Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

Zoonotic nematode in the city of La Plata, Argentina: Report of a case of Calodium hepaticum in Rattus rattus.

This study describes a case of Calodium hepaticum (Trichinellida: Capillariidae) infection in an adult rat (Rattus rattus) from the periurban area of the city of La Plata in the province of Buenos Aires, Argentina. The rat was found with neurological signs (ataxia, lethargy, and episodes of unresponsiveness) in the food storage of a goat production facility. The liver was observed with hepatomegaly and diffuse and irregular yellowish-white spots appearing in striae or small nodules on the external surface and inside the liver. Subsequent microscopic and histopathological studies were performed. Eggs were observed by direct microscopy of the impression smear of liver tissue. A multifocal granulomatous tissue reaction with different stages of fibrocellular tissue was observed in the liver parenchyma. The granulomas contained adults and degenerated eggs delimited by an intense infiltrate of mononuclear cells. Macro and microscopic observations and histopathological liver lesions were compatible with C. hepaticum infection. To our knowledge, this is the first confirmation of C. hepaticum infection in R. rattus in Argentina, increasing the host record of this parasite and a new record of distribution in goat production systems in the country.

Seasonal Variation in Detection of Haemosporidia in a Bird Community: A Comparison of Nested PCR and Microscopy.

In a 2-yr study on prevalence of Haemosporidia in an avian community in Ithaca, New York, USA, we tested the hypothesis that apparent seasonal variation in prevalence is influenced by the detection protocol. We confirmed a higher detection of Haemosporidia using a molecular diagnosis technique (PCR) than by microscopy; this further increased when the PCR test was triplicated. Microscopic examination and PCR techniques have different specificity and sensitivity and therefore different probabilities of detecting hemoparasites. Birds with chronic infections or sampled during winter often have very low parasitemia, and such infections may be missed by microscopy but detected by PCR. Haemosporidian prevalence was higher during the breeding season than during the nonbreeding season regardless of the method used. Detection of Leucocytozoon spp. infection from blood smears using microscopy was challenging.

Multiplex real-time PCR for skin fungal infections: The diagnostic reliability in a one-year non-interventional study.

The skin fungal infection diagnostic workflow currently includes microscopic and culture-based methods as the gold standard. Recent published data described the possible limitations of these conventional techniques documenting the possibility of reducing response time intervals. The present study reports an evaluation of the DermaGenius® (DG) multiplex kit (PathoNostics) for rapid C. albicans and dermatophytes identification directly from skin samples. The investigations involved 90 specimens that underwent DNA extraction and amplification simultaneously to microscopic and culture methods. According to current guidelines, we defined a dermatophytic skin infection as the simultaneous presence of clinical evidence of skin lesions and positive results for dermatophyte elements from microscopy and/or cultures. The collected data remarked on the advantages of the molecular assay, especially in terms of sensitivity and rapidity. A statistical evaluation analysed a comparison between conventional and innovative diagnostic methods. The sensitivity, specificity, positive predictive value, and negative predictive value of DG-PCR in the cutaneous dermatophytosis were, respectively, 94.7%, 78.8%, 88.5%, and 89.6%. Based on our experience, the molecular technique could represent a diagnostic confirmation in the case of previous antifungal treatment, little biological material available, or urgent clinical conditions.

Our study aims to evaluate innovative technologies in diagnosing skin dermatophytosis. The final purpose was to describe an added diagnostic value, aiming to improve patients' outcomes. High sensitivity rates and a restricted turn-around time represent the main advantages.

Development of the first PCR for detection of Psoroptes ovis var. cuniculi infection and its comparison to microscopic examination and serological assay in rabbits.

