Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. 3. The histochemistry of mast cells and globule leucocytes

Comparative studies were made on the structure and histochemistry of mast cells and globule leucocytes in dicrocoeliasis and fascioliasis of the goat. Investigations showed that D. dendriticum and F. hepatica infections in the goat produce an increase in the number of hepatic mast cells and frequent occurrence of globule leucocytes in the epithelium of the bile ducts. The only difference between these infections was that globule leucocytes were more frequent in small bile ducts in dicrocoeliasis than in fascioliasis.Histochemical methods showed the presence of sulphomucins in the granules of mast cells, while both neutral mucins and carboxymucins were revealed in the globules of globule leucocytes. The acidity of the mucosubstances could not be shown to result from hyaluronic acid, chondroitin-4 and -6-sulphates, or sialic acid. The globules of globule leucocytes also contained histochemically identifiable highly basic proteins.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts, mast cell, globule leucocyte     

On the Ultrastructure of Mast Cells and Globule Leucocytes in the Common Bile Duct of Cattle Chronically Infected With Fasciola Hepatica

Mast cells and globule leucocytes in the walls of the common bile ducts of seven uninfected slaughtered cows and of five cows chronically infected with Fasciola hepatica were studied by electron microscopy. The mast cells displayed numerous small intracytoplasmic granules, the matrixes of which were either strongly electron-opaque and homogeneous or less dense with a fine-granular ultrastructure. Unattached ribosomes were abundant in the cytoplasm of the cells. Many mast cells in the walls of chronically infected bile ducts were normal-looking while the cells in the subepithelial tissues often showed striking irregularities in the ultrastructure of the granules. Globule leucocytes were most numerous in the surface epithelium and were not connected by desmosomes to the epithelial cells. The nuclei of the globule leucocytes were similar to those in the mast cells except that indentations caused by the globules were more usually seen. The globules were considerably larger than the mast cell granules but bounded by similar agranular capsules. The matrix of most globules was dense and homogeneous while the other globules showed dense reticulate or lamellate structures. The relationship between the mast cell and globule leucocyte in the liver is briefly discussed with reference to recent reports on these cells in the intestinal wall.     

Failure to induce homologous immunity to Fasciola hepatica in sheep vaccinated with irradiated metacercariae

Vaccination of sheep with either 100 or 1000 γ-irradiated (2.5 krad) metacercariae of Fasciola hepatica, on two occasions six weeks apart, did not generate significant protection against intraruminal challenge with F. hepatica six weeks after the second vaccinating dose as measured by recovery of flukes from liver and bile ducts, twenty weeks after challenge. There was, however, a significant increase in the proportion of flukes retarded in the parenchyma of both vaccinated groups. The percentage of retarded flukes was positively correlated with the degree of liver damage and increased weight of the hepatic lymph nodes. It was not possible to determine if the retarded flukes were derived from the vaccine or challenge infections or both.Challenge infection of both vaccinated and unvaccinated sheep significantly increased the numbers of eosinophils and globule leucocytes in the parenchymal bile duct and the numbers of mast cells and globule leucocytes in the abdominal bile duct. In addition the numbers of eosinophils and globule leucocytes in the parenchymal bile duct were significantly correlated with the percentage of retarded flukes in both vaccinated groups. In the abdominal bile duct, only the numbers of eosinophils in the low level vaccination group were significantly correlated with fluke retardation.Vaccination did not protect against the pathogenic effects of challenge infection as measured by reduced packed cell volumes and weight gain.     

Experimental haemonchosis in goats: effects of single and multiple infections in the host response

Histopathological changes and the distribution of T lymphocytes (CD3), B cells (CD79alpha) and IgG secreting plasma cells were recorded in the abomasum and abomasal lymph nodes of goats during early and late post-infection stages with one to four doses of Haemonchus contortus L3. The infiltration of eosinophils, mast cells, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells in the abomasal mucosa increased dramatically from 10dpi onwards, whereas globule leukocytes were observed only during chronic infection. In late post-infection stages abomasal infiltration of globule leukocytes, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was significantly higher (P<0.05) in reinfected (groups 6-8) than in primarily infected goats (group 5). In the abomasal lymph nodes, marked hyperplasia of lymphoid follicles and medullary cords, with increase of CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was recorded from 10dpi (group 3) onwards. Worm burdens and the severe abomasal response during the late post-infection stages suggests that a rapid expulsion of nematodes did not occur. The prolonged time required for generating globule leukocytes suggested that immune mechanisms dependent of this cell type are of crucial importance in the protective immunity against H. contortus in goats.     

Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge with Haemonchus contortus

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue γδ TCR⁺ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R⁺) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by γδ T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.     

Comparison of four staining methods for detection of mast cells in equine bronchoalveolar lavage fluid

Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The BALF cells from 24 horses were studied. A differential cell count was performed blindly on 400 cells. The percentages of mast cells obtained were analyzed by means of repeated-measures analysis of variance and Fischer's PLSD test. The Bland and Altman method was used to assess agreement among stains. The mean percentage of mast cells in BALF was significantly lower with the fast Romanowsky than with the automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. With the fast Romanowsky stain, the metachromatic granules of mast cells were not stained, and their identification was based on morphologic criteria. Toluidine blue staining allowed detection of the highest mean percentage of mast cells, but was inadequate for performing a differential cell count on other cell types. In conclusion, fast Romanosky stain may be inadequate for detection of mast cells in equine BALF, whereas automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains provide metachromatic staining of mast cell granules.     

Pathological and immunohistochemical study of the abomasum and abomasal lymph nodes in goats experimentally infected with Haemonchus contortus

Histopathological changes and the distributions of T and B lymphocytes and IgG producing plasma cells were recorded in the abomasum and abomasal lymph nodes of goats 3, 7 and 21 weeks post-infection (wpi) after an experimental infection with H. contortus. The low rate of worm recovery by 3 wpi (5.6%) might have been due to larvae death as suggested by the presence of granulomas in the abomasal mucosa at 3 and 7 wpi, or simply due to a poor larval establishment. Marked increase in the secretion of mucus by mucous cells together with an abundant infiltration of eosinophils, mast cells, CD3+ T lymphocytes, CD79a+ B cells, IgG+ plasma cells and globule leukocytes were recorded in the abomasal mucosa, especially at 7 wpi. Except for the globule leukocytes, this reaction decreased substantially by week 21, suggesting this cell type may have been involved in rejection of adult nematodes. The abomasal lymph nodes showed marked hyperplasia, particularly of CD79a+ B cells and IgG+ plasma cells in all infected goats. These reactions may have been responsible for the reduction in the number of worms found in the abomasum between 3 and 7 wpi.     

Bronchopulmonary lavage cytology in the dog: normal findings

Fiberoptic bronchoscopy was used to obtain cytologic specimens from all lung lobes of 9 normal Beagle dogs. Three specimen collection techniques (bronchial lavage, bronchial brushing and bronchial pinch biopsy imprints) and two staining procedures (Wright-Giemsa and Papanicolaou) were used and evaluated. Bronchial lavage was the most satisfactory technique for collection of samples from the deep lung and bronchial brushings were preferred for potential bronchial tree mural lesions. Wright-Giemsa was the stain of choice because mast cells could not be identified and eosinophilic leukocytes could be identified only with difficulty in Papanicolaou stained specimens. Total and differential cell counts were determined on all bronchial lavages from all lung lobes in order to establish baseline reference values. Total nucleated cell counts ranged from 260-120/microliters. There were no significant differences among mean total nucleated cell counts for the different lung lobes. Mean total nucleated cell counts were between 420 and 630 cells/microliters. Approximately 95% of all nucleated cells in normal lavages were undifferentiated alveolar macrophages. Most of the other cells seen were neutrophils, eosinophils, possible globule leukocytes and mast cells. Ciliated and nonciliated epithelial cells comprised less than 1% of the total nucleated cell population.     

Bovine nasal granuloma. Gross and microscopic lesions.

The anterior nasal mucosa of 21 cattle had small closely-packed polypoid nodules. These had a distribution pattern similar to that of inhaled particles. Acute inflammatory changes consisting of eosinophil infiltration, mast cell and globule leucocyte hyperplasia, and oedema were frequent in the epithelium, terminal gland ducts and adjacent lamina propria. Epithelium over nodules was metaplastic to non-keratinising stratified squamous, whilst epithelium of terminal gland ducts was metaplastic to mucus-secreting pseudostratified columnar. Focal accumulation of granulation tissue between gland ducts caused herniations of the superficial lamina propria to form the nodules.     

