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ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 2  |  Page : 92-97  

Morphological and histological analysis of Cotylophoron cotylophorum treated with Acacia concinna


Department of Zoology, Unit of Parasitology, Pachaiyappa's College, Chennai, Tamil Nadu, India

Date of Acceptance22-Jun-2017
Date of Web Publication25-Sep-2017

Correspondence Address:
P Priya
Department of Zoology, Unit of Parasitology, Pachaiyappa's College, Chennai - 600 030, Tamil Nadu
India
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DOI: 10.4103/tp.TP_65_16

PMID: 29114486

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   Abstract 


Objective: Paramphistomosis (stomach fluke disease) is a parasitic infection caused by digenetic trematodes and is considered to be one of the most important parasitic diseases affecting livestock worldwide. This disease is widely prevalent in India, and the highest incidence is reported during monsoon and post-monsoon months. In the present study, in vitro effect of aqueous extract of pods of Acacia concinna (AcP E) on the morphology and the histology of the digenetic trematode Cotylophoron cotylophorum have been investigated.
Materials and Methods: The in vitro effect of Ac PE on the morphology and the histology of a digenetic trematode C. cotylophorum have been examined using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and light microscopic techniques.
Results: The SEM micrograph of treated flukes showed the appearance of few blebs near the oral region and rupture of the dorsal surface of the tegument. The light and TEM observations revealed significant deleterious changes in the internal organization of the fluke. Severe injury to the tegument due to bleb formation, detachment of tubercles, and vacuolization of the subtegumental region was observed. Nuclear indentation, cytoplasmic autolysis, and mitochondrial abnormalities were the other prominent observations.
Conclusion: The results of the present study convincingly showed that Ac PE is an effective anthelmintic causing detrimental effect to C. cotylophorum and appears to be a potent phytotherapeutic agent to control paramphistomosis.

Keywords: Acacia concinna, anthelmintic, Cotylophoron cotylophorum, morphology, paramphistome, ruminant


How to cite this article:
Priya P, Veerakumari L. Morphological and histological analysis of Cotylophoron cotylophorum treated with Acacia concinna. Trop Parasitol 2017;7:92-7

How to cite this URL:
Priya P, Veerakumari L. Morphological and histological analysis of Cotylophoron cotylophorum treated with Acacia concinna. Trop Parasitol [serial online] 2017 [cited 2018 May 24];7:92-7. Available from: http://www.tropicalparasitology.org/text.asp?2017/7/2/92/215515




   Introduction Top


The paramphistome Cotylophoron cotylophorum causes parasitic gastroenteritis in small ruminants resulting in severe morbidity and mortality. Many of the anthelmintic drugs available to combat helminth infections have developed resistance.[1] An alternative to chemotherapeutic drugs is to search for the antiparasitic plant extracts or secondary metabolites derived from them. Several plants have anthelmintic properties and are used to eradicate parasites since ancient time.[2] Acacia concinna is commonly called as soap pod belongs to family Fabaceae. The plant is widely used in traditional system of medicine for control of pathogenic bacterial and fungal diseases.[3] Cercaricidal activity of A. concinna has been reported by Nama and Bhatnagar.[4] Our continuous effort on developing herbal drugs, we have reported the in vitro and in vivo anthelmintic effect of A. concinna against paramphistome.[5],[6] Since the topic is economically and scientifically driven the clues to identify the plausible mode of the action of A. concinna, a thorough study on the morphology and histology of the C. cotylophorum exposed to aqueous extract of pods of A. concinna (Ac PE) would be a feasible investigation.

Parasites have a potential ability to attain wide variety of physiological and biochemical adaptations to survive within the specialized environment of the host.[7] The tegument of the parasite is metabolically active and morphologically specialized to perform various functions such as physical and immunological protection, selective uptake of nutrients, synthesis of secretory substances, osmoregulation, excretion, and the reception of stimuli.[8],[9],[10] Any damage to the tegument will undoubtedly disrupt its physiological functions. Therefore, the present study aimed to investigate the efficacy of Ac PE on the surface topography and various organ systems of C. cotylophorum.


   Materials and Methods Top


Collection and maintenance of parasites

Adult live flukes were collected from the rumen of the sheep slaughtered at the Perambur abattoir, Chennai, Tamil Nadu, India. The worms were washed in physiological saline and maintained in Hedon-Fleig solution.[11]

Preparation of plant extract

Pods of A. concinna were purchased from local authorized dealer. The pods were coarsely powdered. The Ac PE was prepared by boiling 10 g of powdered pods in 100 mL of distilled water for 10 min under constant stirring, which was then made into a fine paste. The prepared paste was then diluted to 0.5 mg/mL of concentration using Hedon-Fleig medium.

