The pathology of cystic echinococcosis and structural details of hydatid cyst and protoscolex Beigh A.B.2,*, Darzi M.M.2, Bashir S.1,2, Shah A.2, Shah S.A.2 2Division of Veterinary Pathology, Faculty of Veterinary Sciences & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology, Srinagar-190006, J&K, India 1Department of Biochemistry, Hamdard University, New Delhi-110062, India *Corresponding author: e-mail: beighab@gmail.com
Abstract Hydatidosis, an important parasitic zoonosis, is a major public health and economic concern throughout the world. A total of 2100 small ruminants (sheep-2052 and goats-48) slaughtered or naturally dead, from various areas of Kashmir valley, were screened for the presence of hydatidosis. Frequently infected organs were lungs and liver. The liver was found to be the most frequently infected organ with prevalence of 61.17% followed by lungs (38.82%). The pulmonary cysts were more fertile (55%) compared to hepatic cysts (45%). Histopathologicallly, the cyst wall consisted of the inner germinal, middle laminated and outer fibrous layer. Inflammatory reaction around the cyst was variable and was characterized by an inner zone of loosely arranged fibroblasts infiltrated with mononuclear cells, followed by densely arranged fibroblasts along with mononuclear cells; and an outer layer of fibrous connective tissue. Hepatocellular degeneration and cirrhosis were observed in liver besides the cellular reaction against the expanding cyst, the severity of which was inversely related to the distance from the cyst. Qualitative increase was observed for acid mucopolysaccharides in fibroblasts, inflammatory cells and bronchial epithelium. Masson's trichrome revealed intense deposition of collagen fibers in the pericystic connective tissue. The structural details of the protoscolices were clearly discernable. The calcareous corpuscles of the protoscolices were distinct when stained with combined Alcian blue PAS and Toluidine stains but were not visible when stained with H&E and Masson's trichrome stains. Top Keywords Histopathology, Histochemistry, Hydatid cyst, Protoscolex, Zoonosis. Top |
INTRODUCTION Cystic echinococcosis (CE) is a zoonotic infection caused by larval forms (metacestodes) of the tapeworm Echinococcus1,2. The World Health Organization has declared echinococcosis as a neglected zoonosis subgroup in its recent strategic plans for the regulation of tropical diseases that are neglected3. CE is still a challenge in the medical field and endemic in many countries across the world4. Echinococcosis has carnivores as definitive hosts and the herbivores and omnivores as intermediate hosts. Humans are infected accidently and are not a part of the natural life cycle of parasite. The adult worm, Echinococcus, develops in the small intestine of carnivores and intermediate stage hydatid cyst develops in the internal organs (mainly the liver and lungs) of humans and herbivores (sheep, horses, cattle, pigs, goats and camels) as fluid-filled bladders which are unilocular in nature5. Its pathogenicity depends upon the severity of the infection and the organ infected6. There are six species of Echinococcus i.e. E. granulosus, E. multilocularis, E. vogeli, E. oligarthrus, E. felidis and E. shiquicus7. Four species among the six known, E. multilocularis, E. granulosus, E. vogeli and E. oligartrus pose a severe threat to the human health8. |
The hydatid cyst of E. granulosus consists of an inner germinal membrane, an acellular laminated layer and an outer adventitial layer. The inner two layers are of parasite origin, while the adventitial layer is host derived. In fertile cysts, buds form in the germinal membrane and develop into brood capsules within which protoscolices are produced. The adventitial layer of hydatid cysts comprises several zones that vary with the host species and the stage of cyst development or degeneration9. Nevertheless, cysts can cause an irreversible damage to the organs, and rupture or puncture of the cyst can infect several organs with their larvae and hence cause the anaphylactic reactions. The animals are sacrificed even before the cysts become mature enough to show various clinical signs, but when their entrails are given to the dogs, it continues the cycle. Animals having a long lifespan such as horses become ill9. |
Echinococcosis presents a serious public health problem especially in the rural areas where the dogs are found in close association with man and other domestic animals, feeding on scraps and intestines of wild herbivores10. The disease is so dangerous to human health, World Health Organization (WHO) specially focused on zoonotic aspects of this disease in one of its active programmes11. Though the disease in domestic animals does not show major clinical signs and is detected only at the time of post-mortem yet it causes great economic losses by way of condemnation of livers and other organs besides lowered meat and milk production12. CE usually develops silently over decades until it surfaces with various clinical signs. Clinical symptoms are directly related to the location, size, and load of cysts present13. |
