The possible transport and exclusion mode of lipofuscin in rat myocardium under electron microscopy.

Lipofuscin accumulation has been observed in human coronary arteries but whether or not myocardial tissue can release lipofuscin generated within cardiomyocytes must be clarified, as this may provide indicators for future anti-ageing research. The hearts of Sprague Dawley rats, aged 6-24 months, were embedded in resin and ultrathin sections cut for electron microscopy. Lipofuscin granules were abundant in cardiomyocytes. Cardiomyocytes were seen to release lipofuscin granules into the myocardial interstitium as cytoplasmic fragments with irregular protrusions on the sarcolemma surface. The cytoplasmic fragments entering the stroma fused directly with the endothelial cells of adjacent capillaries, delivering lipofuscin to the vessel wall. These fragments were also seen to be engulfed by stromal macrophages or fused with fibroblasts, which then combined with capillary endothelial cells to deliver lipofuscin to the vessel wall. Some cytoplasmic fragments disaggregated and formed membrane-like waste, which travelled to the vessel wall from the myocardial stroma as soluble fine particles via diffusion or pinocytosis of capillary endothelial cells. Lipofuscin entered the vascular wall and endothelial cells, forming large and small protrusions or folds that transported the lipofuscin to the vascular lumen and bloodstream.

Cytodifferentiation of pinealocytes (I and II) and astrocyte types of mature male sheep epiphysis cerebri with special emphasis on the presence of neuronal and pigmented-like cells.

Background: The epiphysis cerebri (pineal gland) is a small-sized, photo neuroendocrine organ in the brain of most vertebrates. Their effect is through secretion of melatonin, a serotonin-derived hormone which is stimulated by darkness and inhibited by light and modulates the circadian rhythm; light and dark cycle like a biological clock, sleep patterns (sleep-wake cycle), and sexual development. Aim: This study aimed to identify and differentiate the different cell types filling the pineal gland parenchyma of mature male sheep. Methods: Pineal glands were collected and sliced parasagitally then processed histologically for light and electron microscopic examinations. Results: Two main cell types; pinealocytes and astrocytes were recognized within the gland parenchyma. Pinealocytes were the chief parenchymatus cells that occupied the largest volume of the gland and were classified according to the nuclear pictures (activity status) into two subtypes; pinealocytes I (pale subtype, active) and II (dark subtype, inactive). Astrocyte neuroglial cells had cytoplasmic processes which form a huge supportive framework between the pinealocytes and clarified two types; type I were elongated cells with elongated snake shaped nucleus and type II were smaller in size, with oval nuclei. Another marginal cell type was identified as a neuron-like cell which appeared larger in size than others and distributed sporadically, has eccentric oval nucleus with prominent nucleoli and single, long cytoplasmic process that branched at its terminal forming T-shaped process looks like pseudo unipolar neuron. Moreover, aggregations of pigment granules were markedly observed in the intercellular spaces and also near the blood capillaries. With transmission electron microscope (TEM) a special characteristic feature of pinealocytes; synaptic ribbons were recognized that appeared as bands of electron-dense material with several synaptic spherules; vesicles adjacent to its surface helping in the multivesicular release. Conclusion: The gland parenchyma revealed two main cell types; pinealocytes and astrocytes. Each one was subdivided into two subtypes; I and II. The first one was classified according to their nuclear pictures (activity status) and the second one was according to their shape, size, and cytoplasmic processes. Other cell types were also identified as neuronal and pigmented-like cells in the pineal matrix.

Macroscopic and microscopic comparison of pecten oculi in different avian species.

The current study aims to present differences between the pecten oculi of different avian species through morphologic, macroscopic, light, and electron microscopic examinations. The study is a comprehensive research on seven avian species (sparrowhawk, hawk, magpie, swan, heron, pheasant, duck). The right eyes of the animals utilized in the study were removed for light microscopic examination, whereas their left eyes were removed for electron microscopic and macroscopic examinations. Morphometric analyses, as well as stereo and light microscopic measurements, were carried out on the pecten oculi of the animals. Given all these data, it was determined that the height of the pecten oculi did not differ among the species in the study; however, the pecten oculi were larger in birds with the highest value compared to the other species in the macroscopic measurements. Also, the pecten oculi vessels were larger, and the number of melanocytes was higher in keen eyesight, raptor, and migratory birds with large bulbus oculi. All these data suggest that the pecten oculi not only supplies nutrient to the retina but also contributes to sharp vision during migration and hunting, UV absorption from sunlight, as well as preservation of intraocular equilibrium.