Bovine intestinal cellular responses following primary and challenge infections with Calicophoron microbothrium metacercariae

This study was carried out to establish whether cattle can develop resistance to re-infection by Calicophoron microbothrium by assessing the response of intestinal mucosal globule leukocytes, eosinophils, mast cells and basophils, and the establishment of the parasite in the host. A total of 24 1-year-old Tuli steers were randomly divided into four groups of six animals each and infected with C. microbothrium metacercariae. On the first day of the study, animals in Groups I and II were immunized with 5000 metacercariae and then challenged with 15,000 metacercariae on Day 150 post-immunization. Animals in Group III were immunized with 15,000 metacercariae at the same time that Groups I and II animals were challenged to act as a positive control group. Animals in Group IV were left uninfected and acted as a negative control group. Three animals from each group were slaughtered on Day 28 post-challenge and the remainder were slaughtered on Day 42 post-challenge. The established amphistomes were recovered and histopathological and cytological examinations were done on the jejunum, duodenum, abomasum and the rumen. The establishment rates of the challenge infection in the immunized and challenged groups were lower and ranged from 0 to 0.2% as compared to 6% from naive animals infected as positive controls. Animals immunized and then challenged with C. microbothrium had significantly higher eosinophil, mast cell and globule leukocytes counts in the intestinal mucosa (P < 0.05) as compared to those of the control group. The study indicates that cattle can develop resistance to C. microbothrium re-infection and that eosinophils and mast cells may be important cells in the rejection of the parasite.     

猪子宫大豆素(SBA)受体细胞和肥大细胞观察

本研究用HRP—SBA标记技术、甲苯胺染色法及甲苯胺蓝和HRP—SBA双重染色技术,观察了猪子宫大豆素(SBA)受体细胞和肥大细胞.发现SBA受体细胞和肥大细胞,形态相似,但两者在子宫壁内的分布和数量不同.双重染色证明,SBA受体细胞和肥大细胞是存在猪子宫内膜的不同细胞.     

Regulation of Teladorsagia circumcincta and Trichostrongylus colubriformis worm populations by grazing sheep with differing resistance status

In an experiment lasting 4 years, changes in the Teladorsagia circumcincta and Trichostrongylus colubriformis populations were compared in lambs and adult sheep with differing resistance statuses. Two flocks of 30 rams (resistant R and susceptible S) grazed separate pastures and 8 rams were slaughtered in the middle and at the end of each grazing season. Five groups of tracer lambs were added each year to estimate the pasture infectivity and were killed for worm counts. The availability of animals with differing resistance statuses (rams and tracer lambs) and differing levels of infection made it possible to investigate the number, size and fecundity of worms of these two species. The inflammatory response was measured in the rams by counting the globule leukocytes, mast cells and eosinophils in the fundic, pyloric and intestinal mucosa. In the tracer lambs, the daily egg production by the female worms of both species was negatively correlated with the worm burden. Worm length accounted for 60 and 70% of the variation in the number of eggs in utero for T. circumcincta and T. colubriformis respectively. Worm length was closely associated with the resistance status of the host; there were greater differences between lambs, and S and R rams for T. colubriformis. T. circumcincta worm lengths were not affected by the worm number. Globule leukocyte counts were related to the worm burdens, and mast cell counts to worm length in the R and S rams. The number, size and fecundity of the worms may well be regulated by similar mechanisms in both species, but T. colubriformis seemed to be more intensively regulated than T. circumcincta. This finding could be useful in devising more effective methods of parasite control.     

Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. I. The histopathology of the liver and bile ducts

Comparative studies were made on the histopathology of the liver and bile ducts in dicrocoeliasis and fascioliasis of the goat. Investigations revealed certain similarities in the nature of the pathological phenomena produced by D. dendriticum and F. hepatica. However, a feature peculiar to fascioliasis was the migratory tracks of immature flukes in the liver parenchyma. Further, the lesions produced by fascioliasis were distinctly more pronounced than those present in dicrocoeliasis.In both parasitic diseases the bile-duct walls showed glandular hyperplasia, increased frequency of goblet cells and globule leucocytes, and extensive fibrosis in the outer layers. The desquamation produced by Dicrocoelium remained superficial whereas Fasciola was capable of causing deep erosions in the walls of the main bile ducts. The bile-duct walls did not become calcified. Increased fibrosis was present in the interlobular areas, too, but only fascioliasis produced extensive disorganisation of the hepatic architecture, due to the scars originating from repaired migratory tracks. The liver parenchymal cells were degenerated, showed decreased glycogen but were increasingly infiltrated by neutral fats.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts     

Grey eosinophils in a Miniature Schnauzer with a poorly differentiated mast cell tumor

