Doctor of Philosophy in Zoology

Biochemical, Histopathological and Immunological Studies in White Rats infected with Setaria cervi

Thesis

Submitted For the Award of the Degree of

Doctor of Philosophy In Zoology

Sharba Kausar

Under The Supervision of

Prof. Wajihullah

DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 2017

Scanned by CamScanner Acknowledgement

First and foremost I am extremely thankful to Almighty Allah, the most beneficent and the most merciful, whose benign blessings gave me required strength and devotion for the completion of this work.

Next I would like to thank my family. My parents, who left no stone unturned to make me the person I am today. The whole credit of my achievement goes to their prayers and support during my moments of crisis. They form the backbone and origin of my happiness. My mother in the first place is the person who boosted my morale and inculcated in me the ability to take failure in stride and bounce back higher and stronger, it’s her prayers which are bound in this thesis. My father has always been a pillar of support for me. I owe to his deep feeling for bringing cheer at times of apparently sturdy situations during this work. He always supported my dreams and inspirations.

I thank them for their never ending concerns, their caring and gentle love which has enabled me to bear all the storms of life.

I would like to mention a note of appreciation for my sisters Tanzeem Jabbar and Tabassum Jahan, who always stood beside me, lended their helping hands during the hours of need. Their dedication and persistent confidence in me has taken the loads off my shoulders. Thanks for being compassionate and caring siblings.

With sincere feeling, I offer my heartiest gratitude to my proficient supervisor, Prof. Wajihullah, who has supported me throughout my study programme with patience and knowledge. His invaluable wisdom, enthusiasm as a scientist, wide knowledge and logical way of thinking inspires me immensely. His encouragement, parental affection, sense of forgiveness, scholarly guidance, painstaking efforts and trust lead to the preparation of this manuscript. One simply could not wish for a better advisor.

I would also like to thank Prof. Waseem Ahmad, Chairman, Department of Zoology for providing all necessary laboratory facilities, and I am also thankful to Prof. Iqbal Parvez (former Chairman, Department of Zoology). A special mention to Prof. S.M.A. Abidi, Prof. Malik Irshadullah and Dr. Khalid Saifullah. It’s my pleasure to thank them for their support and kind suggestions. I am highly delighted in paying my gratitude to Prof. Irfan Ahmad, Prof. Mukhtar Ahmad Khan, Prof. Qudsia Tahseen, Prof. Javed Musarrat, Prof. Bilqees Bano (Department of Biochemistry, AMU) and Prof. Ameer Azam (Department of Applied Physics, ZHCET) for their help whenever needed.

It gives me an immense pleasure to express my greatest thanks to all my labmates viz., Dr. Umm-e-Asma, Khadija Khan, Sana, Ansari Naheda who deserve the credit of enlivening the environment and enlightening my burden with their briskness. I specially want to thank Dr. Fakhra Amin for always being there in my good and bad times.

Cooperation of other seniors viz., Dr. P.A. Shareef, Dr.Yasir A. Khan, Dr. Sourabh Dwivedi, Dr. Abdul Hannan Khan, Dr. Shabnam Khan, Dr. Nuzhat Parveen, Dr. Arif Ahmad, Sabiha Khatoon, Dr. Sadia Rashid and Dr. Shabi Fatima Abidi has been noteworthy and are acknowledged with thanks. Moreover a sincere thanks to the research scholars of Section of Parasitology, Department of Zoology for their endless cooperation.

Hearty thanks to all my dear friends Malka Mustaqim, Sayma Samreen, Arjumend Shaheen, Prince Tarique Anwar, Ashiq Rafiq Malik, Md. Fareed, Sakil Ahmed, Syeda Uzma Usman, Yogita Varshney, Dr. Sumaya Ahad, Sabah Malik, Taiba Saeed, Dania Ahmed, Dr. Rushda Sharf, Ab latif Wani and Lubna Rehman for their valuable presence and timely support.

I am very thankful to the non-teaching staff Mr. Zaheer Ahmad, Mr. Raees and Mr. Mushahid for their cooperation. Lastly, thanks to the staff of the Department of Zoology and staff of the dean office of the Faculty of Life Sciences..

I am greatly indebted to the University Grants Commission (UGC), New Delhi for providing financial support in the form of Maulana Azad National Fellowship (MANF) for my research work.

Sharba Kausar Abstract

Setaria cervi is a bovine filariid which is transmitted by Aedes aegypti in India. S cervi, which is generally non pathogenic in its natural hosts, in which prevalence rate is around 80%, may cause serious and often fatal cerebrospinal nematodiasis in unnatural hosts. Larvae of S. cervi may enter cerebrospinal region resulting in lumbar paralysis. Animal models have been widely used for understanding the pathogenesis of the disease, immunomodulations, screening of different drug formulations and other biochemical aspects. S. cervi, which has resemblance with Wuchereria bancrofti in having almost similar nocturnal periodicity; besides sharing a few common antigenic components, is used for the diagnosis of human filariasis. Aim of this study was to see the persistence and longevity of microfilariae and adult worm following infusion and intraperitoneal implant in white rats. Host response by the inflammatory cells and antibodies was also observed along with the changes in TCA cycle, antioxidant and liver enzymes in response to oxidative stress caused by the circulating microfilariae in untreated control rats and those treated with DEC, NTZ and nanocomposite of NTZ+AgNPs.

Adult Setaria cervi worms were collected from the peritoneal cavity of freshly slaughtered buffaloes and microfilariae were recovered by dissecting uterus of gravid females and incubated in Ringer’s solution at 37 °C. Laboratory-bred white rats were used for this study. Microfilariae were infused in the peritoneal cavity of all the rats which were divided into four groups, each having 5 rats. Groups 1, 2 and 3 were given diethylcarbamizine (DEC), nitazoxanide (NTZ) and (NTZ+AgNPs), following the appearance of microfilariae in the peripheral circulation. These drugs were given orally at a dose of 100 mg/kg/day for 6 days. Group 4 served as control which was untreated but infected. In another group of 5 rats a total of 5 adult worms each were implanted intraperitoneally to see the host reaction. Microfilarial density and their longevity was recorded in treated and untreated rats every third day until they disappear from the peripheral circulation. Differential leucocyte count was recorded to see the changes in the blood pictures of infected and uninfected rats during the course of infection. Peritoneal fluid was also aspirated, smeared and stained with Leishman’s stain to observe the host response against the microfilariae in the peritoneum. Pathological changes were observed in the tissues of vital organs such as mesenteries, liver, lungs and spleen.

Abstract

Drugs used during this study were DEC, NTZ and NTZ+AgNPs. AgNPs and NTZ+AgNPs were prepared and characterized by UV-Visible spectral analysis, Fourier transform infrared spectroscopy, scanning and transmission electron microscopy. Ten concentrations ranging from 10µg/ml to 100µg/ml of DEC, NTZ and NTZ+AgNPs were prepared in 200 µl of Ringer’s solution and tried against the microfilariae in vitro at 37 oC in 96-well titer plate. Controls were run by incubating microfilariae in Ringer’s solution to compare the results. Images of treated and untreated microfilariae were taken by scanning electron microscopy to see the possible effect on their body surface. TCA cycle enzymes such as succinate, malate and isocitric dehydrogenase were localized in adult and microfilariae of S. cervi by incubating them for 24 and 6 hours, respectively at 37 oC in 100 g/ml of DEC, NTZ and NTZ+AgNPs by the method as described in Theory and Practice of Histological Techniques by Bancroft and Gamble (2002).

Blood was collected to observe enzyme activities in untreated and treated rats every tenth day. Blood was left at room temperature to clot for 30 minutes and then transferred to the refrigerator at 4 0C for 2 hours so that serum could be obtained. Then it was transferred to 1.5 ml vials and centrifuged at 1000 xg at 4 0C for 5 minutes. Serum was collected and stored at -80 0C. Quantification of total protein was done by the method of Bradford (1976) as modified by Spector (1978). The level of malondialdehyde (MDA), a marker for lipid peroxidation process was determined by the procedure described by Buege and Aust (1978). Glutathione-S-transferase (GST) activity was assayed by the method of Habig et al., (1974), while Superoxide dismutase (SOD) was assayed according to the method of Marklund and Marklund (1974). Catalase (CAT) and Glutathione peroxidase (GPx) activities were measured as per the methods of Aebi (1984) and Flohe and Gunzler (1984) respectively. Liver markers such as Aspartate aminotrasferase (AST), Alanine aminotrasferase (ALT) were estimated by the method of Reitman and Frankel (1957), while Alkaline phosphatase (ALP) was estimated following the manufacturer’s protocol as given in the kit (Kind and King’s 1954).

After the intraperitoneal infusion of microfilarie they appeared in peripheral circulation of white rats after 8±2 days and persisted for 54 days. Peak of microfilaraemia of 20/mm3 was observed on 31st day, followed by a decline and disappearance after 55 days. Nanocomposite of NTZ+AgNPs proved most effective 2

Abstract

as it cleared microfilariae within 18 days of infection followed by DEC and NTZ which took 24 and 33 days, respectively. Eosinophils, basophils, monocytes and neutrophils infiltrated the infected tissues to trap the microfilariae and adult worms for piece meal destruction in the peritoneum. During late phase lymphocytes get activated and multiplied to neutralize the infection by secreting antibodies. Pathological changes were seen around the microfilariae and adult worms which were spotted in the tissue sections of mesenteries, lungs, liver and spleen. SEM images of microfilariae treated with NTZ+AgNPs in vitro, showed ruptured sheath at few places along with nanoparticles sticking on their body surface, while no morphological changes were visible in DEC and NTZ treated microfilariae.

NTZ+AgNPs was the most effective synergistic combination against the TCA cycle enzymes. The nanocomposite almost completely blocked the malate and isocitrate dehydrogenase activities, while activity of succinate dehydrogenase was much reduced in the microfilariae and adult worms of Setaria cervi. Nanoparticles ruptured the sheath which made NTZ accessible to the main body of the microfilariae and produced maximum effect by penetrating through the body surface and acting on the TCA cycle enzymes, which play a vital role in the energy metabolism and survival of both the microfilariae and adult worms.

Malondialdehyde is an end product of lipid peroxidation which showed progressive increase in both treated and untreated rats for the first 20-30 days, indicating an increase in oxidative stress due to the increase in microfilarial density, thereby generating reactive oxygen species which cause lipid peroxidation and increase the level of MDA. Infiltration of the immune cells around the microfilariae at the site of infection cause pathological changes which might be the reason of increase in oxidative stress which was evidenced by increase in MDA level. In treated rats the tissue damage was minimized with decrease in circulating microfilariae that resulted in decreased level of MDA..