A lower motor neuron disease in takahe (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease.

AIMS: To investigate the pathogenesis of a disease in takahe (Porphyrio hochstetteri) with intracytoplasmic inclusion bodies in lower motor neurons. METHODS: Four birds aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the spinal cord from the bird with spinal dysfunction were examined by electron microscopy. RESULTS: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D, so were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method.A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. CONCLUSIONS: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Histological, immunohistochemical and ultrastructural features of polyglucosan bodies in uterine smooth muscle of pet rabbits (Oryctolaguscuniculus).

We document the frequency and morphological and immunohistochemical features of inclusion bodies in uterine smooth muscle cells in 56 (76%) of 74 investigated pet rabbits (Oryctolagus cuniculus). Inclusion bodies began to appear at the age of 2 years and their frequency increased with age (P = 0.047, r = 0.33). They ranged from 5 to 20 µm in diameter, were slightly basophilic to amphophilic with well-delimited oval bodies in haematoxylin and eosin-stained tissue sections and formed in the cytoplasm of the uterine smooth muscle cells with displacement of the cell nuclei. The inclusion bodies were positive with periodic acid-Schiff, Best's carmine, Lugol's iodine and Grocott's methenamine silver methods. They were immunoreactive to a monoclonal antibody raised against human polyglucosan and negative with monoclonal antibodies for several intermediate filament proteins. Electron microscopy revealed that they were non-membranous structures composed of electron-dense amorphous material. The morphological, histochemical, immunohistochemical and ultrastructural features of the inclusion bodies in the rabbi uteri were similar to those of human polyglucosan bodies (PGBs). PGBs appear to occur at a high frequency in the uterus of rabbits, which are known to be susceptible to uterine diseases.

Canine intracranial meningioma with rosette-like collagen deposits.

A 13-year-old female Miniature Dachshund showed seizures and an intracranial mass was seen in the left temporal lobe. Three months after first surgical resection, a recurrence lesion was recognized. Histopathologically, proliferation of spindle cells with diffuse rosette-like eosinophilic deposits, which stained blue with Masson's trichrome stain, was observed. In electron microscopy, the rosette-like deposits were consisted of bundles of minute filaments which were assumed to be collagenous fibrils. Immunohistochemically, the neoplastic cells were positive for vimentin and cytokeratin, and negative for E-cadherin, S100, glial fibrillary acidic protein (GFAP), oligodendrocyte transcription factor (Olig2), and CD204. The rosette-like collagen deposits were positive for type 4 collagen and negative for type 1 collagen. In this report, we describe histopathological features of a canine meningioma with the rosette-like collagen deposits.

A systematic analysis of ultrastructural lesions in the Plasmodium coatneyi splenectomized rhesus macaque model of severe malaria.

Plasmodium falciparum remains one of the world's deadliest diseases and with ongoing concerns of evolving drug resistance, there is a need for continued refinement of the Plasmodium coatneyi infection model in macaques to study severe malaria. As such, the systemic ultrastructural lesions associated with P. coatneyi infection in splenectomized rhesus macaques was evaluated in 6 animals. Autopsy samples from multiple areas of the central nervous system (CNS), kidneys, heart, liver, and lungs of all 6 animals were processed for electron microscopy. A systematic analysis of the ultrastructural changes associated with the plasmodium was undertaken by multiple pathologists to ensure consensus. All tissues exhibited marked sequestration of infected red blood cells comprised either of cytoadherence to endothelium or rosette formation, associated with variable degrees of host cell damage in a range of tissues that in severe cases resulted in necrosis. This is the first complete systemic evaluation of ultrastructural tissue lesions in P. coatneyi-infected rhesus macaques, and the findings have important implications evaluating of the use of this model for the study of severe malaria caused by P. falciparum in humans.

Morphological, histological and ultra-structural studies on the ultimobranchial body of goat (Capra hircus).

