ACANTHOCEPHALA) with the HOST SYNAPTURA ORIENTALIS Sheema SH

International Journal of Scientific Research and Review ISSN NO: 2279-543X

HISTOPATHOLOGY OF ECHINORHYNCHUS VELI (ACANTHOCEPHALA) WITH THE HOST SYNAPTURA ORIENTALIS Sheema SH

Research Division, Department of Zoology, Mar Ivanios College, Nalanchira, Thiruvananathapuram-695 015, Kerala, India.

Abstract

The study elucidated the histopathological manifestations in the intestine of the brackish water edible fish, Synapturaorientalis, naturally infected with the acanthocephalan parasite, Echinorhynchusveli. For histopathology examination the relevant parts of uninfected and infected hosts, were processed following routine histology protocols. Heavy infections were observed in many hosts at the middle section of the intestine. The deep penetration of worms elicited intense host response. The host’s immune reactions were characterized by inflammatory responses involving infiltration and aggregation of inflammatory cells and hyperplasia. Formation of a dense capsular structure, made of inflammatory cells, macrophages and tissue debris, was also observed. In most cases, the capsules were detached from muscularis clearly depecting the degeneration of tissues. It was seen that the intestinal wall was thicker at the point of attachment than the unattached areas. All the layers of the intestine showed inflammation and degeneration. Necrosis of the tissues, inflammation, granulation and structural irregularities were generally found in the infected intestine. The general histopathology revealed the breakdown of tissues which implied the disrupted absorptive mechanism that could result in the restrictive absorption of nutrients by the host.

INTRODUCTION

Histopathology is the study of the diseases affecting the tissues and organs of an organism. With reference to the parasitic infection, histopathology deals with the tissue damage caused by parasitic organisms on specific organs like intestine. Tissue damage could be traumatic due to physical pressure exerted by the parasites or may be caused by the toxic secretory or excretory products of the parasites which may sometimes lead to hypersensitivity reactions. Many abnormal conditions are encountered in various organs due to parasitic infection. Parasitic diseases, either alone or in conjunction with other environmental stresses, may influence weight or reproduction of the host, alter its population characteristics and affect its economic importance (Rohde, 1967).

In fish, as in other vertebrates, the digestive tract is the favoured habitat for several worms and this is likely due to the relative ease of pathogen access via the oral route, ready availability of attachment sites and nutrients, and a relatively nonaggressive immune response

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(Secombes and Chappell, 1996;Ringoet al., 2007). Often, infection of the gastrointestinal tract by helminths has detrimental effects on digestive function (Fairweather, 1997).

Acanthocephalans attach to the host intestinal wall using the hooked proboscis, causing extensive tissue damage and potential fatality in various vertebrates (Nickol and Crompton, 1985). The spines on the proboscis of some acanthocephalans are known to cause lesions in the intestinal wall of host and ultimately affect growth (Buchmann, 1995). The attachment of the armed proboscis causes irreversible mechanical damage to the intestinal tissues and leads to pathological changes. Damage of the intestinal villi, formation of granular tissues and capsule formation associated with host immune responses may seriously affect the digestive and absorptive efficiency of the host (Ali Othman and Al Ghamdi, 2013).

In great majority of previous studies on host-parasite interactions in fish, there has only been descriptions of the physical damage caused by the parasites. The attachment organs of endoparasitichelminths frequently induce inflammation of the alimentary tract (Dezfuliet al., 2011). However detailed information is lacking on the inflammatory responses. Inflammation is a protective reaction of the host in response to injury, resulting in specific chemical and morphological alterations in cells and tissues (Suzuki and Iida, 1992).

A preliminary study on the histopathology of the gut of flat fish S.orientalisinfected with E.veliwas carried out by George and Nadakal (1982). Since then no detailed work has done on this aspect, and hence the present study.

MATERIALS AND METHODS

Uninfected as well as infected intestines with parasites in situwere fixed in 10% neutral buffered formalin for 48 hours. After fixation, the samples were processed following routine histology protocols and paraffin sections of 5 to7 μm thickness were cut and stained with haematoxylin and eosin and mounted in Canada balsam. The stained sections were examined under light microscope and microphotographs taken.

RESULTS

In the infected hosts, the worms were mostly found localized in the middle region of the intestine 10 cm anterior to the cloaca and were remarkably few in duodenal and posterior part of the intestine. However, in heavy infection, worms were found to extend towards the posterior region of the intestine.A smooth external surface was observed throughout the length

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of the intestine of the uninfected fish. But in the infected intestinethere were numerous nodules indicating the sites of attachmentand the penetration of worms (Fig.1).

Histology of uninfected host intestine

Distinctive four layers were observed in the cross section (Fig. 2). The outer most layer was serosa with thick multicellular connective tissues comprising blood vessels, nerves and lymphatics. Just beneath,a muscular layer with outer longitudinal and inner circular layers.Inner to the muscularis, the submucosal layer with coarse areolar connective tissues and elastic fibres.This layer also contained blood vessels, fat cells and nerves. The innermost layer was mucosa made of columnar epithelial cells with flattened basal nuclei and with villi and micro-villi. Interspersed among the columnar cells were a few goblet cells. The mucosal layer was supported by loose connective tissue, the lamina propria just below the basal lamina of the epithelial membrane (Fig. 3).