SOD activity was significantly decreased in microfilaraemic rats during early phase of infection that got elevated afterwards in all the groups except those treated with NTZ which was least effective against the microfilariae. NTZ+AgNPs treated rats showed maximum activity of this enzyme on the 20th day of infection and then it declined afterwards when microfilarial density decreased. But in DEC treated rats SOD activity

Abstract remained elevated for a comparatively longer duration. In the untreated control group there was a constant decline in SOD activity throughout the infection. The increase in the level of SOD was inversely proportional to the microfilarial density. Since SOD catalyzes the dismutation of superoxide to H2O2 and protects tissue from harmful effects of superoxide radicals, it gets elevated when microfilariae were more in blood circulation, indicating its protective role.

Significant increase in catalase activity was observed in the sera of uninfected and all infected groups of rats up to 20th day then showed slight decrease, except in NTZ treated rats which showed decrease 20th day onwards. Since catalase is required for the conversion of H2O2, its level was elevated. Its decreased level in NTZ treated rats may be correlated with its anti-inflammatory properties. In microfilaraemic rats, slight increase in GPx activity was observed in the sera of all groups of treated rats up to 10th day which declined slowly during the later phase of infection, except in DEC treated rats. In untreated rats enzyme activity showed slow progressive increase which was little higher than that of the treated groups. Since GPx play a protective role in reduction of H2O2 to H2O and O2, besides conversion of alkyl hydroperoxides, its increase was obvious in both untreated and treated groups of rats. Significant decline of GPx in NTZ+AgNPs treated rats on 20th day onwards may be due to the affinity of silver nanoparticles with thiol group of glutathione which deactivate this enzyme.

There was a marked increase in GST activity in the sera of all infected rats on the 10th day. Its level was high in DEC treated rats up to 20th day which declined afterwards. NTZ and NTZ+AgNPs treated rats showed slow decline in GST level 10th day onwards. But in untreated rats enzyme level was elevated up to 30th day then declined. The increase in enzyme level was proportionate to the microfilarial density which exerts oxidative stress, in response to which secretion of this enzyme gets accelerated to neutralize the infection. Enzyme levels decreased in NTZ and NTZ+AgNPs treated rats as NTZ hampers the activity of GST 1 by inhibiting the coupling of GSTPis to glutathione which reduces the chemo resistance and affinity of silver nanoparticles to thiol group of glutathione. Increase of enzyme in DEC treated rats may be assigned to its detoxifying property. Level of ALP, AST and ALT was low in all the treated microfilaraemic rats when compared with untreated rats. Enzyme levels were proportionate to the microfilarial density in infected rats. Since there was a progressive decrease in the microfilarial density in the treated rats, the level of liver 4

Abstract markers too decreased accordingly. Increased ALP, AST and ALT in untreated rats indicated degeneration of liver cells caused by microfilariae.

Prominence in protein bands and antibody titer in untreated and treated white rats was due to the increase in the protein fractions especially gamma globulins which were produced in response to the antigenic stimulus of the microfilariae circulating in the peripheral blood. Nanoparticles which are known to stimulate lymphocytes might have enhanced the secretion of antibodies and were also responsible for highest antibody titer in rats treated with nanocomposite of NTZ+AgNPs.

CONTENTS

Acknowledgement

List of Abbreviations

List of Tables

List of Figures

1-6

Introduction

Historical Review 7-32

Chapter 1: Histopathological observations in white rats infected with 33-51 Setaria cervi.

Chapter 2: In vitro efficacy of diethylcarbamazine, nitazoxanide and 52-78 nanocomposite of nitazoxanide and silver nanoparticles against the adult and microfilariae of Setaria cervi.

Part I: Preparation and characterization of NTZ+AgNPs. 54-59

Part II: Drug screening against microfilariae and adult Setaria 60-63 cervi in vitro.

Part II A: Scanning electron microscopy of microfilariae of Setaria 64-67 cervi.

Part II B: Histochemical localization of TCA cycle enzyme in control 68-78 and treated microfilariae and adult Setaria cervi.

Chapter 3: Assessment of biochemical parameters in the sera of 79-103 microfilaraemic rats in response to drugs.

Chapter 4: Immunological observations in the sera of untreated and 104-113 treated white rats infected with Setaria cervi.

Summary 114-118

References 119-181

Publications

Abbreviations

LIST OF ABBREVIATIONS

AgNPs- Silver Nanoparticles

AuNPs- Gold Nanoparticles

ALB- Albendazole

ALP-Alkaline Phosphatase

ALT- Alanine Transaminase

ANOVA- Analysis of Variance

AST- Aspartate Transaminase

BSA- Bovine Serum Albumin

CAT- Catalase

CFA-Circulating Filarial Antigen

CNS- Central Nervous System

CSAs- Cuticle-Specific Antigens

CSF- Cerebrospinal Fluid

CIEP- Counter-current Immuno-Electrophoresis

DEC- Diethylcarbamazine

DLC- Differential Leucocyte Count

DNA- Deoxy Ribonucleic Acid

DPX- Distyrene, a Plasticizer, and Xylene

EBC- Eosinophil Blood Count

EDAX- Energy Dispersive Analysis of X-rays

ELISA- Enzyme-Linked Immunosorbent Assay

ES- Excretory Secretory

FAO- Food and Agricultural Organisation

FSI- Filarial Serum Immunoglobulins

FTIR – Fourier Transform Infrared Spectroscopy Abbreviations

GI- Gastro-Intestinal

GPX- Glutathione Peroxidase

GSH- Glutathione

GST- Glutathione S-Transferase

GSTP1- Glutathione S-Transferase Pi

ICDH- Isocitrate Dehydrogenase

ID- Immuno-Diffusion

IFAT- Indirect Fluorescent Antibody Test

IHAT- Indirect Haemagglutination Test

IOP- Isosmotic Percoll

JNK- C-Jun Kinase

LFT- Liver Function Test

LP- Lipid Peroxidation

MDA- Malondialdehyde

MDH- Malate Dehydrogenase

MMPs- Matrix Metaloproteinase

MPCA- Methyl Piperazine Carboxylic Acid

MTT- 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl Tetrazolium bromide

NADP- Nicotinamide Adenine Dinucleotide Phosphate

NPs- Nanoparticles

NTZ- Nitaxoxanide

OD- Optical Density

PAGE- Poly Acrylamide Gel Electrophoresis

PBST- Phosphate Buffered Saline, Tween-80

PDI- Protein Disulphide Isomerase

PEP- Phospho-Enol-Pyruvate

PFOR- Pyruvate Ferredoxin Oxidoreductase Abbreviations

PCA- Passive Cutaneous Anaphylaxis

PZQ- Praziquantel

ROS- Reactive Oxygen Species

SD- Standard Deviation

SDH- Succinate Dehydrogenase

SEM- Scanning Electron Microscopy

SOD- Superoxide Dismutase

SPR- Surface Plasmon Resonance

SPSS- Statistical Package for the Social Sciences

STH- Soil-Transmitted Helminthes

TBARS- Thiobarbituric Acid Reactive Substances

TCA- Tri-Carboxylic Acid

TEM- Transmission Electron Microscopy

Th2- T-Helper type 2

TPE- Tropical Pulmonary Eosinophilia

TS – Transverse Section

TZ- Tizoxanide

UV- Ultraviolet

UV-Vis- Ultraviolet Visible

WHO- World Health Organisation

WBC- White Blood Cell List of Tables

Table Title Page

Chapter 1. Histopathological observations in white rats infected with Setaria cervi.

Table 1.1: Effect of DEC, NTZ and NTZ+AgNPs on the longevity 40 of the microfilariae of Setaria cervi in untreated and treated rats.

Chapter 2. In vitro efficacy of diethylcarbamazine, nitazoxanide and nanocomposite of nitazoxanide and silver nanoparticles against the adult and microfilariae of Setaria cervi.

Table 2.1: Antifilarial activity of DEC, NTZ and NTZ+AgNPs 61 against adult Setaria cervi in vitro.

Table 2.2: Antifilarial activity of DEC, NTZ and NTZ+AgNPs 62 against microfilariae of Setaria cervi in vitro.

Table 2.3: Succinate dehydrogenase localization in control and 70

treated microfilariae of Setaria cervi. Table 2.4: Malate dehydrogenase localization in control and treated 71 microfilariae of Setaria cervi.

Table 2.5: Isocitrate dehydrogenase localization in control and 72 treated microfilariae of Setaria cervi.

Table 2.6: Distribution of succinate dehydrogenase activity in 74 control and treated adult Setaria cervi.

Table 2.7: Distribution of malate dehydrogenase activity in control 75 and treated adult Setaria cervi. Table 2.8: Distribution of isocitrate dehydrogenase activity in 76 control and treated adult Setaria cervi.

Chapter 3. Assessment of biochemical parameters in the sera of microfilaraemic rats in response to drugs.

Table 3.1: Malondialdehyde levels in the sera of untreated and 91 treated microfilaraemic rats.

Table 3.2: Superoxide dismutase levels in the sera of untreated and 92 treated microfilaraemic rats.

Table 3.3: Catalase levels in the sera of untreated and treated 93 microfilaraemic rats.

Table 3.4: Glutathione peroxidase levels in the sera of untreated and 94 treated microfilaraemic rats.

Table 3.5: Glutathione S-transferase levels in the sera of untreated 95 and treated microfilaraemic rats.

Table 3.6: Alkaline phosphatase levels in the sera of untreated and 96 treated microfilaraemic rats.

Table 3.7: Aspartate aminotransferase levels in the sera of untreated 97 and treated microfilaraemic rats.

Table 3.8: Alanine aminotransferase levels in the sera of untreated 98 and treated microfilaraemic rats.

List of Figures

Figure Title Page

Chapter 1. Histopathological observations in white rats infected with Setaria cervi. Fig. 1.1. Effect of DEC, NTZ and NTZ+AgNPs on the longevity of 41 the microfilariae of Setaria cervi in untreated and treated rats. Fig. 1.2. Differential leucocyte count in white rats infected with 42 Setaria cervi. Fig. 1.3. Differential leucocyte count in microfilaraemic white rats 42 treated with DEC. Fig. 1.4. Differential leucocyte count in microfilaraemic white rats 43 treated with NTZ. Fig. 1.5. Differential leucocyte count in microfilaraemic white rats 43 treated with NTZ+AgNPs. Fig. 1.6. Microfilariae of Setaria cervi in the process of cell 44 adhesion and destruction by the leucocytes in the peritoneal exudates. Fig. 1.7. Sections of mesentery (A), lung (B), spleen (C) and liver 45 (D) showing inflammatory cells in the vicinity or around the trapped microfilariae in these viscera. Fig. 1.8. Encapsulation and leucocyte infiltration showing 46 aggregation, attachment, inpocketing and plug formation around the worm embedded in the mesenteries

Chapter 2. In vitro efficacy of diethylcarbamazine, nitazoxanide and nanocomposite of nitazoxanide with silver nanoparticles against the adult and microfilariae of Setaria cervi.

