Eosinophilic crystalline pneumonia as a major cause of death in 129S4/SvJae mice

Eosinophilic crystalline pneumonia is an idiopathic disease that occurs in many strains and stocks of mice, more commonly in strains on a C57BL/6 background. The disease occurs sporadically in most strains of mice and varies from mild and subclinical to severe and fulminating, sometimes resulting in respiratory distress and death. In this study, 94 aged male and female 129S4/SvJae mice were evaluated for eosinophilic crystalline pneumonia lesions. There was an 87% incidence, with females overrepresented. Histologically, there were multifocal to coalescing inflammatory infiltrates composed of numerous large eosinophilic macrophages and multinucleate cells admixed with eosinophils, neutrophils, lymphocytes, and plasma cells within alveolar and bronchiolar spaces, associated with refractile, brightly eosinophilic, angular crystals. Alveolar macrophages and multinucleate cells contained fine needlelike to rectangular intracytoplasmic crystalline material. Similar crystals were often free within alveoli and conducting airways, often associated with mucous metaplasia of bronchiolar epithelium. This disease may occur spontaneously or in concert with other pulmonary lesions, such as pulmonary adenomas, lymphoproliferative disease, allergic pulmonary disease, and parasitic or fungal infections. The characteristic crystals morphologically resemble Charcot-Leyden crystals, which represent eosinophil breakdown products in humans with eosinophil-related disease. However, crystals in eosinophilic crystalline pneumonia are composed predominantly of Ym1 protein, a chitinase-like protein associated with neutrophil granule products and secreted by activated macrophages. The function of Ym1 protein is not fully understood but is believed to be involved in host immune defense, eosinophil recruitment, and cell-cell and cell-matrix interactions consistent with tissue repair. The mechanism of induction of eosinophilic crystalline pneumonia with Ym1 crystal formation is unknown.     

Ultrastructure of Developing and Mature Caprine Leukocytes

Transmission electron microscopy demonstrated that, based on the structural uniqueness of the granules, caprine granulocytes are easily distinguishable from each other from the promyelocyte stage onwards. The neutrophils had the smallest granules which varied in size and were, in mature cells, either spherical to dumb-bell in shape; in mature cells the granule contents were compact and finely granular. The primary granules were smaller than the secondary granules. The eosinophil granules were large and typically had internal crystalloid structures; a second group of spherical granules with moderately coarse non-crystalloid sub-structure was present in smaller numbers in promyelocytes and myelocytes only. The basophil granules were also large, lackes crystalloids but showed variation in coarseness of granule substance, ranging from finely granular to markedly coarse. Mature granulocytes lackes Golgi apparatus and rough endoplasmic reticulum and ribosomes which were present in promyelocytes, myelocytes, metamyelocytes and bands. The monocytes had moderate numbers of spherical granules, rough endoplasmic reticulum, mitochondria and ribosomes, as well as prominent Golgi apparatus, and the cytoplasm had many small vacuoles.     

Blood and bone marrow findings in two pups with mucopolysaccharidosis type VII

Routine blood smear findings in two of four 11‐day‐old mixed‐breed dog littermates were suggestive of a lysosomal storage disease (LSD) that was documented to be mucopolysaccharidosis type VII (MPS VII) by molecular testing. In this condition, a functional β‐glucuronidase deficiency results in the accumulation of glycosaminoglycans (GAGs) in cells and tissues where β‐glucuronidase is important in GAG degradation. Most neutrophils and a moderate number of lymphocytes within the blood had atypical cytoplasmic magenta inclusions. The bone marrow assessment from one of the two affected pups at 24 days of age revealed similar magenta granulation in myeloid precursor cells that was most prominent in promyelocytes and myelocytes. Moreover, atypical magenta material was present within vacuoles as well as extracellularly in some osteoblasts and macrophages. Histologic bone marrow sections revealed prominent vacuolation of osteoblasts, and some osteoclasts appeared separated from the bone by layers of osteoblasts or hematopoietic cells. At 2 months of age, the second affected dog showed moderate growth retardation and had similar but more prominent hematologic findings that extended to monocytes, eosinophils, and eosinophil precursors. It had an increased number of bone marrow macrophages with many vacuoles that could be seen cytologically to contain magenta material, and there was mild nonselective phagocytosis of hemic cells. Of the hematologic cells, inclusions were most prominent in promyelocytes, myelocytes, and macrophages, cells with relatively high β‐glucuronidase activity, and GAG exposure within lysosomes or lysosome‐like primary granules of granulocyte precursors.     