Ultimobranchial body (UBB) remnant was considered as an enigma till the last few years, then it was recognized as a necessary organ where it is the origin of the parafollicular cells. The samples were fixed and processed for the histological and electron microscopic examination. Macroscopically, the UBB remnant appeared as a white mass at the end of the cranial one-third of the thyroid lobe. It was composed of solid cell nest, cluster of cells and small thyroid follicles. Transmission electron microscope showed some round cells containing euchromatic nuclei, numerous parafollicular cells with darkly stained granules and paler ones. It also showed some mast cells with heterochromatic nuclei and large darkly stained granules. The parafollicular cells were distributed throughout the thyroid gland but concentrated within the UBB remnant. To our knowledge, our findings represent very unique histological manifestations specially the ultra-structural ones which revealed an original finding about the new clear type of cells suggested to be a UBB remnant and ensure those of the light microscope.

Cone distribution and visual resolution of the yellow-legged gull, Larus michahellis (Naumann, 1840).

The morphological characteristics of the yellow-legged gull's photoreceptors and cone distribution were studied using light and electron microscopy. In wholemount fresh retinas, five different coloured oil droplets located in the cone inner segments could be seen and characterized by colour, diameter and stratification. The photoreceptors were classified by comparing the fresh and fixed vertical sections under a light and electron microscope. Rods were easily distinguished from cones based on the outer segment morphology and the absence of oil droplets in their inner segments. Four types of single cones were associated with red, yellow, colourless and transparent oil droplets. Unequal double cones comprised a long principal member with a green oil droplet and an accessory short member containing a green microdroplet which was highly electron-dense under electron microscopy. The different types of oil droplets were counted from microphotographs of fresh retinal samples in 20 regions. The density, percentage and diameter of the oil droplets were determined. The results showed that central regions had the highest oil droplet density which decreased towards the retinal periphery in all quadrants. Moreover, the oil droplet density was higher in the dorsotemporal quadrant than in other retinal regions. The average density of the red oil droplets was highest in the central areas, whereas colourless oil droplets had the highest density throughout the retina. In contrast, transparent oil droplets had the lowest density across all the regions of the retina. Finally, the retinal resolution was 52.61 cycles/degree. It was calculated using the posterior nodal distance and the oil droplet diameter. The work concludes by discussing the significance of the relative proportion of different cone types across the retina.

Light and transmission electron microscopic evaluation of the lamina epithelialis mucosae in the ileum of pre- and post-hatch broiler chicken.

Morphological development of the lamina epithelialis mucosae (LEM) of the ileum of broiler chicken was evaluated using light and transmission electron microscopic techniques. Ileal sections were collected on embryonic days (ED) 14, 17 and 19, as well as post-hatch days (PD) 1, 3, 5, 7, 14, 21, 28, 35, 42 and 56. The result showed that the ileal LEM, which were poorly defined at embryonic days 14 and 17, consisted of enterocytes and some atypically shaped goblet cells. Apico-lateral tight junctions and irregularly shaped microvilli were associated with the enterocytes at ED 14 and 17. The enterocyte microvilli were more uniform in shape and distribution at ED 19. The embryonic goblet cells were varied in shape and possessed basally displaced, star-shaped nuclei and small apical cytoplasmic vacuoles. During the post-hatch ages, the LEM was a typical epithelium with a single layer of columnar-shaped enterocytes that became highly elongated at post-hatch day (PD) 7. The goblet cells were characteristically 'wine-glass' shaped. Follicle-associated epithelium (FAE) showing numerous lymphocytes among the enterocytes occurred in the post-hatch LEM. The intra-epithelial lymphocytes (IEL) were first encountered at PD 1, but increased several folds within the first two weeks post-hatch. Entero-endocrine cells were observed in the epithelium from PD 21. Finally, from this study, it is obvious that enterocytes and small atypically shaped goblet cells occur in the ileal LEM during the pre-hatch period, but these cells assume adult morphological characteristics after hatch. Thus, the cells of the ileal LEM play strategic absorptive, secretory and protective roles in the gut.

Electron microscopy reveals viral-like particles and mitochondrial degradation in scombrid puffy snout syndrome.

