A preliminary study of the localization of infectious laryngotracheitis virus glycoprotein E within specific peripheral blood lymphocytes.

Infectious laryngotracheitis virus (ILTV) DNA has been detected in blood fractions, but the cell phenotype with which the virus is associated is unknown. This study investigated the presence of ILTV antigen in peripheral blood cells of six acutely ILTV-infected chickens (5 or 9 days post ocular inoculation with a virulent isolate) and three sham-inoculated chickens using immunofluorescent staining. Blood fractions were separated by Ficoll-Paque density gradient centrifugation, and smears were prepared from erythrocyte and leukocyte fractions. The smears were stained for ILTV glycoprotein E and the leukocyte markers CD4, CD8, Bu-1 (B cell), KUL01 (monocyte/macrophage), TCRγδ, and TCRαß/Vß2 and examined under a confocal microscope. In samples from infected birds, ILTV gE-specific fluorescence was localized in B cells and all evaluated T cell types, but not in monocytes and erythrocytes. The percentage of CD4, CD8, TCRγδ, TCRαß/Vß1, TCRαß/Vß2 and B cells positive for ILTV antigen ranged from 13.3% to 22.3%. None of the samples from the sham-inoculated chickens exhibited fluorescence for ILTV gE. The results of this pilot study suggest that ILTV has a tropism for peripheral blood T and B cells. Further research is required to investigate whether these cells support ILTV productive replication. RESEARCH HIGHLIGHTSSelective tropism of ILTV for peripheral blood cells was demonstrated in acutely infected birds.The ILTV antigen gE was detected in blood CD4, CD8, TCRγδ, TCRαß and B cells but not in monocytes and erythrocytes.The highest percentage of ILTV antigen was observed in CD4 cells (22.3%) followed by TCRαß/Vß1 (20.6%), CD8 (15.4%), TCRαß/Vß2 or B cells (14.4%) and TCRγδ cells (13.3%).

Evaluation of autofluorescence quenching techniques on formalin- fixed chicken tissues.

Autofluorescence (AF) in formalin-fixed and paraffin-embedded tissues limit their use in immunofluorescence staining techniques. Various methods have been used to reduce AF in human and animal tissues but no protocol has been optimized for avian tissues. The present study was undertaken to evaluate different treatment methods including ammonium chloride, glycine, Trypan blue, sodium borohydride, Sudan Black B, potassium permanganate, LED light, cupric sulphate combined with glycine, ammonium chloride and cupric sulphate in reducing AF in FFPE chicken tissues for the detection of FITC labelled antibodies against immune cell markers. Chicken tissues including conjunctiva, trachea and Harderian gland presented intense non-homogenous AF in cells resembling erythrocytes, connective cells and melanocytes. Only Sudan Black B effectively reduced AF in FFPE tissues; however, no specific fluorescent signal was observed for six FITC labelled antibodies against immune cell markers. Specific fluorescent signal from the FITC-labelled antibodies was observed in frozen chicken tissue sections with minimal AF, suggesting that the AF in FFPE tissues is related to the use of formaldehyde fixatives. In conclusion, this study demonstrates for the first time that AF quenching methods commonly used for other animal species are not appropriate for use in avian tissues and that frozen tissue sections are recommended for immunofluorescence staining techniques in poultry.

Marked differences in virulence of three Australian field isolates of infectious laryngotracheitis virus in meat and layer chickens.

The objectives of this study were to compare the virulence of contemporary infectious laryngotracheitis virus (ILTV) field isolates of classes 9, 10, and 14 in meat and layer chickens, and to evaluate cloacal and oropharyngeal swabs and dust as sample types for ILTV detection. A total of 211 chickens were divided into groups and inoculated with ILTV class 9, 10, or 14, or sham-inoculated via eye drop at 15 or 22 days of age. Chickens were euthanized at 5 and 9 days post-infection. Virulence was assessed by scoring of clinical signs (conjunctivitis, dyspnoea, and demeanour), ILTV genomic copies (GC) in oropharyngeal and cloacal swabs, mortality and microscopic lesions in conjunctiva and trachea. Class 14 caused subclinical infection, while inoculation with class 9 or class 10 resulted in severe clinical signs and microscopic lesions. Compared to class 14 (2.25 ± 0.36 log10 GC), higher viral load was observed in oropharyngeal swabs of classes 9 (7.86 ± 0.48) and 10 (7.53 ± 0.36), with a higher proportion of positive oropharyngeal and cloacal swabs in the latter groups (P < 0.0001). Viral detection in cloacal swabs was delayed at early stages of infection compared to oropharyngeal swabs. Dust samples from class 9- and class 10-inoculated groups showed a trend towards higher GC than that of class 14. Overall, clinical scores, mortality, viral load, and microscopic lesions were similar for classes 9 and 10, but class 9 caused more severe disease in layer chickens than meat chickens. In summary, ILTV classes 9 and 10 exhibited severe virulence, while class 14 exhibited very mild virulence. RESEARCH HIGHLIGHTS Wide variation in the virulence of three field Australian field ILTV strains. Class 9 and class 10 strains were highly virulent, while class 14 was mildly virulent. The highly virulent strains were associated with significantly higher viral genome copies in various sample types than the mildly virulent strain.

A Melanin bleaching method to prevent non-specific immunostaining of chicken feathers.