The possible fate of crystal harbouring larvae of Haemonchus contortus.

An examination of the occurrence of crystalline inclusions in the intestinal cells of fourth and fifth stage Haemonchus contortus revealed that a high percentage of inhibited early fourth stage larvae (L4) and developing L4 and a lower percentage of early fifth stage larvae (L5) harboured these crystals. Before the fourth moult crystals were falling into pieces, indicating that worms harbouring crystals can develop further. The presence of crystals may be associated with a retardation of development.     

Amylopectinosis in fetal and neonatal Quarter Horses

Three Quarter Horses, a stillborn filly (horse No. 1), a female fetus aborted at approximately 6 months of gestation (horse No. 2), and a 1-month-old colt that had been weak at birth (horse No. 3), had myopathy characterized histologically by large spherical or ovoid inclusions in skeletal and cardiac myofibers. Smaller inclusions were also found in brain and spinal cord and in some cells of all other tissues examined. These inclusions were basophilic, red-purple after staining with periodic acid-Schiff (both before and after digestion with diastase), and moderately dark blue after staining with toluidine blue. The inclusions did not react when stained with Congo red. Staining with iodine ranged from pale blue to black. Their ultrastructural appearance varied from amorphous to somewhat filamentous. On the basis of staining characteristics and diastase resistance, we concluded that these inclusions contained amylopectin. A distinctly different kind of inclusion material was also present in skeletal muscle and tongue of horse Nos. 1 and 3. These inclusions were crystalline with a sharply defined ultrastructural periodicity. The crystals were eosinophilic and very dark blue when stained with toluidine blue but did not stain with iodine. Crystals sometimes occurred freely within the myofibers but more often were encased by deposits of amylopectin. This combination of histologic and ultrastructural features characterizes a previously unreported storage disease in fetal and neonatal Quarter Horses, with findings similar to those of glycogen storage disease type IV. We speculate that a severe inherited loss of glycogen brancher enzyme activity may be responsible for these findings. The relation of amylopectinosis to the death of the foals is unknown.     

Characterization of the Blood Cells of Australian Crocodiles (Crocodylus porosus [SCHNEIDER] AND C. johnstoni [KREFFT])

Circulating blood of eight crocodiles (four Crocodylus porosus [SCHNEIDER], four Crocodylus johnstoni [KREFFT]) was examined by light microscopy, cytochemistry and transmission electron microscopy. The results were correlated and on that basis erythrocytes, thrombocytes, lymphocytes, monocytes and three main types of granulocytes were characterized. Erythrocytes, thrombocytes, lymphocytes and monocytes were similar in appearance to those described for other reptiles. In addition, lymphocytes and monocytes had ultrastructural details similar to those described for the cells in man. The three main granulocytes were designated types I, II and III. Type III had characteristics which were similar to those described for the basophil. Types I and II were eosinophilic on blood films stained with Giemsa. On ultrastructural detail, and by comparison with ultrastructural reports for the granulocytes of the fowl and lizard (Agamia stellio), type I appeared to be similar to the heterophil but type II could not be characterized definitely. However, the crystalline array present in granules of type II has been recorded for eosinophil granules in man.     