Aquaculture is an increasingly important food resource, but its sustainability is often limited by disease. In Scombridae fishes, puffy snout syndrome (PSS) is a debilitating condition where tumor-like collagenous growths form around the eyes, nares, and mandibles which impair vision and feeding and frequently lead to mortality. While PSS is considered an infectious or metabolic disease, no disease agents or promoters have been identified. Here, we used electron microscopy (EM) to describe the cellular pathology and search for etiological agents of PSS in Pacific mackerel Scomber japonicus, the first use of this approach for PSS. We examined aquaculture specimens across a range of apparent PSS severity, comparing the results to both wild and aquaculture asymptomatic mackerel. EM imagery consistently revealed viral-like particles in PSS samples, as well as the uniform absence of bacteria, protists, fungi, and other multicellular parasites. In addition to viral-like particles, symptomatic fish had a higher mean percentage of swollen and disintegrating mitochondria than both asymptomatic aquaculture and wild mackerel. This suggests that degraded mitochondria may be related to PSS and could be important to further understanding the origin, promoters, and prevention of PSS. This study serves as a first step in identifying the etiological agents of PSS.

Histomorphological, ultrastructural and morphometrical age-related changes of fundic region of New Zealand rabbits (Oryctolagus cuniculus).

This study investigated the histomorphological, ultrastructural and morphometrical postnatal developmental changes in the rabbit fundic region, especially during changing of the feeding intake. Seventy-two New Zealand rabbits (V-Line breed) at the ages of 1, 7, 15, 23, 30 and 60 days were obtained for light and electron microscopy and morphometric studies of the fundic region. The newborn rabbit's fundic wall was thin and organized into mucosa, submucosa, musclosa and serosa, with a significant increase in thickness with ageing. The fundic glands were few at the first week of life, then increased in length and diameter compared to the preceding age with prominent zonation at 23 days. The gastric pits appeared wide and deep at the first week of life then became typically narrow and shallow at the third week. The mucous cells were the main cell types lining the fundic glands in the first week of life. These cells showed remoulding with a marked increase in Periodic Acid-Schiff reactivity with age. Parietal cells were differentiated earlier (on the first day of life) than the chief cells and distributed at the neck and basal zones. Chief cells differentiated at 15 days old at the base of the glands, followed by an increase in the number and activity. Few active enteroendocrine cells were first seen at 15 days old and then widely distributed throughout the glands. Conclusion: Pronounced histomorphological changes in the fundic mucosal layer, especially the surface and glandular epithelium, correlate with the postnatal rabbit-feeding intake changes.

Ultrastructural identification of developing proximal tubules based on three-dimensional reconstruction.

BACKGROUND: The cellular mechanisms involved in the development of proximal tubules are not only associated with morphogenesis in fetal life, but also with restoration of damaged tubules in adulthood. Knowledge about morphological features of cell differentiation and proliferation along the developing tubule is insufficient, which hinders identification of the cellular origin. OBJECTIVES: This study aimed to investigate ultrastructures of the proximal tubule at different stages of nephrogenesis. METHODS: Electron microscopy was used and guided by computer-assisted tubular tracing to identify the cellular structures. RESULTS: Renal vesicles and S-shaped bodies revealed more proliferative features, such as densely-packed fusiform-shaped cells with numerous protein-producing organelles than membrane specializations typical for mature tubules. At the capillary-loop stage the proximal tubules demonstrated all characteristics of the mature tubules, but not as developed, including shorter but densely packed microvilli, fewer lateral processes with cell-cell contacts, lower basal membrane infoldings, and lower mitochondrial volume density. However, they exhibited an elaborated endocytic system above the nucleus, indicating a membrane transport is being established. Abundant free- and endoplasmic reticulum-adhered ribosomes and Golgi complexes reflected active protein synthesis for cell growth and proliferation. Interestingly, electron dense cells were occasionally intermixed with electron lucent cells characterized by various organelles in less cytosol and a larger nucleus with abundant euchromatin, which is a feature of active proliferation. CONCLUSIONS: These ultrastructures indicate that the morphogenesis of the developing proximal tubule corresponds to the gradually established physiological activities. The two different cellular electron densities may suggest distinctive differentiation of the cells along the tubule.

Electron microscopy and phylogenetic analysis of Bovine papillomavirus infection in cattle from four Egyptian governorates.