A 10‐year‐old female spayed Miniature Schnauzer was presented for investigation of an intra‐nasal mass. The mass was diagnosed by histopathologic examination as an undifferentiated round cell neoplasm with an infiltrate of segmented leukocytes, interpreted as neutrophilic inflammation. The mass was treated with palliative radiation and systemic chemotherapy due to the presence of regional lymph node metastasis. During subsequent monitoring over several months, the peripheral leukocyte concentration was repeatedly within reference intervals to slightly increased with low numbers of toxic neutrophils. Four months after the initial diagnosis, there was a significant leukocytosis of 66 100 cells/μL, and 39 700 cells/μL of the leukocytes had variably mature, lobulated, and hypolobulated nuclei, and grey cytoplasm with clear vacuoles, resembling grey eosinophils. To further characterize these cells, peripheral blood smears from the patient and a canine control with eosinophilia were stained for alkaline phosphatase (AP), peroxidase, and esterase activities, and with Luxol fast blue (LFB). Histopathologic sections of the nasal mass were stained with LFB and immunohistochemically for tryptase. On blood smears, the cytoplasm of the suspected grey eosinophils stained for AP and granules stained with LFB confirmed that there was an eosinophilic lineage. Peroxidase staining was weak, and esterase staining was absent. On histopathologic sections from the nasal mass, the segmented leukocytes contained LFB‐staining granules, indicating an eosinophilic infiltrate was present. Neoplastic cells expressed tryptase, which confirms a mast cell lineage. Our findings suggest that grey eosinophils might be under‐recognized and interpreted incorrectly as toxic neutrophils. This report expands the canine breeds in which these eosinophils have been identified.     

Mast cells of the bovine trachea: staining characteristics, dispersion techniques and response to secretagogues.

Sections of the lower trachea of cattle, fixed in either Carnoy''s or formalin, were stained with toluidine blue, alcian blue, or alcian blue and safranin O to study the mast cell population. After toluidine blue staining, about twice as many cells in tissue fixed in Carnoy''s contained dark blue granules compared with tissue fixed in formalin. In addition, for the first time in cattle, a population of cells containing red granules was identified after staining with alcian blue and safranin O. Most of these red granules were formalin sensitive. An enzymatic dispersal technique for mast cells is described that yielded 9.4+/-0.4% mast cells (percentage of nucleated cells) with a viability of 92.3+/-0.6%. Spontaneous histamine release was 3.3+/-0.8%. Dispersed mast cells were challenged with various immunological and nonimmunological secretagogues. The calcium ionophores, A23187, ionomyocin, and BrX537A, were effective in releasing up to 94% of histamine in mast cells in a dose-response relationship. Pasteurella haemolytica culture supernate caused about 10% histamine release at a dose of 0.5 mg/mL after correction for spontaneous release. The average histamine content of the mast cells was 6.6+/-1.0 pg/cell. Cytospins of dispersed cells fixed in Carnoy''s and stained with alcian blue and safranin O contained mast cells with blue and red granules, and a few cells with a mixture of both granule types. Based on the effects of type of fixation, staining characteristics and histamine content, a mix of subtypes of mast cells is present in the bovine trachea. However, functionally they respond to secretagogues differently than rodent mast cells. Without an immunological secretagogue, studies to determine compounds that will be effective in blocking mast cell degranulation will be limited.     

Ultrastructural Features of the Bovine Cecal Mucosa

The cecal surface epithelium of cattle was lined entirely by columnar cells except near the openings of the glands where a few partially depleted goblet cells were encountered. Surface columnar cells with pale cytoplasm, indented (sometimes dome-shaped) apical border and irregular microvilli, were also found near the gland orifices. Surface columnar cells -near the openings of the glands contained large cytoplasmic vacuoles and lysosomal-like bodies. Near the extrusion zone, however, the columnar cells contained highly indented and apically displaced nuclei. The glands were usually long and straight and lined by an epithelium concisting mainly of undifferentiated and goblet cells. There were few entero-endocrine and non-epithelial cells (intra-epithelial lymphocytes and globule leukocytes). Paneth cells were absent. The undifferentiated cells contained many free ribosomes and apical secretory granules. Some of these cells were undergoing mitotic division. Goblet cells exhibited a typical brandy-glass appearance and showed dark and light mucigenous granules. Endocrine cells with polymorphic secretory granules and cells with more uniform secretory granules were identified in the basal part of the epithelium. Globule leukocytes and intra-epithelial lymphocytes were usually encountered near the basal part of the epithelium. The lamina propria was highly cellular. It contained many plasma cells, mast cells and small lymphocytes, and few eosinophils, neutrophils and globule leukocytes. Some of the plasma cells contained large, dense Russell bodies within the cisternae of the rough-surfaced endoplasmic reticulum. The neutrophils displayed distinct populations of small and large cytoplasmic granules. Some of the eosinophilic granules showed narrow crystalline cores. Large lymphocytes were found in the lamina propria, but occurred less frequently than small lymphocytes.     