Characterization of an erythrocytic virus in the family Iridoviridae from a peninsula ribbon snake (Thamnophis sauritus sackenii)

A wild peninsula ribbon snake (Thamnophis sauritus sackenii) in Florida was found to have hypochromic erythrocytes containing two different types of inclusions: purple granular inclusions, and pale orange or pink crystalloid inclusions that were round, oval, rectangular, or hexagonal in shape. Transmission electron microscopy revealed hexagonal or pleomorphic, homogenous inclusions and enveloped particles morphologically consistent with a member of the Iridoviridae. Histopathology of the animal revealed necrotizing hepatitis consistent with sepsis. Consensus PCR was used to amplify a 628-bp region of iridoviral DNA-dependent DNA polymerase. Bayesian and maximum likelihood phylogenetic analysis found that this virus was distinct from other known iridoviral genera and species, and may represent a novel genus and species.     

The ultrastructural morphology of the camel eosinophil

The ultrastructural morphology of the eosinophil was studied in specimens of peripheral blood from normal adult camels and those with eosinophilia. Specific granules were extremely polymorphic. The specific granules exhibited the basic structure of an electron-dense crystalloid core surrounded by a lighter, homogeneous matrix. The crystalloid cores were extremely variable in size and shape, often were segmented and demonstrated a variety of lamellated patterns that were transverse, longitudinal or concentric to the long axis of the core. It was not uncommon to observe multiple crystalloid cores in a single granule. In addition to large specific granules, microgranules and specific microgranules were observed. The extreme polymorphism of the specific granules and variety of lamellated patterns differentiate camel eosinophils from those of other species.     

Crystalline inclusions in erythrocytes parasitized with Babesia equi following treatment of ponies with imidocarb

Four splenectomized Welsh ponies were infected with Babesia equi. Two ponies were treated with imidocarb dipropionate, and two were not treated. By light microscopic examination, 1% to 2% of the parasitized erythrocytes of treated ponies contained crystalline inclusions. The crystals were rectangular, diamond, or burr shaped. They occupied most of the erythrocytic cytoplasm, and, as a result, the remainder of the pale staining cytoplasm was inconspicuous in Wright-Giemsa-stained blood smears. The size and shape of intraerythrocytic inclusions varied when examined by electron microscopy, but in most instances they were either adhered to or were located close to the parasite. The sides of crystals were either smooth or serrated, and corners were either sharp or notched. Fractures or faults were common in large crystals. Parasitized erythrocytes of nontreated ponies and nonparasitized erythrocytes of treated ponies did not contain crystals. Four hemoglobulin types were identified in five noninfected, nontreated Welsh ponies from the same herd.     

The effects of Strongylus vulgaris parasitism on eosinophil distribution and accumulation in equine large intestinal mucosa

REASONS FOR PERFORMING STUDY: Eosinophilic granulocytes have been associated with parasite or immune-mediated diseases, but their functions in other disease processes remain unclear. Cause and timing of eosinophil migration into the equine gastrointestinal mucosa are also unknown. OBJECTIVE: To determine the effects of intestinal parasitism on eosinophils in equine large intestinal mucosa. METHODS: Large intestinal mucosal samples were collected from horses and ponies (n = 16) from the general veterinary hospital population, ponies (n = 3) raised in a parasite-free environment, ponies experimentally infected with 500 infective Strongylus vulgaris larvae and treated with a proprietary anthelmintic drug (n = 14), and a similar group of ponies (n = 7) that received no anthelmintic treatment. Total eosinophil counts and eosinophil distribution in the mucosa were determined by histological examination. A mixed model analysis was performed and appropriate Bonferroni adjusted P values used for each family of comparisons. P<0.05 was considered significant. RESULTS: There was no difference in large intestinal mucosal eosinophil counts and eosinophil distribution between ponies infected with S. vulgaris and those raised in a parasite-free environment. Experimental infection with S. vulgaris, with or without subsequent anthelmintic treatment, did not change eosinophil counts, and counts were similar to those for horses from the general population. CONCLUSIONS: Migration of eosinophils to the equine large intestinal mucosa appears to be independent of exposure to parasites. Large intestinal mucosal eosinophils may have more functions in addition to their role in defence against parasites.     