Psoroptes mites are the common ecto-parasites of wild and domestic animals worldwide, which causes considerable economic losses in livestock industry. Microscopy is deemed to be the 'gold standard' for the diagnosis of Psoroptes mite infection but it has poor sensitivity for low mite infections and/or sub-clinical infections. To overcome these shortcomings, we screened four genes to develop a sensitive and specific PCR for the detection of Psoroptes mite infection in rabbits, and confirmed its practicability in detecting early infection and monitoring treatment outcome with traditional microscopy and serological tests. Results showed that PCR assay targeting ITS2 (ITS2-PCR) had a high specificity and sensitivity (detection limit: 40.3 pg/µL DNA) for detecting P. ovis DNA. In rabbits artificially infected with P. ovis, all three diagnostic tests showed the same detection rate from 14 days post infection (dpi) to 42 days dpi. However, these diagnostic tests behave differently at 7 dpi and after treatment: at 7 dpi, the detection rate of ITS2-PCR was higher than rPsoSP3-based iELISA and traditional microscopy (ITS2-PCR: 88.9%, rPsoSP3-iELISA: 77.7%, microscopy: 33.3%); at 7 days post treatment (dpt), positivity rates of ITS2-PCR and microscopy rapidly decreased to 0.00% and 11.1%, whereas rPsoSP3-iELISA remained 100% positive rate. Furthermore, the comprehensive comparisons of diagnostic performance and features of three diagnostic tests at 7 dpi were performed. Compared to ITS2-PCR or rPsoSP3-iELISA, microscopy had the lowest sensitivity, and the agreement between these assays was low (κ < 0.3). Field study showed that ITS2-PCR showed a higher detection rate than microscopy (19.4% and 11.1%, respectively). Our results suggested that the ITS2-PCR developed in this study provided a new laboratory tool for diagnosis of P. ovis var. cuniculi infection, and it had advantages over microscopic examination in detection low-level mite infections and serological assay in monitoring treatment outcome.

Pattern analysis for the diagnosis of inflammatory skin lesions in domestic animals: An overview.

Pattern analysis of inflammatory skin diseases is a technique that offers a systematic approach to the histologic diagnosis of skin diseases. First introduced to human dermatopathology in the 1970s, it was widely adopted by veterinary pathologists for the histologic diagnosis of skin diseases in animals. As the inflammatory pattern reflects, to varying extents, aspects of the underlying disease pathogenesis, its use has contributed to the recognition of novel skin diseases in domestic animals, particularly in dogs and cats. Alternative diagnostic approaches used in human dermatopathology, such as "tissue-reaction pattern" and a purely "anatomic approach" have not been as widely used in veterinary pathology. However, veterinary pathologists often combine pattern analysis with anatomic and etiologic factors. This overview outlines the technique, introduces the patterns, and discusses advantages and limitations of pattern analysis in veterinary diagnostic dermatopathology. While molecular analytic techniques and image informatics will undoubtedly prove to be revolutionary in many areas of diagnostic pathology, it is recognized in both human and veterinary arenas that the light microscopic interpretation of hematoxylin and eosin-stained tissue sections will remain the mainstay of routine dermatopathology diagnosis for the foreseeable future.

Morphology of the syrinx of three species of birds from Brazilian cerrado (Psittacara leucophthalmus, Rhynchotus rufescens and Cariama cristata): Gross anatomy and light microscopy study.

The aim of this study was to describe the morphology of the trachea and syrinx at macroscopic and light microscopy levels of three species of birds from different orders that inhabit the Brazilian cerrado. For that, five adult specimens (three males and two females of each species) of white-eyed parakeet (Psittacara leucophthalmus), red-winged tinamou (Rhynchotus rufescens) and red-legged seriema (Cariama cristata) were used. The trachea and syrinx of the birds were collected and destined for anatomical and histological studies. The trachea of the studied birds presented an elongated path and originated in the larynx and extended caudally to the syrinx. No sexual dimorphism was observed in the syrinx of the studied species, probably because it is associated with their song, which is very similar between males and females of these species. The findings of this study allowed us to classify the syrinx as tracheal in the white-eyed parakeet and tracheobronchial in the red-winged tinamou and red-legged seriema. In general, the morphological features of the trachea and syrinx were similar to those described for other species of birds, such as the presence of intrinsic and extrinsic syringeal muscles, and the lateral and medial tympaniform membranes, which would represent important anatomical structures in sound production through vibration during expiration and eventual inspiration. The morphological structure of the syrinx in the three avian species of the Brazilian cerrado is consistent with the ability of these avian species to perform a potential vocalization, especially the red-legged seriema that emits characteristic sounds very loud and can carry several kilometres.

Comparative performance evaluation of blood film microscopy for the diagnosis of bovine trypanosomosis by some laboratories in North-central Nigeria.

Background: Due to its affordability in disease-affected communities and suitability for field application, microscopy has historically been considered the gold standard for field diagnosis of trypanosomosis in rural settings. Aim: This works aims to compare the performance of microscopists on bovine trypanosome microscopy by organizing the first comparative assessment on a correct reading of slides by laboratory professionals using the read slide results and a structured interviewer-administered questionnaire in North-central Nigeria. Methods: Ten participants were addressed, as they were sent a panel of two slides (Slide 1: No Trypanosome present; Slide 2: Trypanosome present) and a questionnaire. Results: All participants greater than 41 years old reported correctly the presence and absence of parasites on slides. Only 3/8 of microscopists from routine diagnostic laboratories reported correctly the presence of the parasite. Conclusion: Our study confirmed errors in reading slides. Therefore, training of microscopists besides a nationwide quality assessment is recommended.

