Laser Capture Microdissection, Culture Analysis, and Bacterial Sequencing to Evaluate the Microbiota of Focal Duodenal Necrosis in Egg Layers.

Focal duodenal necrosis (FDN) is a common intestinal disease of table egg layers. In this research we aimed to identify the bacteria commonly found in FDN lesions as seen with histopathological analysis. Fifty-nine ethanol-fixed duodenum samples were collected from egg layers on eight FDN-affected farms, and 42 samples had typical FDN lesions. Excision of bacteria-containing lesions using laser capture microdissection was performed, followed by 16S rRNA gene sequencing of extracted DNA for bacterial identification. Bacterial sequencing analysis revealed no consistent bacterial species identified from samples with FDN. However, analysis of the relative phylum abundance revealed differences in the duodenal microbiota between layers with FDN and healthy birds. There were differences in the abundance of Proteobacteria, Firmicutes, and Actinobacteria between FDN-positive and FDN-negative control samples compatible with intestinal dysbiosis. In addition, 10 duodenal samples with FDN lesions were collected for bacteriological analysis, yielding 47 colonies on tryptone soy agar, MacConkey agar, and blood agar plates. Using 16S rRNA gene PCR, 39/47 (53.8%) colonies were identified as Escherichia coli. PCR for E. coli virulence genes identified 21/39 (53.8%) E. coli isolates as avian pathogenic E. coli-like. PCR analysis for 19 E. coli virulence genes associated with intestinal disease strains including inflammatory bowel disease found 11/39 (28.2%) isolates containing more than 10 of these virulence genes. In conclusion, FDN appears to be a multifactorial inflammatory intestinal disease associated with intestinal dysbiosis, and Gram-negative bacteria including E. coli may contribute to the pathogenesis of this disease.

Microdisección por captura láser, análisis de cultivos y secuenciación bacteriana para evaluar la microbiota de la necrosis duodenal focal en aves de postura de huevo comercial. La necrosis duodenal focal (FDN) es una enfermedad intestinal común en las gallinas de postura de huevo comercial. En esta investigación, el objetivo fue identificar las bacterias que se encuentran comúnmente en las lesiones provocadas por la necrosis duodenal focal tal como se aprecian con el análisis histopatológico. Se recolectaron 59 muestras de duodeno fijadas con etanol de gallinas de postura de ocho granjas afectadas por necrosis duodenal focal, y 42 muestras tenían lesiones típicas de dicha enfermedad. Se realizó la escisión de las lesiones que contenían bacterias mediante microdisección por captura láser, seguida de la secuenciación del gene 16S rRNA del ADN extraído para la identificación bacteriana. El análisis de secuenciación bacteriana no reveló especies bacterianas consistentes identificadas a partir de muestras con necrosis duodenal focal. Sin embargo, el análisis de la abundancia relativa del phylum reveló diferencias en el microbiota duodenal entre gallinas de postura con necrosis duodenal focal y aves sanas. Hubo diferencias en la abundancia de Proteobacteria, Firmicutes y Actinobacteria entre las muestras controles positivas y negativas para la necrosis duodenal focal compatibles con disbiosis intestinal. Además, se recolectaron 10 muestras duodenales con lesiones de la necrosis duodenal focal para análisis bacteriológico, lo que produjo 47 colonias en placas de agar triptona soya, agar MacConkey y agar sangre. Utilizando un método de PCR para el gene 16S rRNA, 39/47 (53.8 %) colonias se identificaron como Escherichia coli. El método de PCR para genes de virulencia de E. coli identificó 21/39 (53.8 %) aislados de E. coli como similares a E. coli patogénica aviar. El análisis de PCR para 19 genes de virulencia de E. coli asociados con cepas que provocan enfermedades intestinales, incluida la enfermedad inflamatoria intestinal, detectó 11/39 (28.2 %) aislados que contenían más de 10 de estos genes de virulencia. En conclusión, la necrosis duodenal focal parece ser una enfermedad intestinal inflamatoria multifactorial asociada con disbiosis intestinal, y las bacterias Gramnegativas, incluida E. coli, pueden contribuir a la patogenia de esta enfermedad.