Exposure of adult live parasites to aqueous extract of pods of Acacia concinna

The concentration of 0.5 mg/mL was selected based on the quantitative measure of the motility of the parasites. Adult live flukes numbering to ten were incubated in 25 mL of 0.5 mg/mL of Ac PE for 8 h. Appropriate control without the plant extract was also maintained simultaneously under same environment. All the flukes treated with Ac PE survived till 8 h with reduction in motility compared to the control flukes.

Sample preparation

The effect of Ac PE on the tegument of C. cotylophorum was studied using a scanning electron microscopic (SEM). For SEM studies, the control and treated parasites were washed in phosphate buffer saline (pH 7.4) and fixed in 2.5% glutaraldehyde (pH 7.4) at 4°C for 18 h. They were washed using distilled water and dehydrated through graded alcohol series. The specimens were then kept in a desiccator, air dried, and coated in vacuum with gold using IB2 ion coater (Model IB2 Eiko Engineering Co. Ltd.) and observed with a Hitachi S415A SEM. Photomicrographs were taken at various magnifications with an Exakta exa 1 camera, using Nova FP4 120 type film.

Histopathological changes of Ac PE-treated parasites were observed using light and transmission electron microscopy (TEM). For light microscopic (LM) studies, the control and treated parasites were washed in physiological saline and fixed in Bouin's fixative for 12 h. These fixed flukes were washed in distilled water and dehydrated through graded alcohol series, cleared in xylene, and embedded in paraffin wax. Sections were cut at 5 mm thickness and stained in hematoxylin and eosin and were observed under binocular compound microscope for various cellular details.[12] Photomicrographs were taken at various magnifications using Leitz (model: Diaplan) photomicroscope.

For TEM studies, the control and treated parasites were fixed in 3% glutaraldehyde and postfixed in 1% osmium tetroxide. Double fixation gives stability during dehydration, embedding, and also during electron bombardment. The specimens were dehydrated by ascending graded alcohol series and cleared by propylene oxide. Tissues were embedded in silico nized rubber mould with epoxy resin and kept in incubator at 60°C for 48 h. One micron thick sections were cut, before trimming of the blocks for ultramicrotomy. This helps for marking the areas of interest. Further, ultrathin sections (1 nm) were cut using leica ultramicrotome. These ultrathin sections were mounted on the copper grid and stained with double stain, metallic uranyl acetate, and Reynold's solution. The sections were observed using Philips 201C TEM and photographed at different magnifications.


   Results Top


Scanning electron microscopic observation

[Figure 1] shows the SEM images of control [Figure 1]a and Ac PE-treated C. cotylophorum [Figure 1]b,[Figure 1]c,[Figure 1]d,[Figure 1]e,[Figure 1]f. The SEM image of untreated C. cotylophorum showed a smooth tegument [Figure 1]a. Ruptured teguments with blebs were observed on the dorsal surface of Ac PE-treated flukes after 8 h of incubation [Figure 1]b. The oral region of the untreated fluke is provided with sensory papillae [Figure 1]c. Numerous smaller blebs were observed near the oral region of Ac PE-treated fluke [Figure 1]d. The posterior sucker of untreated fluke appeared smooth [Figure 1]e. Blebs were found near the posterior sucker region of Ac PE-treated fluke with rough tegument [Figure 1]f.
Figure 1: Scanning electron micrographs of tegument, oral sucker, and posterior sucker of untreated Cotylophoron cotylophorum and those treated with aqueous extract of pods of Acacia concinna (0.5 mg/mL) after 8 h exposure. (a) Untreated fluke showing smooth tegument (t) (Bar-100 μm). (b) aqueous extract of pods of Acacia concinna-treated showing ruptured tegument (arrowheads) and blebs (arrows) (Bar-100 μm). (c) Untreated fluke showing mouth (m) and sensory papillae (sp) (Bar-20 μm). (d) aqueous extract of pods of Acacia concinna-treated showing rupture of blebs (arrows) near the oral sucker (os) region (Bar-25 μm). (e) Untreated fluke showing posterior sucker (ps) (Bar-35 μm). (f) aqueous extract of pods of Acacia concinna-treated showing rough tegument (rt) and blebs (arrows) in the posterior sucker × (Bar-35 μm)

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Light microscopic observation

[Figure 2] and [Figure 3] show the LM pictures of control and Ac PE-treated flukes.
Figure 2: Photomicrographs of the tegument, posterior sucker and parenchyma of untreated Cotylophoron cotylophorum and those treated with aqueous extract of pods of Acacia concinna (0.5 mg/mL) after 8 h exposure. (a) Untreated fluke showing smooth tegument (t), followed by subtegumental layer (st) ×140. (b) aqueous extract of pods of Acacia concinna-treated showing detachment of the tegumental layer (arrows) and exposing subtegumental (st) region ×160. (c) Untreated fluke showing posterior sucker (ps) with strongly developed musculature ×180. (d) aqueous extract of pods of Acacia concinna-treated showing appearance of bleb (arrow) in the tegument of posterior sucker region ×180. (e) Untreated fluke showing intact parenchyma (p) ×170. (f) aqueous extract of pods of Acacia concinna-treated showing intense vacuolization (v) in the parenchyma ×170