Macroscopic and histological aspects of the pharynx and larynx of the giant anteater (Myrmecophaga tridactyla Linnaeus, 1758).

Giant anteater (Myrmecophaga tridactyla) is an endangered species that resides in much of Latin America, but it has been losing its habitat, especially in the Cerrado biome, where it constantly suffers traumas resulting from fires and roadkill. The anatomical knowledge of structures of the respiratory system is important for a better morphophysiological understanding of the species. Thus, this study aimed to perform the macroscopic and histomorphological description of the pharynx and larynx of the giant anteater. Twelve adult giant anteaters were used, three of them fixed in buffered formalin for further dissection and pharynx and larynx macroscopic analysis of structures. From the other animals, samples of the pharynx and larynx were collected and prepared for histological evaluation under optical microscope. Macroscopically, their pharynx and soft palate are extensive, and the anatomical location of these structures and the larynx differs greatly from that described in other species. The larynx, although more caudal, was similar to that of other animals. Histologically, the epithelium of these regions varied between the pseudostratified ciliated columnar and the non-keratinized stratified squamous epithelium. Laryngeal cartilages were composed of elastic (epiglotti) and hyaline cartilages (arytenoid, cricoid and thyroid cartilage), with an ossification process and glandular clusters around the hyaline cartilage. The distinct anatomical location of the pharynx and larynx of Myrmecophaga tridactyla is the main macroscopic finding of this study, besides the length of the pharynx and soft palate of these animals.

An international collaborative approach to learning histology using a virtual microscope.

Histology is often taught in higher education settings using online virtual microscopes (VM). This study aimed to develop and evaluate the use of VM in teaching on a BSc degree at the University of Nottingham by surveying students and staff. A key development was the use of an e-workbook so that students were actively engaged in creating their own bespoke revision material. Subsequently, this approach was used in a second study evaluating the use of VM in teaching the histology and pathology of the gastrointestinal (GI) tract via group work with students from two BSc courses at the University of Nottingham; one based at Derby (RDHC) and the other in Malaysia (UNMC). Students worked together in groups to complete an e-workbook, develop a presentation, and decide how to collaborate and communicate. An evaluation of these activities revealed advantages in developing transferrable skills, and good engagement with both the histology topic and group work. Analysis of assessment of the module at UNMC showed that student performance improved in the histology-based module after the intervention (p < 0.01) and that this improvement was not evident in other modules taken by the cohort. Furthermore, when interrogating the questions from the examination paper that asked students to identify features from histological images, fewer questions were seen as 'difficult' (p < 0.001) and more were seen as 'average' (p < 0.01). This study demonstrates that the use of VM in histology combined with active learning in creating a revision resource enhances engagement and depth of learning. When further combined with collaborative active group work, students developed a range of histology knowledge and transferrable skills, with notable improvement in examination performance relative to other contemporaneous modules.

MR microscopy of the developing upper extremity of the chicken in ovo using 7 Tesla MRI.

MR microscopy (MRM) is known as ultra-high-field (UHF) magnetic resonance imaging with an in-plane spatial resolution of <100 µm, yields highly resolved non-invasive anatomical imaging and allows longitudinal assessment of embryonic avian development. The aim of the present study was to evaluate the feasibility of in vivo anatomical MRI assessment of the developing upper extremity of the chicken. Thirty-eight fertilized chicken eggs were examined at 7 Tesla acquiring high-resolution T2-weighted images with an in-plane resolution of 74 × 74 µm. To reduce motion artefacts, the eggs were moderately cooled before and during MRI. Development of the upper extremity was anatomically and quantitatively assessed. Chondrification and ossification on MRI were correlated with histological examination. MRM allowed the identification of the embryo from stage D5 onwards. First chondrification of the upper extremity was visible at stage D7, and the differentiation of the forearm was possible from stage D9 throughout the developmental period with excellent correlation to histology. MRM also allowed the differentiation between cortical and medullary bone as well as the detection of chondrified areas. UHF MRM allows the in vivo and in ovo evaluation of the upper limb during embryonic development and provides non-invasive longitudinal anatomical information. This technique allows longitudinal studies of the same embryo during the developmental period and may therefore provide further insights into the development of the upper extremity. With improved coil technique and increasing availability of UHF MR systems, there is great potential regarding several research topics in experimental musculoskeletal radiology.