Light and electron microscopic studies on alpha naphthyl acetate esterase activity of peripheral blood T lymphocytes in chicken.

The purpose of this study was to determine the percentage of peripheral blood T lymphocytes and the localization of Alpha Naphthyl Acetate Esterase (ANAE) enzyme at the electron microscopic level in healthy adult chickens by an alpha naphthyl acetate esterase procedure. Peripheral blood samples taken from 30 adult Leghorn breed chickens were used. The percentage of ANAE positive lymphocytes was 56.4 +/- 2.82% (SD). Pseudoeosinophil and eosinophil granulocytes showed a granular positivity. In the electron microscopic examination, ANAE positive reactions were seen in lysosomal granules found in T lymphocytes, pseudoeosinophil, and eosinophil granulocytes.     

Fine structure of secondary granule inclusions in fowl heterophils after ruthenium tetroxide fixation

Bone marrow from domestic fowls was initially fixed for electron microscopy in glutaraldehyde and post-fixed with either ruthenium tetroxide or osmium tetroxide. Inclusions with distinct outlines were revealed in the secondary granules of heterophil leucocytes after ruthenium tetroxide but not with osmium tetroxide fixation. In longitudinal orientation, these inclusions were rod-shaped and composed of microfilaments measuring 3.7 nm in diameter. In transverse section, the outline of some of these inclusions was hexagonal and therefore the inclusions may be crystalline in nature.     

Morphology of blood cells in carp (Cyprinus carpio L.)

The morphology of blood cells in the carp was investigated by light and electron microscopy. Erythrocytes, thrombocytes, lymphocytes, granulocytes and monocytes were identified as the peripheral blood cells. Thrombocytes were round to long oval, each containing vesicular and microtubular structures and an oval nucleus with abundant heterochromatins. Lymphocytes were divided into three types in size, small, medium and large. Some of the small and medium lymphocytes were alpha-naphthyl-acetate esterase (ANAE) positive, while large lymphocytes were pyroninophilic. Granulocytes were distinguished into three types (type I, type II and type III) according to the morphology of the nucleus and granules. Type I granulocytes possessed lobulated nuclei and a large number of cytoplasmic granules, each of which was oval and contained electron-dense materials and a crystalloid. Type II granulocytes had small eccentric nuclei and were subdivided into IIa and IIb granulocytes by electron microscopic analysis. Granules of type IIa granulocytes were furnished with an electron-dense rim. Granules of type IIb granulocytes were larger than those of type IIa, containing randomly distributed electron-dense and electron-lucent materials. Type III granulocytes possessed round nuclei and a few large granules. The granules were filled with regularly arranged fibriform materials and some needle-like structures. Monocytes were morphologically similar to those of mammals.     

Distribution of eosinophil granulocytes and mast cells in the reproductive tract of female goats in the preimplantation phase

Changes in eosinophil granulocytes and mast cells post-insemination may affect conceptus implantation, but information regarding the numbers of such cells in the mammalian reproductive tract is limited. This study investigated the preimplantation distribution of eosinophil granulocytes and mast cells (MCs) in the reproductive tract organs of female goats. Uterus, uterine cervix and uterine tubes samples were obtained at slaughter. Cornu uteri were washed in phosphate buffer solution (each animal contained at least one embryo). Tissues were fixed in 10% neutral buffered formol, Carnoy solution and Mota’s fixative (basic lead acetate) for 48 h and embedded in paraffin. Six-micrometre-thick sections were stained with Congo red (for eosinophil granulocytes) and toluidine blue in 1% aqueous solution at pH 1.0 for 5 min (for MCs). In the uterus, MCs occurred in highest numbers in the myometrium. Higher MC numbers were observed in uterus, uterine and uterine tubes in the preimplantation (experimental) group (cycle synchronised through 7 days intravaginal sponge with 0.3 g P₄) compared with the control group (P < 0.05). Eosinophil granulocyte numbers were significantly higher in the experimental group than in the control group (P < 0.05). These results indicate preimplantation-related changes in numbers of eosinophil granulocytes and MCs in goat reproductive tract organs.     