Echinococcosis is diagnosed by different ways using X-ray, CT scan, immunological and serological tests including modern diagnostic technique i.e polymerase chain reaction (PCR). Its larval stage forms can usually be detected visually in organs. Special care has to be taken for a specific diagnosis of E. granulosus in instances where Taenia hydatigena in sheep is also a problem. Microscopic examination of the tissue may confirm the diagnosis after the formalin-fixed tissue is processed by various conventional staining methods. The presence of a PAS positive and acellular layer with or without a cellular, germinal membrane that is nucleated can be considered as a characteristic of metacestodes of the Echinococcus14. The present study was envisaged to study histopathological and histochemical structural details of hydatid cyst and protoscolex. |
Top MATERIALS AND METHODS Study material The present study was conducted from the year 2013–2016 on locally reared sheep and goat, including both slaughtered and naturally dead cases in different regions of Kashmir valley. Out of total 2100 heads screened, only 85 cases showed one or more cysts in lungs and livers. Gross and histopathology The affected organs were examined for any gross alterations associated with the cysts. Representative tissue samples associated with hydatid cyst in different organs were collected and preserved in 10% formalin. The fixed tissue samples were processed by routine paraffin embedding technique. Briefly the samples were cut into pieces of thickness 2–3 mm and washed under water for few hours prior to dehydrating in ascending grades of alcohol and later cleared in benzene and embedded in paraffin. Tissue sections of 4–5 μm thickness were stained with Harris haematoxilin and eosin standard method/protocol15. Histochemistry Parallel tissue sections, on the basis of histological examination, were selected and then stained for neutral or acid mucopolysaccharide by PAS technique using Combined Alcian blue15, connective tissue by Masson‘s trichrome stain15 and mast cells by Toluidine blue stain15. Top RESULTS Gross pathology Grossly, the lungs revealed single to multiple hydatid cysts of varying sizes. These were usually of table tennis ball shape but were occasionally as big as a cricket ball. The cysts were either fully embedded in the lung parenchyma or were partially embedded when they were visible from the lung surface. Both, dorsal and ventral aspects of the lungs were affected. Diaphragmatic lobe of the lung was frequently affected. Single to multiple cysts of varying size were observed from the visceral and/or parietal surfaces of liver. In general the cysts were soft and doughy to touch and were filled with clear to slightly turbid fluid. On aspiration of fluid, the cyst collapsed and the cyst membrane, appearing creamy white, could be easily removed from the organ and its fluid contents were found clear to slightly turbid. However, some cysts were appearing firm and contained inspissated contents. Also, some cysts were calcified, gritty and hard to cut.The liver was observed to be the most frequently infected organ with relative prevalence of 61.17% followed by lungs (38.82%). The pulmonary cysts were mostly fertile (55%) compared to hepatic cysts (45%). Histopathology The cysts consisted of a thin inner germinal layer, an eosinophilic laminated layer and an outer adventitial layer. Parasitic membranes (laminated membranes and germinal layers) were obvious in most examined sections, some were continuous and intact containing brood capsules with protoscolices (Fig. 1). Occasionally the cyst membrane was disrupted while in others only remnants of the membranes and degenerated protoscolices were observed. In fertile hydatid cysts the germinal layer consisted of thick eosinophilic layer from which buds of brood capsules, containing potoscolices, projected into the cyst lumen. The structural details of the protoscolices were clearly discernable, comprising of hooks, tegument, invagination, rostellar anlage and wall of brood capsule (Fig. 2). The inflammatory reaction, consisting of eosinophils, mononuclear cells, were observed immediate to the cyst layer and often extended into the surrounding alveoli and even to the adjacent terminal and small bronchioles. Occasionally, severe congestion, interalveolar and alveolar hemorrhages were seen. Immature protoscolices were sometimes observed in lung parenchyma (Fig. 3). In some segments attenuated or dysplastic pulmonary parenchyma was contiguous with palisading macrophages with foamy cytoplasm, eosinophils, epithelioid cells and multinucleated giant cells (Fig. 4). In some cases, there were foci of mineralization in the adventitial layer of the cyst (Fig. 5). The histological picture of the hydatid cyst in liver resembled to that of the lung. Sometimes, immature protoscolices were traced in the central veins (Fig. 6). Occasionally in chronic cases fibroplasia was more evident adjacent to the cysts, when the disrupted hepatocytes were evident in between the proliferating fibrous tissue. In such cases the fibroplasia was even seen in the portal triads resembling the portal cirrhosis. The hepatocytes revealed severe degenerative changes and pyknotic nuclei. Biliary hyperplasia and degenerative changes in biliary epithelium along with infiltration of inflammatory cells were observed in some sections of liver affected with hydatidosis. Histochemistry Combined PAS-Alcian blue staining revealed that the laminated membranes, germinal layers and brood capsules took very deep magenta colour positive for neutral mucopolysaccharides, however adventitia showed areas of positivity for acid mucopolysaccharides. Protoscolices also showed positive specks of acid mucopolysaccharides corresponding to the areas of calcareous corpuscles (Fig. 7). Sometimes the pericystic area showed a linear area of acid mucopolysaccharides but the adventitia continued