Histopathological observations

The worms penetrated upto the muscularis layer (Fig. 4) leading to the formation of a dense capsular structure made of inflammatory cells, macrophages and tissue debris. The intestinal wall was thicker at the points of attachment than the unattached areas. Here, the mucosal and submucosal layers were replaced by granulation tissues and hence thicker. The parasites elicited an inflammatory response in the host tissues resulting in dense capsules. The upper layer of the muscularis showed marked degenerative changes (Fig.5). Another important feature observed was the complete destruction of the columnar epithelium in mucosal layer andhypertrophy of lamina propria. The inflammatory cells infiltrated upto the atrophied muscle coats and sub-serosal connective tissues (Fig. 6).

All the layers of the intestine showed inflammation and degeneration in the heavily infected cases (Fig. 7). The longitudinal section of the infected intestine showed thick capsular structure between mucosa and sub-mucosa. Sub-mucosa was found capsulated and detached from the muscularis layer clearly depicting the degeneration of tissues (Figs. 8 and 9).Necrosis of the tissues, inflammation, granulation and structural irregularities were generally noted in the infected host intestine.

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II MuL

MuL

SML

Fig. 1 Intestines of infected and uninfected S.orientalis (20 x) Fig. 2 C. S. of uninfected intestine of S. orientalisshowing different layers (10 x)

SL-Serosal Layer MuL-Mucosal Layer ML-Mascularis Layer SML-Sub Mucosal Layer II-Infected Intestine UI-Uninfected Intestine N-Nodule

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3 EL

MuL

Fig. 3 C. S. of uninfected intestine of S. orientalisshowing mucosal layer (45 x)

CL-Columnar Cell V-Villus EL-Epithelial Cell G-Goblet Cell LP-Lamina Propria MuL-Mucosal Layer

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SML

P DML

NT IC

Fig.4 C. S. of intestine of S. orientalis showing worm penetration (45 x) Fig.5 C. S. showing degenerative changes in the intestine of S. orientalis (45 x)

H-Hook ML-Mascularis Layer C-Capsule IC-Inflammatory Cells P-Parasite NT-Necrotic Tissue SL-Serosa Layer SML-Sub Mucosal Layer EL-Epithelial cells DML-Detachment of Mascularis Layer

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AML

7 C

DBV

Figs. 6 & 7 C. S. of intestine of S. orientalis showing cell infiltration (45 x)

C-Capsule IC-Inflammatory Cells P-Parasite DBV-Dilated Blood Vessel GT-Granulation Tissue AML-Atrophied Muscularis Layer SM-Separation of Muscle Fibres

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9 P

SML

Figs. 8 & 9 C. S. of intestine of S. orientalis showing worm penetration and capsule formation (45 x)

SL-Serosal Layer C-Capsule IC-Inflammatory Cells P-Parasite ML-Mascularis Layer SML-Sub Mucosal Layer

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DISCUSSION

Histopathology is a powerful tool in determining the extent of damage caused by parasites on their hosts. Histological analysis of the digestive system is considered a good indicator of the nutritional status of fish. The intestine and liver are the most important organs in digestion and absorption of nutrients from food, and therefore monitoring of these organs is considered necessary.

The general histopathology revealed the breakdown of tissues which implied the disrupted absorptive mechanism that could result in the restrictive absorption of nutrients by the host. Generally in parasitic infections, the host immune system reacts in different ways on the parasite and the cellular responses invariably attempt to isolate and destroy the parasites. Usually histopathological changes in fish intestines due to acanthocephalan infections depend on various factors such as species of parasite and host-parasite interactions. Length of the neck and proboscis and presence or absence of a proboscis bulb affect the true pathological outcome observed in the host fish.Accordingly, the attachment organs of endoparasitichelminths frequently induce inflammation of the alimentary tract (Taraschewski, 2000; Dezfuli and Giari, 2008; Dezfuliet al., 2008a).

In the present study the histological observations revealed that there were four layers of different cell composition in the uninfected host fish intestine. Many authors carried out this type of study earlier and observed that fish intestinal epithelia were expanded through folding, but lack the typical crypts of the mammalian villi. The epithelium, together with the underlying lamina propria constitutes the mucosa. Adjacent to the lamina propria is the connective and contractile muscularis mucosa which separates the lamina propria from the submucosa. Within the submucosal layer, the submucosal nerve plexus is found. Further away from the lumen, the circular muscle layer is followed by the myenteric nerve plexus and the longitudinal muscle layer. The perimeter of the intestine is lined by the serosa, a connective tissue layer attached to the mesenteric tissue (Jutfelt, 2006; Clements and Raubenheimer, 2006).

