and by virus isolation. In general, higher numbers of ILTV-positive samples were detected by both assays from conjunctival swabs. For the non-vaccine strains, detection by dot-blot hybridization was equivalent to that for virus isolation. However, for the two vaccine strains, there was some lack of correlation between the dot-blot results and the virus-isolation results. The kappa values between virus-isolation results and dot-blot results for the tissue-culture-origin vaccine, egg-origin vaccine, Ont 1598 field isolate, and virulent strain were 0.00, 0.16, 0.39, and 0.24, respectively, for pharyngeal samples and 0.19, 0.29, 0.58, and 0.48, respectively, for conjunctival samples.