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Figure 3: Photomicrographs of the gastrodermis, testis, ovary, and egg of untreated Cotylophoron cotylophorum and those treated with aqueous extract of pods of Acacia concinna (0.5 mg/mL) after 8 h exposure. (a) Untreated fluke showing lumen lined by columnar epithelium (gastrodermis, gd) and rests upon basement membrane (bm) ×120. (b) aqueous extract of pods of Acacia concinna-treated showing lesions in the gastrodermis (arrows) ×120. (c) Untreated fluke showing testis (tst) with germinal epithelium (ge) (arrow) ×120. (d) aqueous extract of pods of Acacia concinna-treated showing vacuoles (v) and disruption of germinal epithelium (arrow) ×120. (e) Untreated fluke showing intact ovary (ov) lined with germinal epithelium (ge) (arrow) ×100. (f) aqueous extract of pods of Acacia concinna-treated showing vacuole (v) due to destruction of germ cells × 100. (g) Untreated fluke showing intact eggs (e) ×60. (h) aqueous extract of pods of Acacia concinna-treated showing atypical egg with destruction of yolk cells (arrows) ×60

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Tegument

Tegument of the control flukes shows smooth spineless tegument followed by subtegumental layer [Figure 2]a. Complete detachment of tegumental layer was observed in Ac PE-treated flukes [Figure 2]b.

Posterior sucker

The musculature of the posterior sucker of control fluke is strongly developed and is used for the attachment of the fluke to the host ruminal wall [Figure 2]c. In Ac PE-treated flukes, after 8 h and exposure blebs appeared in the tegument of posterior sucker [Figure 2]d.

Parenchyma

The parenchyma of untreated fluke appeared intact [Figure 2]e. Severe lesions appeared in the parenchyma of Ac PE-treated flukes [Figure 2]f.

Intestine

Intestinal cecum of untreated fluke has a thin wall and large lumen. The lumen is lined by a columnar epithelium (gastrodermis), which rests upon a basement membrane [Figure 3]a. Severe lesions were observed in the gastrodermis of Ac PE-treated flukes [Figure 3]b.

Testes

The walls of the testes were lined by germinal epithelium where from the spermatocytes were formed and liberated into the lumen [Figure 3]c. Disintegration of germinal epithelium of testes along with occurrence of vacuoles was observed in Ac PE-treated flukes [Figure 3]d.

Ovary

The ovary is rounded, median, and posttesticular and is lined by germinal epithelium in the untreated flukes [Figure 3]e. Inner medulla consisted of germ cells. Degenerating germ cells were seen in the vacuolar spaces [Figure 3]f.

Eggs

The eggs of the untreated flukes are oval in shape, with distinct yolk cells [Figure 3] g. Profound damage to the eggs such as disintegration of the yolk cells was observed in Ac PE-treated flukes [Figure 3]h.

Transmission electron microscopic observations

The tegument of the untreated fluke consists of compact tubercles, and tegumental syncytium is intact [Figure 4]a. Detachment of tegumental layer was much profound in Ac PE-treated flukes [Figure 4]b.
Figure 4: Transmission electron micrographs of the tegument and gastrodermis of untreated Cotylophoron cotylophorum and those treated with aqueous extract of pods of Acacia concinna (0.5 mg/mL) after 8 h exposure. (a) Untreated fluke showing compact tubercles (tu) (arrows) and intact tegumental syncytium (ts) (Bar-3 μm) (b) aqueous extract of pods of Acacia concinna-treated showing detachment of tegumental layer (arrows) and Subtegument (st) exposed with few vacuoles (v) (Bar-3 μm). (c) Untreated fluke showing intact nucleus (n) in a gastrodermis cell. The cytoplasm (c) is intense and evenly distributed. Granular endoplasmic reticulum (ger) is also seen (Bar-0.5 μm). (d) aqueous extract of pods of Acacia concinna-treated showing swollen granular endoplasmic reticulum (ger) (arrow) and vacuoles (v) in the cytoplasm. Disruption of perinuclear membrane with spillage of chromatin material (arrowhead) (Bar-0.22 μm). (e) Untreated fluke showing mitochondria (m) (arrows) in gastrodermis cells (Bar-1 μm). (f) aqueous extract of pods of Acacia concinna-treated showing swollen and abnormally shaped mitochondria (arrows) (Bar-1 μm)

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Nucleus with intact cytoplasm and granular endoplasmic reticulum is found in the gastrodermis region of the untreated fluke [Figure 4]c. Severe indentation of the nucleus with vacuoles in the cytoplasm was observed in Ac PE-treated flukes [Figure 4]d. Ac PE was much effective in disrupting the nuclear membrane.