to show focal distribution of the same (Fig. 8). Toluidine blue stained sections revealed presence of numerous mast cells in the fibro-cellular reaction adjacent to the cyst wall. The mast cells revealed metachromaticallystained granules both within and outside the cells adjacent to these cells (Fig. 9). Lung sections stained with Masson‘s trichrome revealed intense formation of collagen fibers in the pericystic connective tissue (Fig. 10). In case of liver, extensive fibrosis around cyst wall was evident which had involved the surrounding parenchyma. Comparative staining affinity of the protoscolices revealed calcareous corpuscles to be positively stained for both with combined Alcian blue-PAS and Toluidine blue, but was not stained with H&E and Masson‘s trichrome methods (Fig. 11). Top DISCUSSION Grossly, the lungs revealed single to multiple hydatid cysts of variable size, either fully or partially embedded in the lung parenchyma especially in the diaphragmatic lobes16. Lung parenchyma being spongy with greater capillary bed, besides favouring a greater distribution of onchospheres provides a more space for development of larger embedded cysts6. The cysts were soft with clear to slightly turbid fluid. While others were inspissated, caseated or calcified. It is opined that doughy cysts with inspissated contents observed in some cases probable reflect an attempt on part of the host to contain the cyst development16. Such efforts might have favoured the degeneration and calcification of the cyst with advanced age. Single or multiple, mostly small to medium sized, partially or fully embedded cysts were observed on both parietal and visceral surfaces of liver. Liver has been regarded as one of the most favoured sites for development of hydatid cyst6. However, depending upon the immune competence of the host, the compact tissue resists development of larger cysts and favours cyst calcification12. Calcifications, blood patches, inflammatory zone around cysts and pale margins of liver were reported17. |
In case of lungs, hydatid cyst wall, from inside to outside was composed of endocyst (proligerous membrane), ectocyst (laminated membrane) and pericyst18. There is a space between pericyst and ectocyst through which the tissue fluid and the nutrient medium flows. This space is the place of precipitation for neutral and acid polysaccharide9. |
The inflammatory reaction, consisting of infiltrating eosinophils, mononuclear cells, epithelioid cells and the multinucleated giant cells, were seen immediate to the cyst wall and often extended into the surrounding alveoli. Adjacent to the cysts lung parenchyma was atelectatic or emphysematic and showed congestion and haemorrhage as well18. Granulocytes, mainly derived from infiltrating eosinophils, were in the border zone between the laminated and adventitial layers and the adventitial layer surrounding the echinococcal cyst comprised an anuclear fibrous zone and a connective tissue zone were reported19. Histopathological picture of the cysts in liver was similar to that observed in lungs16,19. Liver sections prepared from the areas neighbouring the cyst wall showed congestion, haemorrhage, hepatocyte necrosis and atrophy together with fibrosis and cellular infiltration of mainly macrophages, lymphocytes and plasma cells were reported18. There was dilatation of sinusoids, and fibrosis was, seen in the portal area16. Further, the extensive fibroplasia and cirrhosis observed in some cases was attributed to immunological reactions of the host tissue18. Biliary hyperplasia and degenerative changes in biliary epithelium along with infiltration of inflammatory cells were observed in some affected livers9. |
The acellular laminar layer is a carbohydrateprotein complex with galactose, galactosamine and glucosamine as the principal component of the polysaccharide portion20. This layer is not present in young cysts until below 14–18 days old, it later appears as a thin, clear layer on its outer margin21. The germinal layer consists of distal cytoplasmic syncytium and a perinuclear layer containing tegument, glycogen, undifferentiated cells22. The laminated layer of the hydatid cyst stains strongly by Schiff‘s reagent (PAS) and is an excellent diagnostic marker in various histological studies21. The cystic wall consisted of three layers which were germinal, laminated and fibrous layer, respectively. The glycogen and mucopolysaccharide content increases in these layers in infected sheep19. The components of the brood capsule are positive for PAS following their extraction using amylase23. Qualitative increase in both acid and neutral mucopolysaccharides in and around the lesions may be attributed to inflammatory process16. The pericytic area showed a linear area of acid mucopolysac-charides. Between the pericyst and the ectocyst there is a space through which tissue fluid and nutrient medium flows18. The space between the pericyst and the ectocyst is the place where precipitation of neutral and acid polysaccharide was observed. |
Masson‘s trichrome staining demonstrated collagen fibers in cyst wall in both lung and liver sections. The fibrous tissue associated with hydatidosis in sheep. The occurrence of collagen reflects the inflammatory response to persistent irritation which may be ascribed to the consistent, slow exosmosis occurring in the hydatid cyst16,19. This is essentially the basic protective response aimed at containment of the parasite which leads to formation of cyst wall. The pronounced fibrosis and cirrhosis observed in certain cases may be due to the immunological reactions of the host tissue. |