Histopathological changes in the alimentary canal of fish infected with Acanthocephala varied depending on the species of parasite (George and Nadakal, 1981). Pomphorhynchuslaevis frequently disrupted the gut wall (George and Nadakal, 1981). Mature parasites also provoke a strong host response, and the praesoma becomes invested with a fibrous capsule of host tissue (Wanstallet al., 1988). The present finding on the nature of the pathological changes in the intestinal tissues, via rupture of the mucosal epithelium at the point

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of attachment and the tissue damage to the various layers of the intestine are in agreement with the reports in Sclerocollumsp by Wanstallet al., (1982) and in Procamallanussp by Salah et al., (2014). Capsule formation is not an invariable consequence of acanthocephalan infections although connective tissue proliferation and inflammatory cell infiltration are common (Bullock, 1963; Schelhaas, 1980; George and Nadakal, 1981; Taraschewski, 2000). The capsule may prevent the gut contents from contaminating the abdominal cavity and causing peritonitis (Pippy, 1969). The deep penetration of Sclerocollum sp. considerably damaged the tissue architecture of the host intestine leading to destruction and necrosis of villi and extension of fibroplasia to muscular mucosa (Amin and Heckmann, 1992; de Buron and Nickol, 1994; Salah et al., 2014).

In heavy infections, the intestine in the present study,appeared to be packed with the worms, almost blocking the intestinal lumen, which will adversely affect the movement of digested materials within the intestine and the absorption of nutrients. Occlusion and significant distention of caeca were reported in Lepomiscyanellus infected with the acanthocephalan Leptorhynchoidesthecatus (de Buron and Nickol, 1994).

Increased number of goblet cells, eosinophil aggregation and lymphocyteinfilteration stimulated by infection have been well documented (Hamerset al.,1992; de Buron and Nickol, 1994; Thomas, 2002; Dick and Choudhury, 2006; Salah et al., 2014).Degeneration as well as necrosis of the mucosal epithelium has been reported in Salmogairdneriinfected with P. laevis and excessive mucus secretion in Rachycentroncanadum infected with the acanthocephalan Serrasentisnadakali (George and Nadakal, 1981).Parasite induced fibrosis in the intestinal wall along with the associated biochemical reactions may affect gut motility (Nickol, 2006). The granulation of tissues observed in the present study would, therefore, be regarded as blockades of intestinal free movements.

In L.cyanellus, the proboscis occasionally reached upto the musclaris and mucosa (de Buron and Nickol, 1994). In green sun fish infected with L. thecatus,erythrocytes aggregated at the sites of attachment in villi indicating haemorrhage(de Buron and Nickol, 1994). Inflammation is a protective reaction of the host in response to injury, resulting in specific chemical and morphological alterations in cells and tissues (Suzuki and Iida, 1992). Inflammation comprises of a complex series of homeostatic mechanisms involving the circulatory, immune and nervous systems in response to tissue injury or infection (Sharkey, 1992).

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The present observation of nodules on the external surface of the infected intestine may be due to deeper penetration of proboscis and bulb and can be an indication of the presence of the worms inside. This finding is similar to that reported by Dezfuli (1991) and Dezfuliet al., (2002) in P. laevis infection, on Leuciscuscephalus. Sometimes, the proboscis perforates the capsule and penetrates the liver (Chaicharn and Bullock, 1967; McDonough and Gleason, 1981; Taraschewski, 1989).

The proboscis of E. veli was found deeply penetrated till lamina propria thereby causing degeneration of villi and epithelial layer. Similarly, complete penetration of proboscis and bulb causes fibrosis, haemorrhage, inflammation and cell necrosis in host fishes infected with several acanthocephalan species (Bullock, 1963; Chaicharn and Bullock, 1967; McDonough and Gleason, 1981; Dezfuli, 1991; Taraschewski, 1989; Wanstallet al., 1986, 1988; Dezfuliet al., 2002, 2008a;Ibraq and Fayaz, 2012). In P.laevis infection the deeper penetration of proboscis and bulb might be assisted by proteolytic enzyme as suggested by Polzar and Taraschewski (1994). It was further shown that those acanthocephalan species which lack the activity of this enzyme cannot penetrate the collagenous tissues and take several days to penetrate the intestinal wall (Taraschewski, 1989).

Chronic inflammation, leading to an increased amount of connective tissue, thickening of lamina propria and infiltration of leucocytes, has been reported at the sites of A. jacksoni attachment (Bullock, 1963). Hamerset al. (1992) reported interspecific differences in the response of leucocytes in fish infected with P. ambiguusand suggested that the cellular defencemechanism may be involved in determining the host specificity, since unsuitable hosts expel the parasite within a few days.

Fish parasitology is, nowadays, considered the most inviting field of research because of the importance of fishes as valuable source of protein. The general histopathology revealed the breakdown of tissues which implied the disrupted absorptive mechanism which could result in the restrictive absorption of nutrients by the host. Heavy infections may cause functional disturbances, retard growth, increase the susceptibility of fish to other infections. (Paperna, 1980; Mahmoud, 1983).

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