Numerous mitochondria are observed in the gastrodermis region of the untreated fluke [Figure 4]e. Swelling of the mitochondria with disintegration of mitochondrial cristae was observed in Ac PE-treated flukes [Figure 4]f.


   Discussion Top


The present study delineated the anthelmintic potential of A. concinna against C. cotylophorum. The action of many anthelmintics is reflected in their ability to inhibit the activity of enzymes involved in carbohydrate metabolism. Our earlier report on the in vitro effect of A. concinna on enzymes of glucose metabolism of C. cotylophorum revealed that Ac PE was effective at 0.5 mg/mL after 8 h exposure.[5] Hence, this concentration of Ac PE was used in the present study to analyze the morphological and histopathological changes of treated flukes. The effect of flukicidal agents on the morphological integrity of trematodes usually exhibited the tegumental distortions and vacuole formation.[10],[13],[14],[15],[16],[17],[18],[19]

The present study corroborates with the earlier reports. SEM observation of Ac PE-treated flukes showed ruptured tegument with swelling and bleb formation [Figure 1]b and [Figure 1]d. The external surface of trematodes is composed of multifunctional syncytial tegument, the major interface through which various physiological functions such as digestion and absorption take place.[20],[21] Ac PE affected this normal physiological barrier, which could ultimately result in paralysis and subsequent mortality of parasites.

Remarkable deleterious effect of A. concinna on C. cotylophorum could be clearly seen from the LM and TEM observations. The Ac PE-treated flukes showed severe injury to the tegument due to detachment of tubercles and vacuolization of the subtegumental region as observed from the morphological studies [Figure 2]b and [Figure 4]b. Vacuolization of parenchyma was also prominent [Figure 2]f. Bleb formation may be a result of disruption of the brush border cytoskeleton as suggested by Stoitsova for Hymenolepis fraterna treated with colchicine.[22] Swelling of basal infolds of the tegument eventually leads to sloughing of the tegumental syncytium. The edema spreads internally to affect subtegumental region. At the cellular level of the tegument nuclear, indentation and mitochondrial abnormalities were the conspicuous changes in treated flukes [Figure 4]d and [Figure 4]f. Mitochondrial abnormalities, i.e., change in the size and shape and disintegration of cristae observed which suggests the impairment of carbohydrate metabolism. Further, as observed from the LM and TEM studies, cytoplasmic autolysis and cellular necrosis were prominent, which may lead to death of the parasites.

A number of studies on tegumental pathology induced by anthelmintics have suggested that the drug acts to alter the permeability and osmoregularity properties of the tegument.[23],[24] The severe damage to tegument would undoubtedly lead to the impairment of several important physiological functions such as osmo-ionic regulation that appears to be severely affected due to the characteristic swelling of the basal infolds in Ac PE-treated flukes. The swelling of the intracellular organelles may be attributed to a change in the tegumental permeability of the apical plasma membrane of the fluke as proposed by Anderson and Fairweather in Fasciola hepatica treated with diamphenethide.[10]

The posterior sucker of the parasite is highly muscularized and is used for attachment to the host ruminal wall. Damage to the muscles associated with posterior sucker and those associated with motility may lead to loss of attachment and subsequent expulsion of the parasites from the host's gut. Treatment with the Ac PE induced bleb formation on the tegument of posterior sucker [Figure 1]f and [Figure 2]d. Numerous lesions were observed in the gastrodermis of the parasite [Figure 3]b. These results suggest that the uptake of the plant extract by the parasite was not only through the tegument but also through the alimentary canal as reported by Seo et al.[25] Lesions in the gastrodermis may result in the impairment of feeding mechanism and absorption of nutrients.

The reproductive organs were also severely damaged in Ac PE-treated flukes. The ovary and testes showed disintegration of germinal epithelium and vacuolization [Figure 3]d and [Figure 3]f. Similar phenomenon was observed in nematodes and trematodes treated with praziquantel,[26],[27] ivermectin,[28] niclosamide, and oxyclozanide.[18],[29] Damage to reproductive system may result in reduced egg production and irregular embryonation.


   Conclusion Top


The morphological and histological changes of C. cotylophorum induced by AcPE here in may be summed up in supposition that Acacia concinna is potent anthelmintic to treat paramphistomosis. Furthermore, the detailed study on the separation and structural identification of the bioactive compound responsible for the anthelmintic effect would be necessary for the structure–activity relationship.

Financial support and sponsorship

We gratefully acknowledge the financial assistance provided by Department of Science and Technology (DST), Ministry of Science and Technology, New Delhi.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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