Part I: Preparation and Characterization of NTZ+AgNPs. Fig. 2.1.1. UV–Vis absorption spectra showing surface plasmon 56 resonance (SPR) of AgNPs and NTZ+AgNPs. Fig. 2.1.2. FTIR spectra depicting the vibration of NTZ alone and 57 NTZ+AgNPs. Fig. 2.1.3. TEM images of AgNPs alone and NTZ+AgNPs at x40000. 57

Fig. 2.1.4. SEM images showing NTZ alone and NTZ+AgNPs, 58 marked by arrows (x1000). Fig. 2.1.5. Energy dispersive X-ray spectrum of NTZ+AgNPs. 58

Part II: Drug screening against microfilariae and adult Setaria cervi in vitro.

Part II A: Scanning electron microscopy of microfilariae of Setaria cervi.

Fig. 2.2.1. SEM images of Setaria cervi microfilariae incubated in 65 Ringer’s solution (control) for 6 hours (x4000). Fig. 2.2.2. SEM images of Setaria cervi microfilariae incubated for 6 65 hours in Ringer’s solution containing 100 µg/ml DEC (x4000). Fig. 2.2.3. SEM images of Setaria cervi microfilariae incubated for 6 66 hours in Ringer’s solution containing 100 µg/ml NTZ (x4000). Fig. 2.2.4. SEM images of Setaria cervi microfilariae incubated for 6 66 hours in Ringer’s solution containing 100 µg/ml NTZ+AgNPs (x4000).

Part II B: Histochemical localization of TCA cycle enzyme in treated and untreated microfilariae and adult Setaria cervi

Fig. 2.2.5. Localization of succinate dehydrogenase in control (A), 70 DEC (B), NTZ (C) and NTZ+AgNPs (D) treated microfilariae of Setaria cervi (x400). Fig. 2.2.6. Localization of malate dehydrogenase in control (A), DEC 71 (B), NTZ (C) and NTZ+AgNPs (D) treated microfilariae of Setaria cervi (x400). Fig. 2.2.7. Localization of isocitrate dehydrogenase in control (A), 72 DEC (B), NTZ (C) and NTZ+AgNPs (D) treated microfilariae of Setaria cervi (x400). Fig. 2.2.8. Localization of succinate dehydrogenase in control (A), 74 DEC (B), NTZ (C) and NTZ+AgNPs (D) treated Setaria cervi (T.S.) (x400). Fig. 2.2.9. Localization of malate dehydrogenase in control (A), DEC 75 (B), NTZ (C) and NTZ+AgNPs (D) treated Setaria cervi (T.S.) (x400). Fig. 2.2.10 Localization of isocitrate dehydrogenase in control (A), 76 DEC (B), NTZ (C) and NTZ+AgNPs (D) treated Setaria cervi (T.S.) (x400).

CHAPTER 3. Assessment of biochemical parameters in the sera of microfilaraemic rats in response to drugs.

Fig. 3.1. Malondialdehyde levels in the sera of untreated and treated 91 microfilaraemic rats. Fig. 3.2. Superoxide dismutase levels in the sera of untreated and 92 treated microfilaraemic rats. Fig. 3.3. Catalase levels in the sera of untreated and treated 93 microfilaraemic rats. Fig. 3.4. Glutathione peroxidase levels in the sera of untreated and 94 treated microfilaraemic rats. Fig. 3.5. Glutathione S-transferase levels in the sera of untreated 95 and treated microfilaraemic rats. Fig. 3.6. Alkaline phosphatase levels in the sera of untreated and 96 treated microfilaraemic rats. Fig. 3.7. Aspartate aminotransferase levels in the sera of untreated 97 and treated microfilaraemic rats. Fig. 3.8 Alanine aminotransferase levels in the sera of untreated 98 and treated microfilaraemic rats.

CHAPTER 4. Immunological observations in the sera of untreated and treated white rats infected with Setaria cervi.

Fig. 4.1. Protein profile in the sera of untreated and DEC, NTZ and 109 NTZ+AgNPs treated microfilaraemic white rats. Fig. 4.2. Antibody titer in the serum of untreated microfilaraemic 110 rats. Fig. 4.3. Antibody titer in the serum of microfilaraemic rats treated 110 with DEC. Fig. 4.4. Antibody titer of serum of microfilaraemic rats treated 111 with NTZ. Fig. 4.5. Antibody titer in the serum of microfilaraemic rats treated 111 with NTZ+AgNPs.

Introduction

Parasitic diseases are major threat to the entire mankind, especially in the tropics and subtropics which are not only present in humans, but also found in animals (Renslo and McKerrow, 2006; Tripathy et al., 2006; Sundar and Ravindaran, 2009; Katiyar and Singh, 2011; Bandyopadhyay, 2012). A variety of parasitic diseases are transmitted by vectors, and therefore, attempts to control their transmission became important (Chatelain and Ioset, 2011). Human filariasis caused by the lymph-dwelling nematodes such as Wuchereria bancrofti, Brugia malayi and Brugia timori, is a major cause of global morbidity and is a hindrance to socio-economic development. 947 million people in 54 countries worldwide remained threatened by lymphatic filariasis alone and require preventive chemotherapy to stop the spread of this parasitic infection, and therefore, WHO is planning to eliminate filariasis by 2020 (WHO, 2016). Lymphatic filariasis has been identified by the WHO as the second leading cause of permanent and long-term disability, whereas blindness is caused by Onchocerca volvulus (WHO, 2000). Over a considerable number of years, Food and Agricultural Organization (FAO) has emphasized the magnitude of losses in livestock industry due to filariasis. In India, this disease is confined to geographical areas which are hot and humid. Tarai areas of India provide a congenial environment for the proliferation of vector and favorable conditions for the development of the parasite. The etiological agents causing microfilariasis in animals are larvae of Setaria, Onchocera, Parafilaria, Stephanofilaria, Dipetolonema and Dirofilaria which also have a zoonotic importance (Lec and Wang, 1991; Wahl et al., 1994; Senthilvel and Pellai, 1999). Microfilariasis in cattle is mostly observed in the sub-Himalayan belt of Uttar Pradesh (Sharma et al., 2000).

Setaria cervi is an important filariid inhabiting the peritoneal cavity of buffaloes causing peritonitis and intestinal occlusion. Adult worms are generally considered to be non-pathogenic although they may cause a mild fibrinous peritonitis, but the larval forms caused serious conditions when they migrate erratically into the central nervous system of unnatural hosts such as horses, sheep and goats, leading to serious and often fatal neuropathologic disorder known as epizootic cerebrospinal setariasis, cerebrospinal nematodiasis, kumri and lumbar paralysis (Pachaury, 1972; Baharsefat et al., 1973; Shin et al., 2002; Tung et al., 2003; Bazargani et al., 2008). Neurological disorders in human by Setaria sp. was also reported by Kadenatsii et al. (1974). These worms flourish in the host body at the expense of its tissues and body fluids and may 1

Introduction

prove fatal (Singh et al., 2014). Usually, studies are conducted on the bovine filariid S. cervi in laboratory models, as this worm resembles the human filariid in its periodicity and antigenic components (Singhal et al., 1973; Kaushal et al., 1987; Bal and Das, 1996; Mukhopadhyay et al., 1996; Wijesundera et al., 1996; Mukhopadhyay and Ravindran, 1997; Dalai et al., 1998; Mohanty et al., 2000; Ahmad and Srivastava, 2007).

Williams (1955) succeeded in transplanting these worms from freshly slaughtered cattle to rabbits and showed the presence of microfilariae in their peripheral circulation, but could not incriminate its vector. Later, Nelson in 1962 succeeded in transplanting both adult worms and microfilariae to rabbits and monkeys. Ansari (1964) studied the microfilaraemia and reported nocturnal periodicity in white rats which were implanted with adult S. cervi worms intraperitoneally.

As for pathology and clinical symptoms are concerned, Setaria tundra caused purulent discharge from the eyes along with pale mucous membrane, rough and dry skin coat and a stiff gait in reindeer (Sharma et al., 1981; Kumar et al., 1984; Kumar and Sharma, 1994; Venu, 2000). It has been speculated that when larvae of animal origin are inoculated by the vector to human, they may cause abscesses, lymphadenopathy, eye lesions, tropical pulmonary eosinophilia and allergic reactions, if develop abnormally in the subcutaneous tissue, heart, eyes, lymphatic channels and central nervous system (Nelson, 1966; Orihel and Eberhard, 1998). Cross reactivity has also been observed among the filariids of human and animal origins. Human filariasis can be diagnosed by using antigens of animal filariids, as they share a few common antigens which react with their corresponding antibodies. The phenomenon referred to as “zooprophylaxis” against lymphatic and subcutaneous filariasis was noticed in areas where the common mosquitoes that feed on people were heavily infected with non specific filariids of man and animals (Nelson, 1992).

There are five stages in the life cycle of filarial nematodes, delineated by four complete moults of the cuticle (Scott, 2000; Bennuru et al., 2009). Two of these moults occur in the arthropod vector, which picks microfilariae from the blood and supports its development up to the third (infective) stage. Infective-stage larvae gain access to the mammalian host and continue developing over many weeks by passing through two further moults into adolescent worms which become adult. After mating, 2

Introduction

the gravid females release large numbers of microfilariae which come in the peripheral circulation and continue the cycle when ingested during the blood meal of the arthropod vector. The prepatent period after experimental infection of maral deer (Cervus elaphus maral) with S. cervi larvae was recorded as 224 days (Shol´ and Drobishchenko, 1973). The life spans of S. marshalli and S. labiatopapillosa were reported approximately 12 and 16 months, respectively after the prenatal infection (Osipov, 1972; Fujii et al., 1995).

When microfilariae reach in the mid gut of intermediate host, they exsheath and come out in the haemocoel by penetration and enter in the thorax. During first 24 hours, they shorten and thicken into the sausage-shaped first-stage larva. After the first moult on 6th day, the second-stage larva starts increasing in size and moults on 9th day to become the infective third stage which grows rapidly. On the 11th day, the majority of infective larvae accumulate in the head and proboscis of the mosquito (Anderson, 2000; Wajihullah, 2001; Bain and Babayan, 2003). S. digitata microfilariae grow into infective stage in mosquitoes in about two weeks, while S. labiatopapillosa takes 6-14 days (Innes and Shoho, 1953; Pietrobelli, 1998). However, in some instances larvae develop to an advanced stage, but eventually become encapsulated and destroyed by the host’s defense mechanism (Anderson et al., 2000).