Ultrastructural changes in feline oocytes during ovary storage for 24- and 48-hours.

Despite established microscopic markers of feline oocyte quality, little is known about their ultrastructural traits. To the best of our knowledge, there is no published report analysing the effect of 24 and 48 h ovarian storage time on the domestic cat oocytes characteristics at the ultrastructural level. Oocytes (n = 30) were classified using the light microscopy as good or bad quality and then proceeded for TEM observations. The location, shape, size and distribution of each organelle was noted in each examined oocyte. In in good quality oocytes the cytoplasmic organelles were generally easier to identify, and furthermore its distribution pattern was more obvious to spot than in bad quality ones. Whereas bad quality oocytes were typically characterised by the lower visibility of the cellular structures and cytoplasmic architecture was less apparent and often arranged without a predictable pattern. In good quality oocytes obtained from fresh ovaries cytoplasmic vacuoles (CVs) occupied a significantly larger area (0,72 vs. 0.18 CVs/µm2, respectively) than in bad quality ones, whereas in bad quality and stored oocytes more cytoplasm was occupied by lipid droplets (LDs) than in fresh good oocytes (0,22 ± 0,09 vs. 0,09 ± 0,05 respectively). It can be concluded that ultrastructure changes in feline oocytes during 24 and 48 h ovarian storage cannot be assessed in light microscopy. The ultrastructure of oocytes was seriously disturbed after 48 h of ovary storage, despite being classified as good quality. However, further investigations utilizing more cells are necessary to confirm reported traits of ultrastructure changes in stored and non-stored oocytes of good and bad quality.

Utility of fluorescence imitating brightfield imaging microscopy for the diagnosis of feline chronic enteropathy.

Fluorescence imitating brightfield imaging (FIBI) is a novel microscopy method that allows for real-time, nondestructive, slide-free tissue imaging of fresh, formalin-fixed, or paraffin-embedded tissue. The nondestructive nature of the technology permits tissue preservation for downstream analyses. The objective of this observational study was to assess the utility of FIBI compared with conventional hematoxylin and eosin (H&E)-stained histology slides in feline gastrointestinal histopathology. Formalin-fixed paraffin-embedded full-thickness small intestinal tissue specimens from 50 cases of feline chronic enteropathy were evaluated. The ability of FIBI to evaluate predetermined morphological features (epithelium, villi, crypts, lacteals, fibrosis, submucosa, and muscularis propria) and inflammatory cells was assessed on a 3-point scale (0 = FIBI cannot identify the feature; 1 = FIBI can identify the feature; 2 = FIBI can identify the feature with more certainty than H&E). H&E and FIBI images were also scored according to World Small Animal Veterinary Association (WSAVA) Gastrointestinal Standardization Group guidelines. FIBI identified morphological features with similar or, in some cases, higher confidence compared with H&E images. The identification of inflammatory cells was less consistent. FIBI and H&E images showed an overall poor agreement with regard to the assigned WSAVA scores. While FIBI showed an equal or better ability to identify morphological features in intestinal biopsies, its ability to identify inflammatory cells is currently inferior compared with H&E-based imaging. Future studies on the utility of FIBI as a diagnostic tool for noninflammatory histopathologic lesions are warranted.

Effect of Romanowsky-Stained Concentrated Preparations versus Direct Smears on Veterinary Students' Ability to Identify Bacterial Sepsis in Fluid Cytology Samples from Dogs, Cats, and Horses.

Veterinary students' accuracy, confidence, and time required to diagnose bacterial sepsis in fluid cytology samples was evaluated using two different slide preparation methods: direct smears and cytocentrifuged concentrated preparations. We hypothesized veterinary students would diagnose fluids as septic on concentrated preparations more accurately and quickly than on direct smears. Thirty third- and fourth-year students who had previously participated in a clinical pathology course completed a survey regarding general cytology experience and reviewed 40 randomized Romanowsky-stained slides via microscopy. Slides consisted of 10 septic and 10 non-septic samples with matched direct and concentrated slides, prepared from fluids from dogs, cats, and a horse. Participants' slide evaluation time, diagnosis, confidence, and slide photographs of areas considered septic were recorded. No difference in diagnostic accuracy between direct and concentrated samples was identified (area under the curve: 57% for both preparations, p = 0.77), although students agreed with pathologist-determined diagnoses more often when viewing concentrated samples (M = 63%, SD = 11% for concentrated; M = 56%, SD = 21% for direct, p = .012). A positive relationship existed between accuracy of diagnosis (R2 = .59) and senior status (p = .002), comfort interpreting cytology slides (p < .03), and if the student had taken the senior pathology rotation (p = .02). Only 38% (121/319) of participant photographs correctly identified sepsis. Under experimental conditions, concentrated preparations did not increase the accuracy of veterinary students' bacterial sepsis diagnosis; however, since accuracy did increase with cytology experience and comfort level, additional pre-clinical and clinical cytology training may benefit students before entering practice.