Cutaneous warts are the common clinical feature of infection with Bovine papillomavirus (BPV), and it is commonly known as bovine papillomatosis. It causes significant economic losses, especially in the dairy sector. The aim of this study was surveillance of the circulating strains of BPV in four Egyptian governorates and characterization by electron microscopy. Warts skin lesions and whole blood from seventy-eight native breed cattle were obtained. Molecular detection using two different sets of primers, phylogenetic analysis, and electron microscopy were carried out. The obtained results showed that using FAP59/FAP64 primer set is more sensitive than the MY09/My11 primer set in the detection of the papilloma L1 gene either in the blood or in the skin lesion. Sequence analysis of the partially amplified L1 gene revealed 4 different strains belonging to Deltapapillomavirus 4. Only Alfayoum_Deltapapillomavirus_2018 (accession no: MW018705) was found to be closely related to the strain previously isolated in different Egyptian governorates in 2017, and 2 strains were closely related to an isolate of equine origin. Electron microscopy examination of the skin lesions showed the presence of negatively stained rounded, non-enveloped virus particles with a size of 60 nm in diameter. In conclusion, continuous surveillance and characterization of the circulating strains using multiple sets of primers are important. Efficient biosecurity measures must be applied to decrease transmission of papillomavirus between the different animal species, especially in the mixed management system.

Development and ultrastructure of bovine matured oocytes vitrified using electron microscopy grids.

The aim of this study was to establish a methodology of cryopreservation of cattle oocytes and the quality assessment of oocytes and subsequent embryos produced in vitro under our laboratory conditions. Previously in vitro matured (IVM) oocytes were vitrified in minimum volume by ultra-rapid cooling technique. The oocytes were put into the equilibration solution (3% ethylene glycol in M199-HEPES + 10% foetal bovine serum) for 12 min, transferred to vitrification solution (30% ethylene glycol + 1 M sucrose in M199-HEPES + 10% foetal bovine serum) at room temperature for 25 s, then placed onto nickel electron microscopy grids and plunged into liquid nitrogen. After warming 75% of the oocytes were assessed as viable. Part of viable oocytes was taken for electron microscopy, the remaining oocytes were fertilized in vitro, and the presumptive zygotes were cultured until the blastocyst stage. Embryo cleavage and blastocyst rates in vitrified group after warming were 64.98% and 17.3%, resp. versus 70.72% and 25.54% in the control group (P < 0.05). No significant differences were found in the blastocyst total cell number, TUNEL and dead cell indexes between both groups. Ultrastructure of vitrified oocytes showed damages in smooth endoplasmic reticulum (SER) vesicles and lipid droplets as well as irregular arrangement of solitary cortical granules. Several mitochondria were damaged and the microtubules around the chromosomes were less occurred compared to the control group. However, the extent of injuries was lower than reported by other authors studying the ultrastructure of vitrified bovine oocytes, what is also supported by the better development of our oocytes after IVF. In conclusion, the designed oocyte vitrification technique ensures obtaining the blastocysts of the quality comparable to the fresh oocytes.

Four Cases of the Melanotic Variant of Malignant Nerve Sheath Tumour: a Rare, Aggressive Neoplasm in Young Dogs with a Predilection for the Spinal Cord.

Four cases of a rare melanotic variant of malignant nerve sheath tumour (MNST) in dogs are described. All four cases presented with neurological clinical signs due to multicentric, intradural, intra- and extraparenchymal neoplasms that surrounded the spinal and cranial nerves and infiltrated the adjacent spinal cord and brain. The dogs were young (3 months to 3 years of age), all were female and four different breeds were represented. Characteristic histological features were interweaving fascicles of spindle-shaped cells, sometimes with an architecture reminiscent of Antoni A and B patterns. Some spindle cells showed prominent cytoplasmic melanin pigmentation and such cells were positive by Masson-Fontana stain. Immunohistochemistry performed in three cases was positive for S100 and vimentin, strongly positive for melan A in the melanized cells and negative for glial fibrillary acidic protein and periaxin. Non-melanized cells did not express melan A. Transmission electron microscopy findings in one case were consistent with a peripheral nerve sheath tumour and demonstrated cytoplasmic pre-melanosomes and melanosomes. Melanotic variants of MNSTs are rare in animals with only a solitary report of two previous canine cases in the literature.