Prenatal infection by larval stage was observed in S. marshalli infection where the larvae penetrated the placenta and migrated into the foetus to complete their development (Kitano, 1994). Congenital infection of S. marshalli has also been found in calves and in bovine foetus (Kitano, 1994; Fujii, 1995). S. digitata was reported in an eight months-old bovine foetus from China (Mo et al., 1983). A 5.2 cm long immature female S. yehi was found free in the body cavity of a 31 day old black-tailed deer fawn (Weinmann and Shoho, 1975). But there is no evidence of prenatal infection in temperate zones. This may be due to the fact that filarial infection occurs in warm summer season when arthropod vectors are active and transmission in late autumn and winter is not possible because of the lack of vectors.

Several antigens have been demonstrated which are common for the bovine (S. cervi) and human (B. malayi) filarial nematodes (Kaushal et al., 1987; 1994). S. cervi has been widely used as a test organism for the in vitro screening of prospective

Introduction

antifilarial compounds (Singhal et al., 1973; Anwar et al., 1978; Srivastava and Ghatak, 1983a). The S. digitata–Mastomys coucha model has also been found to be amenable to chemotherapeutic and immunobiological investigations in experimental filariasis (Srivastava et al., 1983; Srivastava and Ghatak, 1983b; Mukhopadhyay et al., 1996). Till date, no effective vaccine is available against the filarial worms and therefore, their infections are controlled with existing antifilarial drugs. Singhal et al. (1969) reported disappearance of S. cervi microfilariae from the peripheral circulation of rats after oral administration of Diethylcarbamazine (DEC). Nitazoxanide (NTZ) is known as a broad spectrum drug against a wide variety of intestinal parasites and enteric bacteria infecting animals and humans (Romero et al., 1997; Brokhuysen et al., 2000; Abboud et al., 2001; Rossignol et al., 2001; Gilles and Hoffman, 2002). Rao et al. (2009) reported the in vitro and in vivo effects of NTZ and tizoxanide (TZ) in gerbils infected experimentally by B. malayi nematode. Control programmes generally rely on sustained delivery of antifilarial drugs, such as ivermectin, albendazole and DEC, which have been the drugs of choice for filariasis control (Gayen et al., 2013; Singh et al., 2013). These drugs disrupt disease transmission to some extent but are associated with systemic and inflammatory adverse reactions

Introduction

(Saini et al., 2012; Singh et al., 2013). The sustained use of these drugs increased the threat of drug resistance which has already been revealed in various veterinary diseases. The use and status of these drugs, including their limitations, have been reviewed by Katiyar and Singh (2011).

Anthelmintic resistance is widespread among nematodes of livestock as a consequence of frequent drug administration of the same class of compounds over long periods (Geerts et al., 1997; Wolstenholme et al., 2004; von Samson- Himmelstjerna and Blackhall, 2005; Bourguinat et al., 2006). Therefore, there is an urgent need to develop backup drugs. In addition to the discovery and development of novel antiparasitic drugs, combination chemotherapy was considered as a powerful strategy to slow the emergence of drug resistance (Barnes et al., 1995; Nyunt and Plowe, 2007). Rapid death of microfilariae following treatment is associated with adverse events that can be severe in lymphatic filariasis and onchocerciasis or even fatal in patients with heavy Loa loa infections (Pani et al., 2002; Supali et al., 2002; Keiser et al., 2003; Hochberg et al., 2006; Basáñez et al., 2006). Therefore, new drug- based formulations are required, especially the synergists to improve their antifilarial effect. Due to the ineffectiveness of vaccines against helminth parasites, the development of effective alternative is important, that can be achieved by nanoparticles based drug formulations (Khan et al., 2015). Recently, it has been studied that due to unique properties and large surface areas, metal oxide nanoparticles possess effective antimicrobial activities (Azam et al., 2012). Among various metal nanoparticles, silver nanoparticles (AgNPs) are particularly interesting because they possess excellent biocompatibility and were able to exert inhibitory effect at a concentration that is below their cytotoxic limits, and hence used as antimicrobial (Pillai et al., 2012; Mohamed et al., 2014). A clear understanding of the mode of action of antifilarials will be helpful to target the biochemical pathways operating in filarial nematodes (Gupta and Srivastava, 2005).

Drug-repurposing (re-profiling, therapeutic switching or drug repositioning) is another area to enhance the efficacy of existing, failed or abandoned drugs or advanced clinical candidates (Sekhon, 2013). It is a useful strategy to accelerate the drug development process due to lower costs, reduced risk and decreased time to market, due to availability of preclinical data (Padhy and Gupta, 2011). This enables

Introduction

not only pharmaceutical companies but also public-sector researchers to engage in drug discovery and development efforts (O’Connor and Roth, 2005). Over the past years, a variety of drug-repurposing initiatives have been launched with particular attention to neglected tropical and rare diseases (Allarakhia, 2013), hence it is likely that these efforts will be quite useful in near future.

Filarial infections are generally specific to their vertebrate hosts, and therefore, ideal conditions for the drug screening may only be provided, if human filariids could be transferred to a closely allied animal host. This is practically not possible in majority of the cases, but infection like B. malayi can be maintained in cats (Edeson et al., 1959; Edeson et al., 1960). Alternatively, screening of antifilarial agents on a much related parasite must be done. In almost all the available models, microenvironment is not completely identical with that of the human parasite. The best model available for the in vivo screening of antifilarial agents involves the use of Litomosoides carinii infected cotton rats (Hawking and Sewell, 1948). The validity of this method can be questioned on the basis that L. carinii infection responds to certain drugs which have no effect on human filarial infection caused by Wuchereria, Loa loa or Onchocerca. The screening method using Dipetalonema witei is further less reliable as its chemotherapeutic correspondence to human infection is lesser than that of L. carinii (Worms et al., 1961). Even DEC, a potent filaricidal agent has no effect on D. witei infection. Furthermore, there is a long incubation period of 50 days for L. carinii (Hawking and Sewell, 1948) and 8 months for Dirofilaria immitis, before microfilariae appear in peripheral circulation (Webber et al., 1955). Singhal et al. (1972) suggested that S. cervi infection in rat model may be the best option to study screening of antifilarial agents as it corresponds to human filariasis in its sensitivity to drugs and no arthropod vector is required, as parasite could easily be implanted in white rats. Keeping this background in mind, biochemical, histopathological and immunological studies were carried out in white rats experimentally infected with S. cervi by intraperitoneal implant and microfilarial infusion. The efficacies of DEC, NTZ and nanocomposite of NTZ and silver nanoparticles (NTZ+AgNPs) were also assessed against the microfilariae along with their role in immunomodulation and pathology of the affected tissue invaded by the adult worms and microfilariae.

Historical Review

Setaria cervi was reported for the first time by Rudolphi in 1819 from red deer (Cervus elaphus) and was named as Filaria cervi. Railliet and Henry (1911) studied this worm in detail and placed it in genus Setaria. In 1931 Mapleston recovered S. cervi from hog deer, Cervus axis. Baylis (1936) suggested that both F. cervi and S. cervi are the same species and should be named as S. cervi. Since then this nematode is known as S. cervi. Other prominent species of this genus are S. digitata, S. labiatopapillosa. S. marshalli, S. tundra, S. equina, S. yehi, etc.

Zhu and Zhang (1981) documented filariasis in 252 sheep in Xinbin, China. The parasite detected in cerebro-spinal cord was identified as Setaria digitata. Quan-qun (1981) conducted the post mortem examination of draught cattle in China and found 42.3% animals positive for Setaria infection. Blood examination in young cattle and buffaloes showed the incidence as 20 and 28.5%, respectively. Out of 284 cattle slaughtered between November 1981 and March 1982 in Philippines, 100 were found positive for Setaria spp. In another survey of 23 farms of dairy cattle, the same workers reported a prevalence of 11.28%. Nakano et al. (2007) conducted an epidemiological survey for bovine Setaria spp. in cattle in Aomori and Kumamoto prefectures of Japan. 35 out of the 50 worms collected from abdominal cavities of cattle of Aomori prefecture were identified as S. digitata, 14 worms as S. marshalli and one worm as S. labiatopapillosa. Out of 847 Setaria worms collected from Kumamoto prefecture, 816 worms were identified as S. digitata and 31worms as S. marshalli. Prevalence of S. cervi in cattle in Surinam and Gambia was 42 and 32.69%, respectively (Frickers, 1948; McFadzean, 1955), which is less when compared to the prevalence of S. digitata which happened to be 56.8% in the study conducted by Sundar and D’Souza (2015). In Korea, 5% prevalence was reported for S. digitata at an abattoir in Jeju by Kim et al. (1968), but a later study described 57% prevalence for this parasite in cattle (Paick et al., 1976). Rhee et al. (1994) reported that 25.1% of cattle were infected with S. digitata and 2.9% with S. marshalli at an abattoir in Jeonju. Infection rate of S. labiatopapillosa varied in different countries. Green and Trueman (1971) reported 75% infection rate from Australia, which has prevalence rate of 50% in France (Brengues and Gidel, 1972), while 58.6% positivity was observed for the same parasite by Ogbogu et al., (1990). But this species has been reported to be the most common species among cattle in the United States and Canada (Willard and Walker, 1969). In India, S. digitata infection is prevalent in an area in