Specular microscopy of the corneal endothelial cells of bovines: an ex vivo study.

Background: The endothelium is the most posterior layer of the cornea and is essential for maintaining corneal transparency. Due to variations in corneal endothelial parameters among different species, knowledge of the normal parameters for each species is crucial. Aim: To evaluate the corneal endothelium of bovines using contact specular microscopy. Methods: Twenty eyeballs from 10 male Brangus (Bos taurus) aged 24 months were evaluated. Contact specular microscopy was performed on the central corneal area. The analyzed parameters were endothelial cell density (ECD) and endothelial cell morphology. Results: The ECD in the central area was 1,277 cells/mm2. Regarding the morphology, mainly cells with six (74.3%), five (14.7%) and seven sides (10%) were found. There were no significant differences in ECD and morphology between left and right eyes. Conclusion: Contact specular microscopy facilitated the analysis and measurement of corneal endothelial parameters in bovines. The data obtained will serve as a reference for the analysis of bovine corneal endothelium.

The importance of appropriate processing and direct microscopic examination for the timely diagnosis and management of invasive infections caused by filamentous fungi.

The gold standard for diagnosis of invasive fungal infections caused by filamentous fungi remains the visualization of fungal elements in fluids, and biopsy/tissue collected from a normally sterile body site. Parallel recovery of viable fungus from the sample subsequently permits antifungal susceptibility testing of the individual isolate. Central to both processes is the appropriate processing of tissue specimens to avoid damaging fungal elements and optimize viable organism recovery. Historically, mycologists have proposed that homogenization (grinding or bead-beating) of tissue should be avoided in cases of suspected fungal infection as it likely damages hyphae, instead preferring to chop tissue into small portions (dicing) for direct microscopic examination and culture. Here, we have compared the two processes directly on material from clinical patient cases of mucoromycosis and invasive aspergillosis. Representative portions of fresh biopsy samples were processed in parallel either by chopping (dicing) in the mycology reference laboratory or by bead-beating in the adjoining general microbiology laboratory. Aliquots of the samples were then cultured under identical conditions and subjected to direct microscopic examination. The results demonstrated that tissue homogenization significantly reduced (i) organism recovery rates in cases of both mucoromycosis and invasive aspergillosis and (ii) the number of fungal elements detectable upon direct microscopic examination. To our knowledge, this is the first study to directly compare these alternative processing methods and despite only employing a limited number of samples the data presented here, provide support for the perceived mycological wisdom that homogenization of tissue samples should be avoided when filamentous fungal infections are suspected.

The gold standard for diagnosis of fungal infections remains the visualization of fungal elements in samples from usually sterile sites. Here we show that certain methods employed for processing biopsy samples significantly impact the ability to detect and grow fungi from genuine cases of infection.

Comparison of the Anvajo Vet Fluidlab 1 urine sediment analyzer to manual microscopy and Idexx SediVue analysis for analysis of urine samples from cats and dogs.

The Vet Fluidlab 1 (Anvajo), a new urine sediment analyzer for use in veterinary medicine, uses holographic microscopy to detect urine sediment particles in uncentrifuged urine. We compared the performance of the Fluidlab to manual microscopy and Idexx SediVue analysis for the detection of RBC, WBC, epithelial cells (EC), struvite crystals (STR), all crystals (CRY), and casts (CST) in urine samples from cats and dogs. The performance of the Fluidlab for the detection of bacteria was compared to bacterial culture. We included 624 urine samples from feline (238; 38%) and canine (386; 62%) patients; 227 samples had been submitted for bacterial culturing. The sensitivity of the Fluidlab compared to manual microscopy was 92.1% for RBC, 90.1% for WBC, 87.5% for EC, 67.6% for STR, 53.9% for CRY, and 12.5% for CST. Specificity was >97% for STR and CST, 90.0% for CRY, 78.4% for WBC, 59.4% for EC, and 55.1% for RBC. Sensitivities and specificities of the Fluidlab for analytes compared to manual microscopy were found to be similar to those obtained by the Fluidlab compared to SediVue analysis. Miscellaneous materials (e.g., lipid droplets, sperm, cell detritus) seemed to be the main reason for the high false-positive rate in RBC and EC classification by the Fluidlab. Detection of bacteria by the Fluidlab compared to bacterial culture had a sensitivity of 89.8% and a specificity of 72.3%. The performance of the Fluidlab is acceptable for the detection of WBC and bacteria; sensitivity for the detection of CRY and CST, and specificity for the detection of RBC and EC, require improvement.