Exogenous Jaagsiekte Sheep Retrovirus type 2 (exJSRV2) related to ovine pulmonary adenocarcinoma (OPA) in Romania: prevalence, anatomical forms, pathological description, immunophenotyping and virus identification.

BACKGROUND: Ovine pulmonary adenocarcinoma (OPA) is a neoplastic disease caused by exogenous Jaagsiekte Sheep Retrovirus (exJSRV). The prevalence of JSRV-related OPA in Eastern European countries, including Romania is unknown. We aimed to investigate: the prevalence and morphological features of OPA (classical and atypical forms) in the Transylvania region (Romania), the immunophenotype of the pulmonary tumors and their relationships with exJSRV infection. A total of 2693 adult ewes slaughtered between 2017 and 2019 in two private slaughterhouses from Transylvania region (Romania) was evaluated. Lung tumors were subsequently assessed by cytology, histology, immunocytochemistry, immunohistochemistry, electron microscopy and DNA testing. RESULTS: Out of 2693 examined sheep, 34 had OPA (1.26% prevalence). The diaphragmatic lobes were the most affected. Grossly, the classical OPA was identified in 88.24% of investigated cases and the atypical OPA in 11.76% that included solitary myxomatous nodules. Histopathology results confirmed the presence of OPA in all suspected cases, which were classified into acinar and papillary types. Myxoid growths (MGs) were diagnosed in 6 classical OPA cases and in 2 cases of atypical form. Lung adenocarcinoma was positive for MCK and TTF-1, and MGs showed immunoreaction for Vimentin, Desmin and SMA; Ki67 expression of classical OPA was higher than atypical OPA and MGs. JSRV-MA was identified by IHC (94.11%) in both epithelial and mesenchymal cells of OPA. Immunocytochemistry and electron microscopy also confirmed the JSRV within the neoplastic cells. ExJSRV was identified by PCR in 97.05% of analyzed samples. Phylogenetic analysis revealed the presence of the exJSRV type 2 (MT809678.1) in Romanian sheep affected by lung cancer and showed a high similarity with the UK strain (AF105220.1). CONCLUSIONS: In this study, we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPA in sheep. Additionally, we described the first report of atypical OPA in Romania, and to the best of our knowledge, in Eastern Europe. Finally, we showed that MGs have a myofibroblastic origin.

Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.

Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.

Cytologic, histologic, microbiologic, and electron microscopic characterization of a canine Prototheca wickerhamii infection.

An adult dog was presented for chronic cough and a recent development of ulcerated, erythematous nares with nasal discharge. Cytology of enlarged peripheral lymph nodes revealed many intracellular and extracellular organisms. These round or rarely oval organisms measured approximately 5-9 µm in diameter and frequently contained several globular structures, ranging from deeply basophilic to magenta. A thin, clear halo was present. Smaller 1-2 µm, magenta forms were also observed. Fungal culture yielded small, wet, raised, irregularly shaped, white to pale tan colonies. Microbiologic staining of cultured material revealed features suggestive of algae. Histopathology of the lymph nodes revealed marked granulomatous inflammation with intralesional algal organisms suggestive of Prototheca. Electron microscopic findings were also consistent with protothecosis. Polymerase chain reaction, followed by direct DNA sequencing, identified the organism as Prototheca wickerhamii. A brief literature review discussing protothecosis in veterinary medicine is included.

The shell gland in laying and natural moulting commercial egg-type chickens: A histomorphological and ultrastructural study.

The purpose of the present study was to determine the histological and ultrastructural changes in the luminal epithelium of the shell gland associated with natural moulting. Samples of the shell gland from laying (32 weeks old) and moulting (75 weeks old) hens were studied using histological, histochemical and electron microscopic techniques. In addition, TUNEL was used to demonstrate the distribution of apoptotic cells in the luminal epithelium of the shell gland. Autophagy, characterized by the presence of autophagosomes and autolysosomes, was evident in the early stages of degeneration in non-ciliated, ciliated and mitochondrial cells. The intermediate and advanced stages of regression in non-ciliated as well as mitochondrial cells occurred via apoptosis, while both apoptotic and necrotic ciliated cells were observed during the later stages of degeneration. The results of the present study suggest that a synergy of autophagy, apoptosis and necrosis is involved in the involution of the shell gland during natural moulting.