Historical Review

Odisha which is endemic for bancroftian filariasis and it was observed that 12.5% cattle harbor both adult worms in the peritoneum and microfilariae in the circulation, while 70% of the cattle harboured adult worms which were amicrofilaraemic (Mohanty et al.,. 2000). Mohan (1975) recorded higher number of S. digitata compared to S. cervi in South India. Cattle have been found to be less affected by S. labiatopapillosa as indicated by few prevalence reports (Rao, 1941; Sarwar, 1946). However, infection rate of this species in cattle from Odisha was recorded as 19.3% by Patnaik (1989). In the study conducted by Sundar and D’Souza (2015), 18.96 % of the cattle revealed S. labiatopapillosa which is low as compared with other countries wherein this species occurred in the regions with temperate weather conditions, except for Nigeria which implied the adoption of the species for cooler climatic conditions. Shastri (1973) recorded an overall incidence of 80% of Setaria spp. in bovines at Parbhani and Aurangabad of Maharashtra. Its low prevalence rates of 3.59 and 9.09% were recorded in buffaloes in Mathura and Jaipur (Chauhan and Pande, 1980; Bhopale et al., 1982). Sastry and Rao (1985) conducted a survey in 12 villages of East Godavari district of Andhra Pradesh and observed microfilaraemia in 4.6% cattle and 5.42% buffaloes. Arun et al. (1988) examined 152 goats and 450 cattle in Tripura out of which, 8 goats were found to be harbouring S. cervi and 92 cattle were found positive for Setaria spp. Examination of blood samples of 353 buffaloes and 79 horses from Nainital of Uttarakhand showed microfilarial positivity in 26.06 and 29.11% of these animals (Sharma and Kumar, 1994). Siddiqui et al. (1996) also studied the prevalence of Setaria infection in Uttar Pradesh by screening the buffaloes and horses in abattoirs for adult Setaria spp. Blood samples of these animals were also examined for the presence of microfilariae which were present in 37.58% buffaloes and 55.3% horses. An incidence of 4.71% microfilaraemia in 653 buffaloes was documented by Umesh et al. (2004) in north India. In another study, 139 buffalo calves were examined in Uttar Pradesh and out of these 5 calves were found to have immature and gravid females of S. labiatopapillosa embedded under the serosa in the region of the small intestine (Chauhan and Pande, 1980). In a survey of 840 cattle, including 274 lactating animals, 302 non-lactating animals, 128 heifers, 82 calves and 68 adult males, an incidence of 4.92% of microfilaraemia was recorded in six districts of north India (Sharma and Joshi, 2002). Incidence of the disease was highest in adult males (12.2 %), followed by heifers (7.64 %), non-lactating (3.82 %) and lactating animals (2.70 %). Blood films from 683 graded murrah buffaloes in and around 8

Historical Review

Tanuku, West Godavari district of Andhra Pradesh were examined by Venu et al. (2000) who observed microfilaraemia in 7.47% buffaloes. The highest prevalence of microfilaraemia was recorded as 11.84% during summer. Gogoi (2002) recorded the common filarial parasites in Indian subcontinent which belong to the genera Parafilaria, Setaria, Onchocera, Elaeophora, Stephanofilaria and Dirofilaria. The overall prevalence of bovine microfilariosis in certain coastal districts and Tirupati of Andhra Pradesh was found to be 4.29% (Pavan et al. 2004). 1051 blood samples in and around Tirupati were analysed by Digraskar et al. (1999) to determine the relationship of bubaline microfilariasis with age, sex, physiological status and breed of animal. They found that adult female buffaloes between 6 and 9 years of age were more susceptible, followed by those between 3 and 6 years and above 9 years of age. The overall incidence rate of bubaline microfilariasis in females was 3.526%. Lactating buffaloes had the highest incidence (3.842%) followed by heifers (3.571%) and non-lactating buffaloes (1.149%). Significantly higher incidence of bubaline microfilariasis was recorded in murrah crosses (5.686%) than in non-descript buffaloes (2.527%). The prevalence of bubaline microfilariasis in East Godavari district of Andhra Pradesh was also studied by Rao et al. (2005) and the overall prevalence was found to be 8.49%. Prevalence of the infection was more during summer which accounted for 11.26%. The animals which were more than 9 years of age were more affected (9.7%). Sundar et al., (2005) conducted a prevalence study on setariosis in cattle and buffaloes in Karnataka state in which 500 cattle were screened. About 27.6% were found positive for adult worms without microfilariae in blood and 9.8% had both worms as well as microfilariae. Moreover, the prevalence of Setaria spp. was higher in males than in females. Seasonal variation in the incidence of infection of S. cervi was observed by Ansari (1977) in the buffalo population of Aligarh district, Uttar Pradesh. Rainy season witnessed the highest incidence rate while the lowest rate was observed in winter season. The periodic concentration of microfilariae was noticed maximum at night time and minimum at about mid day.

S. cervi, is generally considered to be non-pathogenic causing only a mild peritonitis in its specific host, but can cause serious neuropathological disorders in unnatural host (Sarwar, 1945; Innes, 1952, Shoho, 1958; 1968; Soulsby, 1982). Paralysis or incoordination of the limbs in ruminants with cerebrospinal setariasis was found to be caused by the demyelination of the nerves that extend from the spinal cord (Kopcha et

Historical Review

al., 1989). However, the process of demyelination of the CNS nerves was observed by Tung et al. (2003) who studied 2 cases of bovine cerebrospinal setariasis and this correlates well with the notion that the demyelination had been attributed to the action of matrix metalloproteinase (MMPs) which were secreted by the infiltrating inflammatory cells due to migration of parasite and toxic secretory products of larvae. The cause of abnormal functioning of upper and lower motor neurons in cerebrospinal nematodiasis was considered to be the diffuse parasite migration in the CNS of sheep and goats (Proost et al., 1993). Wang et al. (1985) studied the cause of lumbar paralysis in horses, sheep and goats in central and southern China through artificially infected foals, sheep and goats by Setaria spp and by comparing the results of the contribution made by Japanese worker Shoho (1951). Two cases of cerebrospinal nematodiasis in horses in Japan were reported by Kawata et al. (1991), while Bregoli et al. (2006) reported meningeal nematodiasis in a red deer (Cervus elaphus) in north eastern Italy. In a histopathological examination of the spinal cord and brain of goats, microfilariae of different sizes were seen between the meninges and the nervous tissue in Central Saudi Arabia (Mahmoud et al., 2004). The main lesions caused by the microfilariae were migratory traumatic haemorrhagic tracts in the spinal cord and the brain. Malacic lesions and sporadic aggregates of lymphocytes were also noted in the brain. A case report of cerebrospinal nematodiasis in a buffalo with lumbar paralysis due to invasion of the spinal cord by S. cervi was presented by Pachauri (1972). Immature or larval Setaria are capable of penetrating the placenta and migrating into the foetus, where the nematodes can complete their development. Congenital and prenatal infections of S. marshalli and S. digitata have been reported in bovine foetus (Kitano 1994; Fujii 1995; Mo et al., 1983). Congenital infection with morphological features of Setaria species was recorded in Korea (Fujii et al., 1995; Wee et al., 1996; Kim et al., 2010).

The impact of S. tundra microfilariae on cervid health remains unknown and is difficult to separate from the impact of adult worms. The captive reindeer in the experimental zoo with a microfilarial density of 950/ml of blood had S. tundra infection, which showed inexplicable symptoms resembling those described in chronic Setaria spp. in buffaloes (Sharma et al., 1981; Kumar et al., 1984; Kumar and Sharma 1994; Venu 2000). Since these reindeer harboured other parasites as well, it was impossible to fully evaluate the impact of microfilariae on their overall health.

Historical Review

When zoonotic infection of filariids of animal origin occurs in humans, it can cause allergic reactions followed by ectopic lesions by the developing worm during the erratic migration of the parasite in different parts of the body (Nelson 1966; Orihel and Eberhard 1998). Based on extensive data reviewed by Innes and Shoho (1953), it may be assumed that neural nematodiasis also exists in man although its diagnosis is very difficult. There are very clear serological test reactions in patients with onchocerciasis, even when the antigens have been prepared from other filariids which gives an idea of possibility of development of cross-immunity against filarial nematodes in man. The phenomenon of zooprophylaxis against filarial worms causing considerable morbidity has also been noticed in areas where the common mosquitoes that feed on man were heavily infected with other filariids from man and animals (Nelson, 1992). S. digitata was recovered from the cardiac ventricle, eye and cystic corpus luteum of cow (Fujita et al., 1985; Ohtake et al., 1989; Nair et al., 1993; Shin et al., 2002). It was also observed that S. digitata worms migrated to the urethra and passed out through the urine (Thrimurthy et al., 1995). Tung et al. (2003) observed that the larvae of S. digitata first enter the cerebrospinal cavity where they mature and then migrate to peritoneal cavity thus, providing evidence of heterotopic parasitism. They also found that S. marshalli and S. digitata worms cause neurological signs like quadriplegia and lumbar paralysis lead to cerebrospinal setariasis in Taiwan.

Mild leptomeningitis and eosinophilic and lymphocytic encephalomyelitis with numerous haemorrhagic tracts, degeneration and necrosis due to migration of the larvae were found in the histopathological examination of CNS of necropsied cattle, sheep and goats infected by S. digitata and S. labiatopapillosa in Iran (Bazargani et al., 2008). Blazek (1976) reported the presence of S. cervi in the central nervous system of infected red deer (Cervus elaphus hippelaphus) which showed considerable pathological changes that also provides evidence of neurotropism as observed in case of S. digitata in cattle. Few descriptions of filariid cerebrospinal nematodiasis exist in case of cervids. In Taiwan, Wang (1990) reported that S. cervi caused cerebrospinal nematodiasis in deer, while Hai et al. (1995) observed that S. labiatopapillosa caused the paralysis of the hindquarters of sika deer (Cervus nippon). However, no central nervous disorders were associated with the presence of S. tundra in Swedish reindeer (Rehbinder, 1990). According to Nelson (1966), the circulating Setaria microfilariae cause no apparent damage unless it invades CNS. Pursglove Jr. (1977) found an

Historical Review

association between fibrinous peritonitis and severe infection of S. yehi which was evident in a young New Jersey deer. Decreased thriftiness, poor body condition, and an undeveloped winter coat were seen in reindeer calves with heavy S. tundra infections whose histopathology indicated granulomatous peritonitis with lymphoplasmacytic and eosinophilic infiltration (Laaksonen, 2010). These histopathological pictures were different from that reported for sheep with lumbar paralysis attributed to setariasis, in which, eosinophilic cuffing in the white matter and fissures of the cerebellum were observed (Baharsefat et al., 1973). Occular infection was also reported in a child infected with Dirofilaria immitis (Mirahmadi et al., 2017). Migrating Setaria larvae within the CNS and eyes can stir up localized eosinophilic granulomatous inflammation, while sheathed microfilariae were patent in the blood (Jubb et al., 2007). Nygren (1990) documented pathology during a Setaria outbreak in Finnish moose which was found to be associated with granulomatous lesions caused by adult nematodes in the wall of the urinary bladder and uterus. Yoshikawa et al. (1976) also observed similar lesions which were caused by adult S. digitata in the wall of the urinary bladder of cattle. During the examination of the testicle sections in horses, chronic inflammation (orchitis chronica) and focal necrosis perivascularis were observed due to the S. equine infection (Kornas et al., 2010).