Accuracy of the IDEXX SediVue Dx analyzer for quantifying RBC and WBC indices in the urine sediments of cats and dogs compared with manual microscopic evaluations.

BACKGROUND: The IDEXX SediVue Dx (SediVue) is an automated, in-clinic urine sediment analyzer for veterinary patients. The bias between the results from manual microscopy and the SediVue is currently unknown. OBJECTIVES: To assess the diagnostic accuracy of the SediVue, we aimed to determine the bias between the SediVue (index test) and manual microscopy (reference standard) for the quantification of RBCs and WBCs in urine. METHODS: Urine remnant samples were collected from cats and dogs that contained RBCs (n = 462) and WBCs (n = 510). Retrospective analysis of results from urine sediment examinations using both manual microscopy (using a KOVA and DeciSlide system) and the SediVue (1.0.1.3) was performed. Bias was determined with Bland-Altman plots. SediVue-captured images from high-bias samples were reviewed, and biases were compared. RESULTS: The median bias for semi-quantitative RBC and WBC counts was determined for RBC and WBC counts. The cutoffs were RBC ≤ 5/HPF, 0.3; RBC 5.1-10/HPF, 10.1; RBC 10.1-20/HPF, 10.6; and RBC > 20/HPF, 28.93; WBC ≤ 5/HPF, 0.1; WBC 5.1-10/HPF, 2.2; WBC 10.1-20/HPF, 9.4; and WBC > 20/HPF, 26.6. High bias between the methods was identified in 98 samples (21.0%) with RBCs and 77 samples (15.7%) with WBCs. Reviewer-based enumeration of the SediVue-captured images decreased the percentage of samples with high bias to 17.3% for RBCs and to 11.4% for WBCs. CONCLUSIONS: Bias in the RBC and WBC counts between manual microscopy and the SediVue was unlikely to impact clinical interpretations in a majority of cases. Although reviewer enumeration of SediVue-captured images reduced observed bias, inherent differences between methodologies appeared to have a larger impact on the bias.

Elucidation of the neurological effects of clothianidin exposure at the no-observed-adverse-effect level (NOAEL) using two-photon microscopy in vivo imaging.

Neonicotinoid pesticides (NNs) cause behavioral abnormalities in mammals, raising concerns about their effects on neural circuit activity. We herein examined the neurological effects of the NN clothianidin (CLO) by in vivo Ca2+ imaging using two-photon microscopy. Mice were fed the no-observed-adverse-effect-level (NOAEL) dose of CLO for 2 weeks and their neuronal activity in the primary somatosensory cortex (S1) was observed weekly for 2 weeks. CLO exposure caused a sustained influx of Ca2+ in neurons in the S1 2/3 layers, indicating hyperactivation of neurons. In addition, microarray gene expression analysis suggested the induction of neuroinflammation and changes in synaptic activity. These results demonstrate that exposure to the NOAEL dose of CLO can overactivate neurons and disrupt neuronal homeostasis.

Biochemical changes in the cytoplasm of bovine oocytes during the in vitro maturation process: a Raman microscopy study.

The sequence and chronology of the main biochemical changes occurring in the cytoplasm of bovine oocytes during the in vitro maturation process were tracked by Raman microscopy applied to cells previously subjected to enzymatic digestion of the zona pellucida. Specific spectral markers for proteins, lipids and carbohydrates were used to evaluate the developmental status of the ooplasm at four different times. Spectral changes revealed that lipid accumulation was dominant during the first six hours of culture while protein content reached the average levels characteristic of mature oocytes within the last four hours of the maturation process. A time-dependent decrease in carbohydrates was also observed. Finally, the carbohydrate-to-protein (P1037/P1002) ratio proved to be sensitive enough to determine the cytoplasmic maturation state of bovine oocytes and promises to be useful in future research aimed at optimizing culture conditions through the promotion of protein accumulation in the ooplasm.