Metallosis in a Dog as a Long-Term Complication Following Total Hip Arthroplasty.

Metallosis is the accumulation of metallic debris in soft tissues resulting from wear following total joint replacement. A dog was evaluated for lameness 4 years after total hip arthroplasty using a titanium alloy and cobalt chromium total hip system. Radiographs revealed severe acetabular component wear, implant-bone interface deterioration, and peri-acetabular osteolysis. During surgical revision, black periarticular tissue surrounded the implants. Histologically, there was fibrosis and granulomatous inflammation with abundant, intra- and extracellular, black, granular material and smaller amounts of clear punctate to acicular material. Laser capture microdissection followed by x-ray fluorescence microscopy indicated the material contained large amounts of titanium with smaller amounts of vanadium, cobalt, and chromium, confirming the diagnosis of metallosis. The clear material was birefringent under cross-polarized light, stained positive with Oil-Red-O, and thus was consistent with polyethylene. Metallosis exhibits characteristic gross and histologic lesions and is a differential diagnosis for aseptic loosening of hip implants.

Involvement of the different lung compartments in the pathogenesis of pH1N1 influenza virus infection in ferrets.

Severe cases after pH1N1 infection are consequence of interstitial pneumonia triggered by alveolar viral replication and an exacerbated host immune response, characterized by the up-regulation of pro-inflammatory cytokines and the influx of inflammatory leukocytes to the lungs. Different lung cell populations have been suggested as culprits in the unregulated innate immune responses observed in these cases. This study aims to clarify this question by studying the different induction of innate immune molecules by the distinct lung anatomic compartments (vascular, alveolar and bronchiolar) of ferrets intratracheally infected with a human pH1N1 viral isolate, by means of laser microdissection techniques. The obtained results were then analysed in relation to viral quantification in the different anatomic areas and the histopathological lesions observed. More severe lung lesions were observed at 24 h post infection (hpi) correlating with viral antigen detection in bronchiolar and alveolar epithelial cells. However, high levels of viral RNA were detected in all anatomic compartments throughout infection. Bronchiolar areas were the first source of IFN-α and most pro-inflammatory cytokines, through the activation of RIG-I. In contrast, vascular areas contributed with the highest induction of CCL2 and other pro-inflammatory cytokines, through the activation of TLR3.

Laser Capture Microdissection of Feline Streptomyces spp Pyogranulomatous Dermatitis and Cellulitis.

Suspected Streptomyces spp infections were identified in 4 cats at UC Davis Veterinary Medical Teaching Hospital between 1982 and 2011. Three had ulcerated, dark red mycetomas involving the dermis, subcutis, and fascia with fistulous tracts and/or regional lymphadenopathy. One cat had pyogranulomatous mesenteric lymphadenitis. Granulomatous inflammation in all cats contained colonies of Gram-positive, non-acid-fast organisms. All 4 cats failed to respond to aggressive medical and surgical treatment and were euthanized. Laser capture microdissection (LCM) was used to selectively harvest DNA from the affected formalin-fixed, paraffin-embedded (FFPE) tissues. Cloned amplicons from LCM-derived tissue confirmed the presence of Streptomyces spp in the dermatitis cases. Amplicons from the remaining cat with peritoneal involvement aligned with the 16S ribosomal RNA gene for Actinomycetales. Usually considered a contaminant, Streptomyces spp can be associated with refractory pyogranulomatous dermatitis and cellulitis in cats with outdoor access. LCM is useful in the diagnosis of bacterial diseases where contamination may be an issue.

Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis.

REASONS FOR PERFORMING STUDY: Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs. OBJECTIVES: To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis. STUDY DESIGN: Experimental study. METHODS: Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05. RESULTS: Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover. CONCLUSIONS: Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.

Beyond H&E: integration of nucleic acid-based analyses into diagnostic pathology.

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction-based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

Temporal and geographic clustering of polyomavirus-associated olfactory tumors in 10 free-ranging raccoons (Procyon lotor).