A subcutaneous mass on the neck of a horse

A 25-year-old Arabian gelding was presented for investigation of a subcutaneous neck mass. Fine-needle aspirates and impression smears revealed mast cells with widely varying degrees of cytoplasmic granulation and scattered eosinophils. Histopathology revealed a poorly circumscribed mass composed of sheets and bundles of mast cells with a large population of eosinophils. The mast cells were separated into numerous lobules by a heavy collagenous stroma, and multifocal collagen necrosis was present. Strong reactivity of the tumor cells for both Giemsa and toluidine blue stains confirmed the diagnosis of a mast cell tumor, and a Luna stain accentuated the eosinophilic infiltrates. Cutaneous mast cell tumors are found in many domestic animals but are uncommonly encountered in horses. Equine cutaneous mast cell tumors are usually benign, and there are no reports of visceral metastasis. Surgical excision is thought to be curative.     

Relationship of gastrointestinal histology and mucus antiparasite activity with the development of resistance to trichostrongyle infections in sheep

Changes in the numbers of globule leucocytes, mast cells, eosinophils and goblet cells in the gastrointestinal mucosa were examined in relation to the development of resistance and elimination of nematodes in grazing sheep in their firsty year of life. Sheep immunised against Trichostrongylus colubriformis, and sheep treated with dexamethansone were also examined. A strong association between resistance to infection and the presence of globule leucocytes was found. In contrast, the numbers of mast cells or goblet cells were not correlated with resistance. Globule leucocyte and eosinophil numbers were also correlated with antiparasite activity in mucus.

Immunising infections of T. colubriformis given to 10-month-old sheep, their duration limited by thiabendazole treatment, gave rise to considerable immunity to homologous challenge infections. Larvae that developed to the 4th stage were as effective at stimulating immunity as those that developed to the 5th stage. Dexamethasone treatment abrogated resistance to trickle challenge infection with T. colubriformis and reduced mucosal globule leucocyte and mast cell numbers. After cessation of drug treatment, the re-establishment of resistance and adult worm elimination were associated with repopulation of the mucosa with large numbers of globule leucocytes and high antiparasite activities in mucus.     

Response of Corriedale and Crioula Lanada Sheep to Artificial Primary Infection with Haemonchus Contortus

Clinical, parasitological and biochemical parameters were evaluated in Corriedale and Crioula Lanada sheep after a single experimental infection with Haemonchus contortus. Ten 4-month-old worm-free lambs, of each breed, were infected with 200 L₃ H. contortus per kg live weight and four uninfected animals of each breed were used as controls. Every week, the animals were weighed and blood and faecal samples were collected for measurement of packed cell volume (PCV), total serum protein (TSP) and albumin (ALB), and the number of eggs per gram of faeces (EPG), respectively. Twelve weeks after infection, the animals were slaughtered. The worm burden was determined and samples of the abomasal mucosa were processed for determination of the number of eosinophils, mast cells and globule leukocytes. No significant differences in PCV, TSP, ALB, parasite burden or the cell populations of the abomasal mucosa were observed between breeds, but Crioula lambs had a lower EPG count. The comparison of the infected groups with their respective controls revealed significant alterations in PCV, TSP and ALB in the Corriedale lambs and in PCV, TSP, ALB and the density of eosinophils and mast cells in the Crioula lambs.     

Cell proliferation activity of proliferating bile duct after bile duct ligation in rats

The cell proliferation activity of proliferating bile ducts produced by bile duct ligation (BDL) in rats was examined histologically, immunohistochemically, and ultrastructurally. Proliferating bile ducts, which were similar to normal bile ducts, increased with time after BDL. The cell proliferation activity of proliferating bile ducts, measured using proliferating-cell nuclear antigen and 5-bromo-2'-deoxyuridine antibodies, tended to be high at 1 and 3 days after BDL and decreased progressively at 2 to 4 weeks after BDL. On the other hand, alpha-smooth muscle actin-positive myofibroblast-like cells increased continuously after BDL. These findings indicate that there is a negative correlation between the cell proliferation activity of proliferating bile ducts and that of myofibroblast-like cells.