Pronephric leucocytes of Cyprinus carpio: isolation, separation and characterization

Since the teleost pronephros is an important source of diverse immunocytes, suspensions of pronephric cells from young adult carp have been characterized. In freshly prepared suspensions, adherent, spreading cells (macrophages?) constituted less than 3% of the total population. Granulocytes and lymphocytes were co-dominant (less than 80%) leucocyte types. Continuous Percoll density gradient centrifugation yielded discrete subpopulations with these rho values and cytological characteristics: Fraction I & II rho = 1.055-1.070 thrombocytes, monocytes, macrophages, and lymphocytes. Fraction III rho = 1.080-1.090 granulocytes, type 1. Fraction IV rho = 1.105-1.110 erythrocytes and granulocytes, type 2. Fraction V rho = 1.118-1.125 granulocytes, type 3. Fraction VI rho = 1.140-1.150 granulocytes, type 4. Granulocyte motility increased markedly over the first 24 hr in vitro, and was enhanced by components washed from intact yeast. The subtypes of granulocytes were distinguishable by not only the rho values, but also on the basis of cell size, ultra-structure of the granules, and their histochemical and phagocytic characteristics. After simultaneous in vivo injection of Bacillus megaterium (Gram + ve), Aeromonas hydrophila (Gram - ve) and Saccharomyces cerevisiae (yeast), individual pronephric leucocytes were found capable of phagocytosing all three types of particle. Granulocytes which had phagocytosed B. megaterium were slower than macrophages in their ability to kill the bacteria. Encounter with B. megaterium or S. cerevisiae in vitro elicited a clumping reaction which involved mostly the larger leucocytes [granulocytes]. Both adherent cells and non-adherent cells were phagocytic in vitro.     

Adenovirus inclusions in the ileum in coccidiosis in suckling piglets

Numerous intranuclear inclusion bodies in enterocytes were detected exclusively in the ileum of two nine-day piglets coming from a litter infected with diarrhea. The inclusion bodies were homogeneous in hematoxylin and eosine (HE), their staining was not clear enough, was amphophilic and they filled nearly the whole nucleus. They were eosinophil less often and had a halo on the periphery. Their staining was clearly orange-red in Feulgen's nuclear reaction after re-staining with G orange and bright green. Intranuclear inclusions were located exclusively on shortened villi and pseudovilli of ileum above the follicles of activated Peyer's patches. The findings of intranuclear inclusions in ileum demonstrate adenovirus enteral infections in suckling piglets.     

Mucosal distribution of eosinophilic granulocytes within the gastrointestinal tract of horses

OBJECTIVES: To establish reference values for the range of the number of eosinophils found in equine gastrointestinal mucosa and to describe the distribution of this cell within the equine gastrointestinal mucosa. SAMPLE POPULATION: Gastrointestinal mucosal specimens from 14 adult horses euthanatized for reasons other than gastrointestinal disease. PROCEDURES: Gastrointestinal mucosal specimens were collected and grouped according to their anatomic regions. For histologic examination slides were stained with Luna's eosinophil stain to determine eosinophil accumulation and distribution. The mucosa was divided into 5 sections for each anatomic location, and the percentage of eosinophils in each of the 5 sections relative to the total eosinophil count in all sections was determined. Additionally, the number of eosinophils per square millimeter of mucosa was calculated as a measure of the degree of eosinophil accumulation. RESULTS: Lowest numbers of eosinophils were found in the stomach, and numbers increased from there to the cecum, then decreased from the ascending colon (right ventral colon, left ventral colon, pelvic flexure, left dorsal colon, and right dorsal colon) to small colon. In all gastrointestinal sections, most eosinophils were located near the muscularis mucosae and were rarely found near or on the luminal surface of the mucosa. CONCLUSIONS AND CLINICAL RELEVANCE: The distribution of eosinophils in the gastrointestinal tract of horses followed a pattern within the mucosa and between different sections of the gastrointestinal tract. The derived reference values and distribution data could be used to detect changes in eosinophil response in the equine gastrointestinal mucosa caused by diseases states.     