Reports of primary nervous system tumors in wild raccoons are extremely rare. Olfactory tumors were diagnosed postmortem in 9 free-ranging raccoons from 4 contiguous counties in California and 1 raccoon from Oregon within a 26-month period between 2010 and 2012. We describe the geographic and temporal features of these 10 cases, including the laboratory diagnostic investigations and the neuropathologic, immunohistochemical, and ultrastructural characteristics of these tumors in the affected animals. All 9 raccoons from California were found within a localized geographic region of the San Francisco Bay Area (within a 44.13-km radius). The tight temporal and geographic clustering and consistent anatomic location in the olfactory system of tumor types not previously described in raccoons (malignant peripheral nerve sheath tumors and undifferentiated sarcomas) strongly suggest either a common cause or a precipitating factor leading to induction or potentiation of neuro-oncogenesis and so prompted an extensive diagnostic investigation to explore possible oncogenic infectious and/or toxic causes. By a consensus polymerase chain reaction strategy, a novel, recently reported polyomavirus called raccoon polyomavirus was identified in all 10 tumors but not in the normal brain tissue from the affected animals, suggesting that the virus might play a role in neuro-oncogenesis. In addition, expression of the viral protein T antigen was detected in all tumors containing the viral sequences. We discuss the potential role of raccoon polyomavirus as an oncogenic virus.

Pathological pigmentation in cardiac tissues of Atlantic salmon (Salmo salar L.) with cardiomyopathy syndrome.

It is widely accepted that melanin formation may play an immunologic role in invertebrates and ectothermic vertebrates. In farmed Atlantic salmon, cardiomyopathy syndrome (CMS) is a common viral disease associated with severe cardiac inflammation that may be accompanied by heavy melanisation of the heart. By the use of histology, laser capture microdissection and transcription analysis of tyrosinase genes, we here show that this melanisation is linked to de novo melanogenesis by melanomacrophages, suggesting an active part in the inflammatory reaction. No general systemic activation of the extracutaneous pigmentary system in response to viral infections with affinity to the heart was observed.

Technical note: Use of laser capture microdissection for the localization of tissue-specific global gene expression in rumen papillae.

Gene expression profiling of bovine rumen tissue has provided insight into dietary regulation of rumen epithelial function. However, most studies have relied on a heterogeneous sample with multiple tissue and cell types. The objective of this study was to use laser capture microdissection to characterize RNA expression profiling of epithelial and connective tissues of rumen papillae. Papillae were biopsied from 3 lactating dairy cows, frozen in cryomolds, cut into sections, stained, and dehydrated, and epithelial and connective cells were collected using laser capture microdissection. Total RNA was isolated from epithelial and connective tissue and global gene expression was assessed using the Affymetrix GeneChip Bovine Gene 1.0 ST array (Affymetrix, Santa Clara, CA). Data preprocessing was conducted using the robust multi-array average method, and detection of differentially expressed genes (DEG) was determined using ANOVA. The model included the fixed effect of tissue, and a Benjamini-Hochberg false discovery rate of 0.1 was applied to DEG. We found 382 DEG between epithelial and connective tissues. Analysis of these DEG using Ingenuity Pathway Analysis (Redwood City, CA) found that epithelial and connective tissues in rumen papillae expressed distinct RNA profiles (signatures). The epithelial signature was enriched with RNA encoding tight junction and metabolic genes, whereas connective signatures were enriched with RNA encoding proteins involved in cell structure and extracellular matrix composition. The molecular functions enriched within the top networks between the 2 tissues from the Ingenuity Pathway Analysis included connective tissue disorders, dermatological diseases and conditions, gastrointestinal disease, tissue morphology, and tissue development. In summary, it is possible to use laser capture microdissection for the localization of tissue-specific global gene expression in rumen papillae. This approach may be useful to improve the accuracy and interpretation of molecular measurements in future studies.

Male-specific repeats in wild Bovidae.