Three species of Setaria viz, S. digitata, S. cervi and S. labiatopapillosa were observed in cattle and buffaloes in Karnataka by Sundar et al. (2005). These worms were found freely in the peritoneal cavity or attached to the intestines, mesentery, walls of the peritoneum, lungs, liver, heart, urinary bladder, uterus and fascia while a few of them were found embedded in patches of inflammatory tissue attached to the walls of the pelvic peritoneum. Karki (2008) studied the seasonal occurrence of microfilariae of the Setaria spp. in affected goats and confirmed the cerebrospinal nematodiasis locally called ‘Kumri’ in Nepal in which traumatic injury of the lumbar region, parasitism in the spinal cord, nutritional deficiency and non response to supplementation of vitamins and minerals occur. Pathological injuries caused by S. cervi were also studied earlier in laboratory reared rabbits (Khatoon et al., 1983). Systemic pathological changes were revealed by the histological examinations after experimental Setaria infection in lambs and rabbits which included enlargement of the liver, kidney, heart and spleen along with haemorrhagic foci in the brain and

Historical Review

emphysema in the lungs (Kumar et al., 1991; Sharma et al., 1998). Hydropic degeneration, necrosis of hepatocytes, proliferation of Kupffer cells, infiltration of mononuclear cells and polymorphs were seen around the microfilariae in hepatic sinusoids and blood vessels, while lungs showed emphysematous, degenerative, necrotic and infiltrative changes and exudation due to the presence of microfilariae in blood vessels. Microfilariae were also found in the interstitial blood vessels and arterioles in the kidneys showing interstitial connective tissue hyperplasia, compensatory hypertrophy, necrosis, and cellular infiltration. Mohan (1976) observed multiple nodules containing microfilariae associated with eosinophilia, chronic splenitis and degeneration of the liver with bundles of microfilariae in its blood vessels along with localized aggregation of inflammatory cells around the microfilariae in the kidneys and lung lobes which suggested the role microfilariae of Setaria spp in pathogenesis.

Differential leucocyte count (DLC) is a significant parameter in the blood picture of an animal, especially during any kind of stress from disease, trauma, and infection. DLC varies with variation in the kind of parasitic infections (Gretillat, 1976). During a study of immunomodulatory effects of DEC, a remarkable increase in eosinophils and neutrophils in 2nd and 3rd weeks and lymphocytes during 4th - 7th weeks was observed in white rats and mice experimentally infected with S. cervi (Medina-De et al., 2012; Zakai and Khan 2015). Sharma et al. (1985) observed marked lymphocytosis during a chronic infection. Eosinophils that are terminally differentiated granular leucocytes reside primarily in vertebrate mucosal tissues and function in host defense. These cells are involved in the inflammation and histopathology in response to parasitic helminth infection (Wynn et al., 2004). Eosinophilia is defined as the presence of >500 eosinophils per microlitre of blood which is a hallmark of helminth infection (Murrel, 1982). During parasitic infections, the increase in the number of peripheral blood eosinophils is driven by Th2 cell- derived cytokines i.e., IL-5, IL-3 and GM-CSF (Rosenberg et al., 2007). Infections like schistosomiasis, visceral toxocariasis, strongyloidiasis, filariasis, ancylostomiasis, fascioliasis, trichinellosis, and paragonimiasis have highest eosinophil blood count (EBC) as these parasites undergo a phase of development that involves migration through the tissues (Tefferi, 2005). However, patients with asymptomatic filariasis and acute gastroenteritis showed DLC within normal range (Sneha and Chavarkar,

Historical Review

2017). Natasha (2015) reported that a patient suffering from filariasis showed no abnormality in the blood count. Sharma and Tyagi (2010) found coincidental microfilaria in a case of AML-M4 with eosinophilia. Dhameja and Bhatia (2014) reported a case of cervical swelling and mild fever whose haematological investigations showed 10% eosinophils. Sarojini and Senthilkumar (2013) observed increase in WBC count in lymphatic filariasis in Arakkonam area, Tamil Nadu, India. Basophils release histamines and leukotriene that contribute to the symptoms of allergic inflammation and have been proposed to play a key role by inducing class switching to IgE in B cells (Fallon et al., 2000).

Neutrophils are the most numerous among the leucocytes and have a circulating half- life of only 6–8 hours which is shorter than that of eosinophils. They are the first cell type recruited to the site of an acute inflammatory response and have the ability to act as phagocytic cells, to release lytic enzymes, and to produce reactive oxygen species with anti-microbial potential (Mollinedo et al., 1999; Cassatella et al., 1997). Neutrophils have often been overlooked in helminth infections as they are predominantly involved in the phagocytosis of microbial pathogens (Tkalcevic et al., 2000).

Lymphocytes are the immunocompetent cells that bring about the immune response in the host. Increase in the lymphocyte count was observed during Hymenolepis nana infection in experimental mouse (Parvathi and Karemungikar, 2011). After the phagocytic neutrophils fail in checking the invading parasites, it is the lymphocytes that are responsible for the host’s response to overcome the parasitic stress. They are also essential for the repair mechanism. The increase in monocytes i. e., monocytosis, too, is a result of immunological response of the infected host to combat helminth infection. The infected host adopts various defense mechanisms to dislodge helminth infection which is indicated by the variations in its white blood parameters that include leucopenia, neutropenia, lymphocytosis, eosinophilia and monocytosis (Parvathi and Karemungikar, 2011). Monocytosis was reported in a 37 years old male who was diagnosed with chronic myeloid leukemia associated with filariasis (Pahwa et al., 2015).

Hewitt et al. (1947) discovered Diethylcarbamazine (DEC) which has been used as an effective therapeutic agent against lymphatic filariasis over the last five decades. It is

Historical Review clinically used as a dihydrogen citrate salt and is given orally. It reaches all parts of the body within 25 minutes after its intake. ELISA can determine accurate blood levels of DEC as observed by (Mitsui et al., 1998). The plasma half-life of DEC varies from 6.1 to 8.1 hours and its concentration in blood reaches zero within 48 hours. Excretion of this drug is mainly renal. Although DEC has been found to be a highly effective microfilaricidal drug by Jing-yuan et al. (1991), varying degree of its activity has also been reported at different doses (MacKenzie, et al., 1985; Weil et al., 1988; Ismail et al., 1996; Eberhard et al., 1997; Noroes et al., 1997). The standard DEC treatment regimen has been suggested to be 6 mg/kg per day over a period of 10 to 20 days (Campbell, 1986; WHO, 1993). An effective regimen used in mass treatment programs for lymphatic filariasis is the medication of common cooking salt with DEC in concentrations ranging from 0.1% to 0.6% (WHO, 1993; Gelband, 1994 and Shenoy et al., 1998). However, current practice in mass drug treatment with DEC is a single annual dose of 300 mg of DEC for adults and 150 mg for children (often combined with Ivermectin, and sometimes with Albendazole also) (Kimura et al., 1996; Taylor and Turner, 1997).

Although it is widely administered in endemic regions, its mechanism of action remains unknown. However, Maizels and Denham (1992) gave experimental evidences that suggest its involvement in the modification of the host’s innate immunity while Kanesathasan et al. (1991) suggested its interference with prostanoid metabolism which appears to affect the clinical outcome. It has been observed in the studies conducted earlier that DEC therapy leads to improved function of both neutrophils and eosinophils which results in significantly better adherence and cytotoxicity in the treated host (King et al., 1983; Chandrashekhar et al., 1984). Mc Garry et al. (2005) performed experiments on mammalian models to observe the action of DEC and found that its activity was mediated by cyclooxygenase pathway and inducible nitric oxide. However, the role of the cyclooxygenase pathway and nitric oxide in the mechanism of action of DEC is controversial, as it is contraindicated by the studies of Rajan et al. (1998). DEC is found to alter the metabolism of arachidonic acid in the microfilariae as well as the host’s endothelial cells thereby leading to the constriction of blood vessels and aggregation of defensive cells including granulocytes and platelets (Tripathi et al., 2006). Johnson et al. (1988) reported that DEC enhances IgG-mediated granulocyte adherence to microfilariae of

Historical Review

B. malayi. This study has led to the suggestion that DEC stimulates the host defense activity as it acts as an opsonin, rendering microfilariae accessible to phagocytes by modifying their surface, though it was ineffective in vitro which was confirmed by earlier workers (Hawking et al., 1948; Hawking and Laurie, 1949). These observations suggest that DEC stimulates the host defense activity. Johnson et al. (1988) detected high titer of antimicrofilaricidal antibodies in the sera of chronic patients which was low in microfilaraemic man as per report of Alves et al., 2001. It has also been indicated earlier that the adaptive antigen specific immune response is not necessary since DEC exhibits its microfilaricidal effect in congenitally athymic nude mice at 100 mg/kg dose (Vickery et al., 1985).

Studies by Hawking (1979) suggested that DEC does not have a direct lethal effect on the microfilarial surface as the microfilariae remained unharmed even after exposure to those concentrations of the drug that exceeded the therapeutic level. After treating with DEC, Gibson et al. (1976) found ultrastructural alterations on the microfilarial surface which was due to the release of acid mucopolysaccharide from the surface of microfilariae, while Staniunas and Hammerberg (1982) suggested the aggravation of polyanionic components on the worm surface which have the ability to fix complement in case of human onchocerciasis.

In vitro studies demonstrated that DEC induces loss of the microfilarial sheath and lysis of the cytoplasm of W. bancrofti which cause reduction in motility and neuromuscular alterations, along with the formation of vacuoles, and dissolution of cytoplasm. This result was replicated in vivo, in microfilaraemic blood retrieved from patients 40 minutes after the administration of DEC (Florencio and Peixoto, 2003; Peixoto et al., 2004). The microfilarial exsheathment is possibly due to the alterations of specific biochemical pathways, such as the release of endoproteases as earlier indicated by Devaney and Howells, (1979). It has also been reported that DEC may reduce the fertility of adult female worms, as it reduces intrauterine embryos which suggests its effects on embryogenesis (Peixoto, 2004; Gunawardena et al., 2005). The use of fluorescent DEC analogs in a B. malayi model has demonstrated that DEC accumulates in adult filarial worms, specifically in the pharynx, oesophagus, nerve ring, testes in male and vulva in female worms (Junnila et al., 2007).

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Horii and Aoki (1997) demonstrated significant killing of microfilariae within 30 minutes of administration of a single dose in animals. It has been found effective against microfilariae of W. bancrofti and Onchocerca volvulus in randomized trials (Diallo et al., 1986; Meyrowitsch et al., 1996; Bockarie et al., 1998). Although the response varies significantly among individuals but there was a significant decline in the microfilarial count after the treatment (Andrade et al., 1995; Stolk et al., 2005). DEC is not effective against the adult worm of W. bancrofti and O. volvulus as microfilariae reappeared after treatment (Ottesen, 1985; Boussinesq, 2006). Therefore, continuous use of this drug increases the threat of drug resistance which has already been reported in many veterinary studies, and is a serious concern for the control of filariasis (Bourguinat et al., 2006). Nonetheless, DEC is effective against both the microfilariae and adult worms of Loa loa, B. timori and B. malayi causing rapid clearance of microfilariae, whereas adult worms remain alive in 20–60% of patients treated with DEC, hence, repeated administrations may be required (Fan, 1992; Hakim et al., 1995; Boussinesq, 2006).