Occurrence of mast cell reaction predominantly in the vicinity of the developing cysts, peribronchial and peribronchiolar regions of lungs, was an indication of local typel hypersensitivity and has also been frequently observed in other parasitic infections of lung24. Mast cells have a significant role in a myriad of inflammatory diseases and are also involved in remodeling of the tissues. Mast cell activation is triggered by tissue hypoxia which in turn releases various proteolytic enzymes, angiogenic factors and growth factors which in turn mediate the tissue destruction and its remodelling in a variety of physiological as well as pathological conditions25. However, in case of hydatidosis, whether this reaction occurs primarily against migrating oncosphere or as a result of exudation of hydatid fluid from enlarging cyst remains to be determined. The hydatid fluid has been found to be highly antigenic leading to acute hypersensitivity reaction26. Anaphylaxis following rupture of cyst has been frequently observed27. Mast cells secrete a huge amount of mediators having proteolytic, growth and proangiogenic effects and hence, they prove to be very important cell type involved in the pathogenesis of many diseases28. In our study metachromatic areas and mast cells accumulation was demonstrated in and around fibroplastic areas around the cyst. There is significant proliferation of perivascular mast cells and their enhanced secretory activity under chronic cases. Mediators that are released from the mast cells regulate the tissue remodelling thereby may also contribute to pulmonary fibroplasia29. The role of interaction between mast cells and fibroblasts during the progression of lung fibrosis and an increased load of active mast cells near the fibroblast foci and alveolar type II cells were reported30. |
In conclusion, inflammatory reaction against the cyst was of variable degree. Further, even within the same animals the pathogenicity to the individual cysts was variable and was related to the age of the cysts. Histopathological picture was almost similar in lung and liver except recruitment of increased numbers of mast cells in lung in the vicinity of the cyst which might be significantly involved in inducing Type-1 Hypersensivity. |
Top ACKNOWLEDGEMENTS Authors thank the Director of Research, Sher-e- Kashmir University of Agricultural Sciences and Technology, Kashmir for providing financial support to conduct this study. We also thank Mr. Ali Mohammad, Mr. Mohammad shafi and staff of histopathology laboratory for their help, cooperation and support extended during the period of study. |
Top Figures | Fig. 1.: Section of the sheep lung affected with hydatidosis revealing parasitic membranes (laminated membranes (LM) and germinal layers (GM)), Adventitial layer (AL), brood capsules with Protoscolices (P). H&E ×100
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| | Fig. 2.: Section of the sheep lung affected with hydatidosisshowing structural details of the protoscolices. (H: Hooks; IN: Invagination; RA: Rostellar anlage; R: Rostellum TG: Tegument; WB: Wall of brood capsule LU: Lumen of brood capsule. H&E ×1000
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| | Fig. 3.: Section of the sheep lung affected with hydatidosisexhibiting immature protoscolices in lung parenchyma. H&E ×100
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| | Fig. 4.: Section of the sheep lung affected with hydatidosisdepicting macrophages, eosinophils and Langhan‘s type giant cells adjacent to the cyst. H&E ×400
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| | Fig. 5.: Section of the sheep lung affected with hydatidosis showing foci of mineralization in the adventitial layer of the cyst (arrow). H&E ×100
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| | Fig. 6.: Section of the sheep liver affected with hydatidosis evincing immature protoscolices in the central vein. H&Ex400
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| | Fig. 7.: Section of the sheep lung affected with hydatidosis exhibiting laminated membranes, germinal layers, and brood capsules positive for neutral mucopolysaccharide and the adventitia positive for acid mucopolysaccharides. Alcian Blue-PASx100
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| | Fig. 8.: Section of the sheep lung affected with hydatidosis revealing the pericysticarea positive for acid mucopolysaccharides. Alcian Blue-PASx100
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| | Fig. 9.: Section of the sheep lung affected with hydatidosis revealing metachromatically stained granules both within and outside the mast cells adjacent to the cyst wall. Toluidine Blue × 1000
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| | Fig. 10.: Section of the sheep lung affected with hydatidosis revealing marked formation of collagen fibers in the pericystic connective tissue reaction. Masson‘s trichrome x100
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| | Fig. 11.: Section of the sheep lung affected with hydatidosis showing protoscolices with H&E stain (A) corresponding Combined Alcian Blue-PAS stained section (B) Toluidine Blue stained section (C) and Masson‘s trichrome stained section (D) x400. Calcareous corpuscles were stained positively both with combined Alcian blue-PAS (arrows) and toluidine blue (arrows) but not stained with H&E and Masson‘s trichrome methods
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