In this study, we investigated repetitive sequences localized on Y chromosomes. Repetitive DNA sequences represent a substantial part of the eukaryotic genome and, among them, a large portion comprises sequences repeated in tandem. Efficient and rapid isolation of repeat units is possible due to a laser microdissection technique used for Y chromosome separation, followed by polymerase chain reaction (PCR), cloning, and sequence analysis. We applied the derived repeat units to members of nine tribes within the Bovidae. Apart from the Y chromosomes of Bos taurus and Bubalus bubalis, where we used known sequences of repetition, the derived sequences were used as probes for fluorescent in situ cross-hybridization to members of the nine tribes of the Bovidae. We investigated the distribution of repeat units within the tribes and their localization on the Y chromosome. Sharing of sequence variants would indicate common descent, while the rapid horizontal evolution should allow discrimination between closely related species or subspecies.

Gene and protein expression of cartilage canal and osteochondral junction chondrocytes and full-thickness cartilage in early equine osteochondrosis.

The objective of this study was to investigate the expression of several regulatory factors associated with cartilage maturation in horses with early osteochondrosis (OC) compared to normal controls. The hypothesis was that expression levels of Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTH-rP), vascular endothelial growth factor (VEGF), platelet-derived growth factor-A (PDGF-A), and matrix metalloproteinase-13 and -3 (MMP-13, -3) would be increased in OC. Articular cartilage and osteochondral samples were collected from the femoropatellar joints from seven OC and eight normal young (1-6 months) horses after euthanasia and snap frozen or suspended in 4% paraformaldehyde. Laser capture microdissection was used to capture cells surrounding cartilage canals and the osteochondral junction. Total RNA was isolated from whole cartilage and laser-captured cells. Equine-specific Ihh, PTH-rP, VEGF, PDGF-A, MMP-13, and MMP-3 mRNA expression levels were evaluated by real-time (RT)-PCR. Spatial tissue protein expression was determined by immunohistochemistry. In laser-captured samples, there was significantly increased MMP-13 and PDGF-A gene expression in chondrocytes adjacent to cartilage canals and increased PDGF-A gene expression in osteochondral junction chondrocytes of OC-affected foals. In full-thickness cartilage samples, there was significantly increased Ihh, MMP-3, and MMP-13 gene expression in OC samples, while PTH-rP protein expression was significantly higher along the osteochondral junction. The results suggest that pathways involving cartilage maturation and ossification are altered in early OC and may be associated with disease pathogenesis.

Technical note: Determination of cell-specific gene expression in bovine skeletal muscle tissue using laser microdissection and reverse-transcription quantitative polymerase chain reaction.

Skeletal muscle is a very heterogeneous tissue consisting of diverse cell types with specific transcription profiles. Therefore, the measured mRNA abundance of a certain cell type marker is influenced by the transcriptional activity as well as by the usually unknown number of contributing cells in the sample. In studies on the transcriptional activity of adipogenic genes, as indicators for the development of intramuscular adipocytes, an altered number of adipocytes or respective progenitor cells can mask changes in transcriptional activity. To overcome this problem, we started to use laser microdissection to isolate RNA of adipocytes and muscle fibers separately for downstream analysis. Even muscle fiber types can be collected and analyzed separately. Laser microdissection in combination with biopsy techniques enables gene expression studies of particular cell types during the life cycle of an animal. First experiences using laser microdissection for adipogenic gene expression studies in bovine skeletal muscle are described, and the influence of sample preparation and future challenges are discussed.

Comparison of aryl hydrocarbon receptor gene expression in laser dissected granulosa cell layers of immature rat ovaries.

In order to understand ovarian toxicity of aryl hydrocarbon receptor (AhR) agonists, in situ gene expression of the AhR was examined during follicle development in immature rats. In situ hybridization on frozen sections of ovaries from 24-day-old Sprague-Dawley rats showed that the AhR mRNA was localized in the granulosa cells and occasionally in the theca cells of the follicles irrespective of the developmental stage. In situ gene quantification on granulosa cell layers collected by laser microdissection further revealed that the granulosa cells expressed less AhR mRNA according to development of belonging follicles, but more ß-subunit of inhibin A mRNA, a quality control gene. These results may help to elucidate vulnerable developmental stages of follicles to toxicities of the AhR agonists.