Rossignol and Maisonneuve (1984) first described nitazoxanide (NTZ) as a human cestocidal drug that was originally developed as a veterinary anthelmintic. Many workers in their clinical trials have reported NTZ as a broad spectrum drug which is effective against intestinal cestodes, nematodes and protozoa (Gilles and Hoffman, 2002; Juan et al., 2002; Ortiz et al., 2002; Stettler et al., 2004; Walker et al., 2004; White, 2004; Hemphill et al., 2006; Anderson and Curran, 2007; Aslam and Musher, 2007; Craigand, 2007; Kappagoda et al., 2011; Singh and Narayan, 2014). The efficacy of NTZ was assessed against nematode parasites like Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura and Strongyloides stercoralis with variable therapeutic effects (Romero et al., 1997; Abaza et al., 1998; Geary et al., 1999; Davila-Gutierrez et al., 2002; Juan et al., 2002; Diaz et al., 2003; Fox and Saravolatz, 2005; Anderson and Curran, 2007; van den Enden, 2009). Fox and Saravolatz (2005) suggested that successful NTZ treatment regimens for nematode parasites typically involve six doses over two days. Mesquita et al., (2014) found NTZ to induce oxidative stress in Leishmania infantum and might be a useful compound for the investigation of new therapeutic targets. It acts against the energy metabolism of lower organisms by inhibiting the activities of enzymes, pyruvate ferredoxin oxidoreductase (PFOR) and protein disulphide isomerase (PDI) (Hemphill

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et al., 2006; Aslam and Musher, 2007; Pal and Bandyopadhyay, 2012; Lloyd-Evans et al., 2014, Abdel-Hafeez et al., 2015). Somvanshi et al. (2014) conducted studies on Caenorhabditis elegans and found that NTZ acts on the nematodes through avr-14, an alpha type subunit of a glutamate gated chloride channel. Therefore, NTZ was considered to be a potential drug development candidate eligible for rapid transitioning into development for human soil-transmitted helminth (STH) infections (Olliaro et al., 2011). Nevertheless, one trial suggested that NTZ does not work well against whipworms at a single-dose as suggested by Speich et al. (2012) while Hu et al. (2013) later suggested that higher dosage of this drug was effective against STHs. Treatment of mice with NTZ significantly reduced the number of live larvae of Toxocara canis found in the brain (Delgado et al., 2008). Tritten et al., (2012) tested NTZ alone and in combination with commercialized anthelmintics on Trichuris muris and Ancylostoma ceylanicum, in vitro and in vivo with variable effects. Identification of new and potent anthelmintic compounds with long term microfilaricidal and/or macrofilaricidal activity for chemotherapy of filariasis, especially lymphatic filariasis was suggested (Vande, 1991; Grove, 1996).

Nanoparticle based drug formulation is an effective alternative against helminth parasites due to the lack of effective vaccines (Khan et al., 2015). Drugs which are most frequently used against filariasis are DEC, ivermectin and albendazole (Gayen et al., 2013; Singh et al., 2013). Smith et al. (1998) reported that the cuticles of

microfilariae and adult worms were resistant to hydrogen peroxide (H2O2) due to the presence of α-tocopherol in the lipid fraction of parasite surface. Moreover, DEC in

combination with H2O2 as well as Albendazole (ABZ), showed synergism against filarial parasites (Molyneux et al., 2003; Rajendran et al., 2004; Sunish et al., 2006; Wamae et al., 2011; Sharma et al., 2014). Thomsen et al. (2016) reported that triple drug therapy that combines albendazole, DEC and ivermectin is more effective in bancroftian filariasis. Thus, strategizing combinations of existing drugs or their combinations with nanoparticles is the present day requirement for developing novel leads in antifilarial therapeutics. Possible advantages of nanoparticles in biomedical and industrial applications for human health and environment are now accepted in the literature (Service, 2003). In the biological field, effects of size, shape, uptake, and distribution are the aspects of nanoparticles on which research is focussed (Patra et al., 2007).

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Major developments in the diagnosis, detection and treatment of various diseases in future can be achieved by implementation of nanotechnological advances in microbial therapeutics (Oka et al., 1994; Curtis and Wilkinson, 2005; Angeli et al., 2008; Debbage, 2009; Swai et al., 2009). The in vitro efficacy of ABZ+CuO nanocomposite as antifilarial agent was studied by Zafar et al., (2006), using S. cervi as a model, both in the dark and under UV light. The antibacterial and antiviral behaviors of silver, silver ions and silver containing compounds have been studied earlier (Tokumaru et al., 1974; Oloffs et al., 1994). For the formulation of new pharmaceutical products, uniform silver nanoparticles need to be prepared with specific requirements in terms of size, shape, physical and chemical properties (Bigger et al., 2002; Merisko- Liversidge et al., 2003). The nanoparticles are known to generate reactive oxygen species (ROS) because of their chemical reactivity to kill the microbes and parasites. Nanoparticles of silver (AgNPs) exhibit their antibacterial potency by inducing p53 expression and apoptosis in eukaryotic cells (Kim et al., 2007; Shrivastava et al., 2007; Gopinath et al., 2008; 2010). Strikingly, AgNPs are reported to induce apoptosis which is associated with the generation of free radicals (Foldbjerga et al., 2009; Miura and Shinohara, 2009; Asha Rani et al., 2009). Shrivastava et al. (2007) observed enhanced antibacterial potency of AgNPs. Morones et al., (2005) made valuable efforts to explore the underlying molecular mechanism of the antimicrobial activity of silver. Dissolved silver nanoparticles are found to interact with the thiol groups of vital enzymes and inhibit them resulting in biological actions against microorganisms (Matsumura et al., 2003; Hwang et al., 2008; Choi et al., 2008). Kittler et al., (2010) demonstrated that the presence of salts and biomolecules in the suspension medium significantly affects the ionisation rate of AgNPs.

McGarry et al., (2005) observed that the host responds to the microbial pathogen through inflammation. Generation of a pro-oxidant state provides a premise for antifilarial drug development as oxidative stress plays a great role in innate immunity (Verhasselt et al., 1998). It has been reported that the smaller AgNPs induced higher levels of mitochondrial ROS which results in reduction of mitochondrial membrane potential, release of cytochrome c into the cytosol, JNK activation and translocation of Bax to mitochondria as observed in mouse fibroblasts and human hepatocytes (Hsin et al., 2008; Piao et al., 2011; Onodra et al., 2015). Ranjbar et al., 2014 found AgNPs a potential synergist in the treatment of diseases as they maintain

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concentration of the circulating drug. The possible efficacy of AgNPs on the microfilariae of B. malayi was investigated by motility assay and it was reported that there is a subtle link between oxidative stress and apoptosis induction (Buttke and Sandstrom, 1994; Singh et al., 2012). Nanosilver is expected to achieve a concentration which has desired local antifilarial therapeutic action in the infected population, provided that AgNPs are distributed widely in the tissues including the lymphatics (Lankveld et al., 2010).

Kumar (1987) studied the in vitro effect of drugs against energy metabolism of S. cervi which involved both carbohydrate as well as lipid metabolism, and observed that DEC, levamisole, mebendazole and thiabendazole influence only carbohydrate metabolism. The antifilarial activity of chalcone derivatives on S. cervi was assessed by viewing motility and viability of parasites and MTT assay. Evaluation of antifilarial activity was done by using S. cervi glutathione S-transferase (GST) as a drug target (Awasthi et al., 2009). Madkoriya et al. (2013) studied the effects of tetracycline, rifampicin and doxycycline on the motility of S. cervi in terms of MTT reduction assay and reported significant decrease in its motility after 24 hours with increasing concentration of tetracycline and rifampicin but with doxycycline the effect was not marked (2- 20 mg/ml).

Gupta and Rathaur (2005) assayed the in vitro effect of DEC, butylated hydroxyanisole and phenobarbitone on the GST of adult female S. cervi and found that GST is inducible in response to the antifilarial drug DEC and may play an important role in the parasite’s survival thus, could be a potential drug target.

The anterior and posterior ends of the adult worms of Setaria spp. such as S. cervi, S. digitata, S. marshalli, S. marshalli pandei, S. equina and S. labiatopapillosa, were studied by scanning electron microscopy at critical points to observe their morphological peculiarities (Shoho and Uni, 1977; Zdarska and Scholl, 1978; Srivastava et al., 1985; Almeida et al., 1991; Yadav and Tondon, 1991; Rhee et al., 1994). Li and Yu (1990) and Das and Das (1995) studied the morphology of the larval stages of S. labiatopapillosa, S. leichungwingi, S. equina and S. digitata, whereas Nikander et al. (2007) studied the morphology of S. tundra obtained from the peritoneal cavity of woodland caribou (Rangifer tarandus). Watermeyer et al. (2013) studied S. graberi by scanning electron microscopy (SEM) and redescribed it. SEM

Historical Review images were quite helpful in differentiating it with other Setaria spp. present in the same host. Kumar and Kumar (2016) studied the ultrastructure of S. digitata (male and female) worms collected from bovine peritoneal cavity from India and used nomarski interference contrast optics along with SEM to study the surface features of the microfilariae. Franz and Zielke (1980) also used SEM to study the larvae of W. bancrofti from the vector and from experimental rodent hosts. Earlier study conducted by Madhathiparambil et al. (2011) demonstrated the effect of Triton X-100 on the surface of S. digitata which showed that Triton X-100 treatment resulted in deepening of the striations and vertical wrinkling of the parasite wall. SEM has been employed by Tippawangkosol et al. (2004) to observe the in vitro effects of ivermectin (IVM), DEC and albendazole (ABZ) against infective third stage larvae of B. malayi, while Piexoto et al. (2003) observed that the microfilariae of W. bancrofti lost their sheath after treatment with DEC. Singh et al. (2012) observed significant ultrastrucural damage by SEM in the microfilarial sheath after incubation in nanoparticles of silver.