Pancreatic necrosis and ventricular erosion in adenovirus-associated hydropericardium syndrome of broilers

Seven 19-day-old broiler chickens affected with hydropericardium syndrome (HPS) with pancreatic necrosis and gizzard erosions were investigated pathologically and virologically. Mortality increased after 13 days of age in a flock on a broiler farm. The mortality rate of the flock reached 10% by 19 days of age. Macroscopically, the chickens had hydropericardium (the characteristic gross change of HPS), pinpoint white foci in the pancreas, and ventricular erosions. Histologically, the chickens had multifocal hepatic necrosis with intranuclear inclusions in hepatocytes, a marked increase of macrophages in the spleen and lung, mild epicardial edema, multifocal necrosis of pancreatic acinar cells with intranuclear inclusions, focal necrosis of the ventricular koilin layer, and degeneration of the ventricular glandular epithelium with intranuclear inclusions. Immunohistochemically, intranuclear inclusions in the liver, pancreas, and ventriculi were stained positively against group I avian adenovirus (GIAAV) antigens. Ultrastructurally, 67-nm diameter viral particles were present in intranuclear inclusions. Virologically, serotype 4 of GIAAV was isolated from the liver, heart, and kidney of affected chickens. The pathologic changes of the present cases differ from previous cases of HPS; therefore, the present strain of GIAAV may have different pathogenicity for chickens than the previous virus strain of HPS.     

Fasciola hepatica products induce apoptosis of peritoneal macrophages

Immunomodulatory properties have been described for Fasciola hepatica excretory-secretory products (FhESP), with their interaction with the innate immune cells being crucial during the early stages of infection. Previously, we demonstrated that FhESP induce eosinophil apoptosis. In this work, the ability of FhESP to induce apoptosis of peritoneal macrophages was evaluated. These parasite products were observed to induce apoptosis in peritoneal macrophages stimulated in vitro with FhESP, as well as in cells recovered from infected mice. The ability of FhESP to modify the viability of macrophages by apoptosis induction may constitute a crucial event for extending its survival in the host.     

Leukokinesis in bovine ostertagiasis: stimulation of leukocyte migration by Ostertagia

A blind-well chemotaxis chamber method was used to indicate migration stimulation of bovine neutrophil and eosinophil polymorphonuclear leukocytes and macrophages as related to ostertagiasis. Live exsheathed Ostertagia ostertagi 3rd-stage larvae (L3) and soluble L3 antigen (SLA), prepared by freeze thawing and sonic disruption of L3, enhanced cellular migration for eosinophils, but not for neutrophils and macrophages. Products of lymphocytes cultured with SLA for 3 to 6 hours were also examined, using lymphocytes from peripheral blood of helminth-free cattle and cattle infected with O ostertagi or Trichostrongylus axei. Lymphokines that enhanced cellular migration of neutrophils, eosinophils, and macrophages were present in culture supernatants of SLA-stimulated lymphocytes from O ostertagi-infected cattle, but not from cattle infected with T axei or helminth-free cattle. Seemingly, L3 and SLA were stimulants of eosinophil migration. Further, neutrophil, eosinophil, and macrophage migration was modulated by lymphokines produced by SLA-stimulated lymphocytes from cattle with ostertagiasis.