High-throughput viral microneutralization method for feline coronavirus using image cytometry.

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.

Detection of porcine epidemic diarrhea virus-neutralizing antibody using high-throughput imaging cytometry.

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry-based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status (n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

Characterization of functional variables in epididymal alpaca (Vicugna pacos) sperm using imaging flow cytometry.

Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model.

Complete blood cell count in psittaciformes by using high-throughput image cytometry: a pilot study.

The avian hemogram is usually performed in veterinary diagnostic laboratories by using manual cell counting techniques and differential counts determined by light microscopy. There is no standard automated technique for avian blood cell count and differentiation to date. These shortcomings in birds are primarily because erythrocytes and thrombocytes are nucleated, which precludes the use of automated analyzers programmed to perform mammal complete blood cell counts. In addition, there is no standard avian antibody panel, which would allow cell differentiation by immunophenotyping across all commonly seen bird species. We report an alternative hematologic approach for quantification and differentiation of avian blood cells by using high-throughput image cytometry on blood smears in psittacine bird species. A pilot study was designed with 70 blood smears of different psittacine bird species stained with a Wright-Giemsa stain. The slides were scanned at 0.23 microm/pixel. The open-source softwares CellProfiler and CellProfiler Analyst were used for analyzing and sorting each cell by image cytometry. A "pipeline" was constructed in the CellProfiler by using different modules to identify and export hundreds of measures per cell for shape, intensity, and texture. Rules for classifying the different blood cell phenotypes were then determined based on these measurements by iterative feedback and machine learning by using CellProfiler Analyst. Although this approach shows promises, avian Leukopet results could not be duplicated when using this technique as is. Further studies and more standardized prospective investigations may be needed to refine the "pipeline" strategy and the machine learning algorithm.

Computer-assisted nuclear morphometry in the cytological evaluation of canine perianal adenocarcinomas.

Stained cytological specimens from 18 canine perianal adenocarcinomas were analyzed by computer-assisted nuclear morphometry in order to evaluate the prognostic value of this technique. In each case, the nuclei of at least 100 neoplastic hepatoid cells were measured and the mean nuclear area (MNA), mean nuclear perimeter (MNP), mean nuclear diameter (D mean), maximum nuclear diameter (D max) and minimum nuclear diameter (D min) were calculated. There were significant differences in all of these parameters when non-metastatic tumours were compared with tumours in which lymph node metastasis had been identified. A statistically significant correlation was found between the survival period of the dogs and MNA, MNP, D max and D mean. The age of the dog, diameter of the tumour and presence of nodal metastasis were also correlated with survival, but there was no correlation with breed, sex or D min. These results indicate that MNA, MNP, D max and D min may be used as prognostic indicators for canine perianal adenocarcinoma.

Nuclear morphometry in squamous cell carcinomas of canine skin.

To investigate the diagnostic and prognostic value of nuclear morphometry in squamous cell carcinomas (SCCs) of canine skin, histological samples from 15 dogs were retrospectively analysed by computerized nuclear morphometry. In each case, the nuclei of at least 100 neoplastic cells were measured, and the mean nuclear area (MNA), standard deviation of MNA (SDa), mean nuclear perimeter (MNP), standard deviation of MNP (SDp), nuclear form factor (FF), and standard deviation of FF (SDf) were calculated. The morphometric data were examined in relation to tumour histological grade. No statistical differences were found between the different grades of SCC in terms of the mean values of SDa, SDp, FF or SDf. The MNA values, however, differed significantly between grades: I and III; I and IV; II and III; II and IV. The MNP values differed significantly between grades: I and III; I and IV; II and IV. The results indicate that nuclear morphometry analysis is a simple and reproducible method that could be used to provide objective diagnostic criteria for cutaneous SCCs in the dog.

Comparison of nuclear morphometric parameters in cytologic smears and histologic sections of spontaneous canine tumors.

BACKGROUND: Nuclear morphometry may provide useful diagnostic and prognostic information for neoplasms in animals. Most available data have been obtained from histologic sections. Nuclear morphometry of cytologic smears may provide important preoperative information. OBJECTIVE: The goal of this study was to compare nuclear morphometric parameters in cytologic smears and histologic sections from spontaneous canine tumors. METHODS: Mean nuclear area (MNA), mean nuclear perimeter (MNP), mean nuclear form factor (FF; nuclear perimeter(2)/4pi nuclear area) and their respective SDs were assessed by image analysis of both hematoxylin and eosin-stained histologic sections and May-Grünwald-Giemsa-stained cytologic smears from the same case in 20 spontaneous canine tumors of different histogenesis. The above parameters were selected as being the best morphometric tools for measuring variation in shape and size in cells after neoplastic transformation. Data were compared by ANOVA with P<.01 considered significant. RESULTS: There was a significant difference between histologic and cytologic specimens for MNA, MNP, and their SDs. Only the differences between FF and the SD of FF were not statistically significant. CONCLUSIONS: Only nuclear morphometric data related to nuclear shape and nuclear shape variability are comparable between histologic and cytologic specimens. Nuclear area and perimeter may be affected by the different fixation and smear preparation techniques used in histology and cytology.

DNA measurement and immunohistochemical characterization of epithelial and mesenchymal cells in canine mixed mammary tumours: putative evidence for a common histogenesis.

DNA measurement by image cytometry, and a detailed immunohistochemical study using monoclonal antibodies directed against different human cytokeratin types, muscle-specific actin, vimentin and S100 protein were carried out on normal canine mammary tissue (n =4), benign canine mammary mixed tumours (n =20) and malignant canine mammary mixed tumours (n =13). The results showed that ductal and alveolar luminal cells in normal and neoplastic tissue were immunoreactive with CAM5.2 and AE1/AE3 antibodies recognizing human keratins.Basal/myoepithelial cells were clearly differentiated from ductal and alveolar epithelial cells, since the latter only expressed cytokeratins, whereas the former also expressed vimentin and muscle-specific actin. This immunohistochemical study showed that there is loss of expression of muscle-specific actin and cytokeratins in areas of myoepithelial proliferation, and enhanced expression of vimentin and S100 protein in proliferative areas with osseous and/or chondroid metaplasia. The ploidy studies revealed that 20% (4/20) of benign and 54% (7/13) of malignant mixed tumours of canine mammary gland were aneuploid and that the epithelial and myoepithelial components of the mixed tumours had identical DNA content. Our results reinforce the role of myoepithelial cells in mesenchymal metaplasia in mixed mammary tumours and suggest the possibility of a common origin of both components from a totipotential stem cell with capacity for divergent differentiation.