Melanin in pigmented organs like the skin is known to react with 3,3'-diaminobenzidine (DAB) to give a brown colour indistinguishable from the colour that DAB imparts to target antibodies bound to specific antigens. This can lead to false positives in chicken feathers during immunoperoxidase staining. Here, we present a simple, fast and practical method for bleaching chicken feathers which can be applied prior to immunohistochemistry staining without affecting specific antigen-antibody binding. To our knowledge, this is the first report of a melanin-bleaching technique prior to immunoperoxidase staining techniques of chicken feathers for detection of pathogens. Optimisations of the method include:•Removal of melanin from tissue sections using a short incubation with potassium permanganate followed by incubation with oxalic acid prior to immunostaining for improved specificity.•This technique did not affect the antigenicity of infectious laryngotracheitis virus antigen and did not cause damage or detachment of tissues from the slides.

Equations defined using gene expression and histological data resolve coeliac disease biopsies within the Marsh score continuum.

BACKGROUND/AIM: The gold standard diagnostic for coeliac disease (CD) is subjective histological assignment of biopsies into the Marsh score categories. It is hypothesized that discrete Marsh score categories can be quantitatively resolved into a continuum using discriminant equations defined using histological and gene expression data. Therefore, the aim of this study was to use a combination of histological and gene expression data to develop equations that classify CD patient biopsies into a quantitative Marsh score continuum which could be used by clinicians to monitor CD treatment efficacy. METHODS: Both empirical and simulated gene expression and histological data were used to define predictive Marsh score equations. The distances of treated sample biopsies from the Marsh score standards were determined using the Mahalanobis distance calculation. RESULTS: Three function, high resolution discriminant equations derived from simulated data were used to accurately classify 99.6% of simulated and empirically derived biopsy data. The first function resolved active (Marsh type 3) CD from mild (Marsh type 1) CD. The second function resolved normal (no specific pathology) biopsies from mild CD. The third function resolved active Marsh score 3 into a and b subcategories. Finally, measuring the Mahalanobis distance enabled the conversion of discrete Marsh score categories into a continuum. CONCLUSIONS: This proof-of-concept study successfully demonstrated that the discrete Marsh score scale can be converted into a quantitative continuum capable of high resolution monitoring of patient treatment efficacy using equations defined by gene expression and histology data.

Celiac disease gene expression data can be used to classify biopsies along the Marsh score severity scale.

BACKGROUND AND AIM: The diagnosis of celiac disease autoimmune pathology relies on the subjective histological assignment of biopsies into Marsh score categories. It is hypothesized that Marsh score categories have unique gene expression signatures. The aims were as follows: first, to develop a celiac disease quantitative reverse transcription-polymerase chain reaction (RT-PCR) array; second, define gene expression signatures associated with Marsh score categories; and third, develop equations that classify biopsies into Marsh score categories and to monitor the efficacy of patient treatment. METHODS: Gene targets for inclusion in the celiac RT-PCR (qRT-PCR) array were identified using systematic analysis of published celiac transcriptomic data. The array was used to assess the gene expression associated with histological changes in duodenal biopsies obtained from adult patients. Finally, Marsh score classification equations were defined using discriminant analysis. RESULTS: The array contained 87 genes. The expression of 26 genes were significantly (p < 0.06) associated with the discrete Marsh score categories. As the Marsh score pathology of biopsies increased, there was a progression of innate immune gene expression through adaptive Th1-specific gene expression with a concurrent decrease in intestinal structural gene expression in high Marsh score samples. These 26 genes were used to define classification equations that accounted for 99% of the observed experimental variation and which could classify biopsies into Marsh score categories and monitor patient treatment progression. CONCLUSIONS: This proof-of-concept study successfully developed a celiac RT-PCR array and has provided evidence that discriminant equations defined using gene expression data can objectively and accurately classify duodenal biopsies into Marsh score categories.

Gut microbiome and depression: what we know and what we need to know.

Gut microbiome diversity has been strongly associated with mood-relating behaviours, including major depressive disorder (MDD). This association stems from the recently characterised bi-directional communication system between the gut and the brain, mediated by neuroimmune, neuroendocrine and sensory neural pathways. While the link between gut microbiome and depression is well supported by research, a major question needing to be addressed is the causality in the connection between the two, which will support the understanding of the role that the gut microbiota play in depression. In this article, we address this question by examining a theoretical 'chronology', reviewing the evidence supporting two possible sequences of events. First, we discuss that alterations in the gut microbiota populations of specific species might contribute to depression, and secondly, that depressive states might induce modification of specific gut microbiota species and eventually contribute to more severe depression. The feasibility of both sequences is supported by pre-clinical trials. For instance, research in rodents has shown an onset of depressive behaviour following faecal transplantations from patients with MDD. On the other hand, mental induction of stress and depressive behaviour in rodents resulted in reduced gut microbiota richness and diversity. Synthesis of these chronology dynamics raises important research directions to further understand the role that gut microbiota play in mood-relating behaviours, which holds substantial potential clinical outcomes for persons who experience MDD or related depressive disorders.

Can the sensitivity of the histopathological diagnosis of coeliac disease be increased and can treatment progression be monitored using mathematical modelling of histological sections? - A pilot study.

PURPOSE: The aim of this pilot study was to attempt to define a set of equations from histological observations of tissue affected with coeliac disease (CD) to predict Marsh score. MATERIAL/METHODS: Tissue from 15 patients with untreated CD, 6 patients with treated CD and 9 healthy control patients were stained using the standard H&E, Giemsa's staining for tissue sections and Alcian Blue protocols. A number of histological measures were then taken from each section and the data was used to ultimately design a set of linear predictive algorithms to calculate Marsh score. RESULTS: Using MANOVA and discriminant analysis, two linear functions were defined which could accurately predict the Marsh score of patients 62.5% (full Marsh score) to 79.2% (simplified Marsh score) of the time. CONCLUSIONS: This pilot study has shown that a set of objective histological measures can be used to define algorithms to predict Marsh score in CD patients and also monitor treatment compliance and progression.