Enzymes are expected to have an important role in host parasite interactions and disease process as they have certain biological functions that make them essential for the survival of the parasite (Bhandary, 2006). Obtaining information about the metabolism of filarial nematodes was considered necessary for a better understanding of the mechanism of action of the available drugs and for the development of new antifilarial drugs, and it was found that among all metabolic pathways, metabolism of carbohydrate plays a significant role in providing energy to filarial species. However, a few reports are available regarding the effects of anthelmintics on the survival and enzyme activities of the S. cervi in vitro and in vivo (Ahmad and Srivastava, 2007; Singh and Rathaur, 2010; El-Shahawi, 2010; Srinivasan, 2011).

Parasites living in the peritoneum, GI tract and tissues of the hosts have been studied for the assessment of the effect of anthelmintics on a number of enzymes of glycolytic and oxidative pathways (Van den Bossche and Janssen, 1969; Gaitonde, 1971; Benedictov, 1975; Reznik, 1977; Anwar et al., 1977; Walter, 1979; Agarwal et al., 1990; Hussain et al., 1990ab; Ahmad and Srivastava, 2007; Srinivasan et al., 2011; Khan et al., 2012). As tricarboxylic acid (TCA) cycle provides intermediates for other pathways, it is considered to be a significant pathway for carbohydrate catabolism. However, much information is not available from the tissue inhabiting nematodes. Filarial parasites are known to have active glycogenic and glycolytic pathways and

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somewhat submissive TCA cycle (Saz, 1981; Barrett, 1983). Earlier studies have shown the presence of TCA cycle enzymes in adult B. pahangi, D. immitis, C. hawkingi, D. viteae, L. carinii, O. volvulus and S. cervi also (Srivastava and Ghatak, 1971; Anwar et al., 1977; Walter, 1979; Middleton and Saz, 1979; Walter and Schultz-key, 1980; Agarwal et al., 1986; Dunn et al., 1988). These findings show that oxygen is quite essential for the maintenance of motility and survival of these filarial worms. Since TCA cycle does not constitute a significant energy-yielding pathway, aerobic requirement may lie completely in the oxidative decarboxylation of pyruvate

to acetate and CO2 (Wang and Saz, 1974).

It has been found that most adult filarial worms use the glycolytic breakdown of carbohydrate to lactate as a preferred route for the supply of their energy requirements (Saz, 1981; Barrett, 1983; Barrett et al., 1986; Dunn et al., 1988; Köhler, 1991). However, Rew and Saz (1977) have suggested that microfilariae exhibit an aerobic carbohydrate catabolism, requiring oxygen at least for motility, but apparently not for survival. Srivastava et al., (1988) suggested minor catabolic routes that are similar to those in adults and result in acetate and succinate formation by the microfilariae. A full complement of TCA cycle enzymes were present in B. pahangi and D. immitis, even though the enzymes involved in the initial steps of the cycle were found with very low activity, while in S. digitata, these enzymes were almost absent (Barrett, 1983; Barrett et al., 1986; Comley and Mendis, 1986; Dunn et al., 1988; Unnikrishnan and Raj, 2000). Khatoon et al. 1984 observed marked activity of glucose-6-phosphatase throughout the body of the microfilaria except for Innenkörper whereas, intense aldolase activity was observed in the excretory pore and G-cells. Studies were done on the distribution of glucose-6-phosphatase, succinate dehydrogenase and adenosine triphosphatase in adult S. cervi worms which were intraperitoneally transplanted in rabbits (Khan et al., 2012).

It is known that anthelmintics inhibit a variety of enzymes in adult nematodes. Subrahmanyam (1987) observed that DEC alters glucose uptake and inhibits phosphoenolpyruvate carboxykinase, fumarate reductase and succinate dehydrogenase. The effects of DEC, tetramisole and chlorpromazine drugs on glucose-6-phosphatase and succinate dehydrogenase in the microfilariae recovered from the peripheral circulation of rats were observed by Zakai and Khan (2015). Barrett (1976) suggested that among the TCA cycle enzymes, the ratio of succinate

Historical Review dehydrogenase to fumarate reductase is supposed to give an idea about the aerobic or anaerobic status of the organisms. Fry and Brazely (1983) observed this ratio in S. digitata 1.75: 1, which is close to the value of facultative anaerobes. Malate dehydrogenase (MDH) and malic enzyme (ME) are commonly found in the cytosol of parasites. In case of S. cervi, the microfilariae and adults resemble each other in their metabolic pattern. MDH has been reported as the most active enzyme in microfilariae, followed by lactate dehydrogenase and fumarase, whereas phosphoglucoisomerase, PEPCK and FBP-aldolase were relatively less active (Ahmad and Srivastava, 2007). DEC has been found to be less effective in inhibiting microfilarial MDH and ME from S. digitata, while filarin, a drug of herbal origin, effectively inhibits MDH (Banu et al., 1992).

Sharma and Rathaur (1999) observed the histochemical localization of acetylcholinesterase in microfilariae, while presence of collagenase was confirmed in adult and microfilariae of S. cervi by Pokharel et al. (2006). Baqui and Khatoon (1982) documented histochemical changes in the activity of alkaline phosphatase and glycogen in S. cervi obtained from levamisole and suramin treated rats.

Oxidative stress is an inevitable consequence of aerobic metabolism which is a major contributing factor to innate immunity, necessary to elicit effective responses against microbial infections (Verhasselt et al., 1998). Nevertheless, filariid worms have evolved strategies to evade host’s hostile environment through a series of enzymatic and non-enzymatic antioxidant system (Chiumiento and Bruschi, 2009; Henkle- Duhrsen and Kampkotter, 2001; Selkirk et al., 1998). A powerful effector tool against parasites is represented by highly reactive oxygen species which are produced during normal cellular metabolism, especially by activated phagocytes, and even by some anti-parasitic drugs. ROS are also produced due to the host response against the parasite and are capable of damaging membranes, proteins and nucleic acids which is sufficient to kill cells or even the entire parasite (Petkau, 1986). Phagocytic cells are the most important effectors of parasite killing in vitro; however, there is a lack of evidence of direct killing in vivo. Phagocytes, including eosinophils and neutrophils and also platelets either secrete toxic proteins or use free radicals in combination with antibodies or complement factors to kill the parasite. Most phagocytes pass through a respiratory burst that results in the release of hydrogen peroxide (H2O2) and

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- superoxide anions (O2 ) which undergo Harber-Weiss reaction to form singlet oxygen 1 * ( O2 ) and hydroxyl radicals (OH).

The parasites also have to deal with the oxidative stress imposed by the host´s immune response in addition to coping with ROS levels generated from intrinsic sources. Moreover, ROS which are produced during the epithelial innate immune response of the vector or by the vector resident gut bacteria or during melatonic encapsulation processes are also faced by the parasite (Kumar et al., 2003; Cirimotich et al., 2011). All protozoan and helminth parasites apparently have one or more antioxidant enzymes that are able to scavenge or neutralise the reactive oxygen species (ROS), and these enzymes play a crucial role in protecting the parasite against the host response. Therefore, antifilarial drug formulations should be designed in such a way that high levels of ROS could be produced independently without involving host cells.

Lipid peroxidation (LP) products are formed as a result of the oxidation of polyunsaturated fatty acids induced by ROS. Malondialdehyde (MDA) is one of the several products formed during lipid peroxidation and is used as ROS biomarkers as its level provides an indication of the extent of lipid peroxidation (Serarslan et al., 2005; Siwela et al., 2013). Significant elevation of MDA level in the blood of the organisms has been reported against various parasitic infections (Erel et al., 1997; Shiono et al., 2003; Rezaei and Dalir-naghadeh, 2006; Saleh et al., 2009; Ince et al., 2010; Kucukkurt et al., 2014). Gomathi, 2000 noticed alterations in lipid peroxidation in the tissues of Mastomys natalensis infected with B. malayi. Significantly higher MDA levels were observed in the tissues of birds and animals infected with helminth and protozoan parasites when compared with that of uninfected animals (Bagchi and Basu, 1993; Gharib et al., 1999; Oliveira and Cecchini, 2000; Ince et al., 2010; Siwela et al., 2010; Siwela, 2013; Fahny et al., 2014). Increase in the MDA levels in the serum of humans infected with helminths has also been noticed by various workers (Kilicet al., 2003; Saraymen et al., 2004; El-Badry, 2006; Kaya et al., 2007; Aloho- Bekong et al., 2011). A significant increase in the MDA level in the serum of Egyptian buffaloes infected with filarial worms was also observed, however, after the co-administration of levamisole and ivermectin its level was reduced compared to untreated buffaloes (Radwan and Talkhan, 2009). Filarial infection is also associated

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with a marked increase in arachidonic acid level that indicates structural and functional disorders of the cellular membranes which is accompanied by an increase in the quantity of thiobarbituric acid reactive substances (TBARS) (Kuchbaev and Basterbekova, 2001; Pompeia, et al., 2002; Huang, et al., 2007). Franca et al., (2012) reported a positive correlation between the degree of parasitemia and TBARS levels and activity of antioxidant enzymes in dog’s blood infected with Rangelia vitalii.

Embedding and penetration of the parasites in the mucosa of the host causes inflammation. But the cells of the host remain protected from oxidative damage, as they are equipped with several antioxidant defense enzymes. However, as soon as the activities of the antioxidant enzyme system are overwhelmed by the generation of reactive oxygen species, the cells no longer remain protected (Siwela et al., 2013). Halliwell (1999) categorized antioxidant enzymes into primary and secondary antioxidants, the former react directly with pro-oxidants (eg. catalase and superoxide dismutase), while the latter are involved in the generation of low molecular weight antioxidant species. Alterations in enzyme biochemistry, biological molecules (ROS) and cell membrane including increase in cellular formation of oxidative stress via creating imbalance between ROS and the antioxidant systems are induced by drug combinations which can act in synergy, potentiation or antagonism (Davies, 1992; Jones, 1996; Escobar-Gracia et al., 2001; Essa et al., 2012).

The activity of superoxide dismutase (SOD) is crucial in the prevention of oxidative damage by scavenging and converting superoxide anions to hydrogen peroxides which acts as a substrate for catalase and is decomposed into water and molecular oxygen thus, protecting the tissues from the actions of the highly reactive hydroxyl radicals (Nelson and Kiesow, 1972; Aebi, 1984; Michiels et al., 1994). Activities of these enzymes are altered during these processes which leads to ROS induced pathological disorders and DNA damage (Ramaiah and Jaeschke, 2007).

In the liver, the production of reactive oxygen or nitrogen species can cause damage which is prevented by SOD, catalase, glutathione, and glutathione peroxidase (Halliwell and Gutteridge, 1989). The first defense against pro-oxidants is the catalysis of dismutation of superoxide to hydrogen peroxide by plasma SOD (Halliwell and Chirico, 1993; Fridovich, 1995; Celi, 2010).

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Helminth infections stimulate the immune system to produce H2O2 whose accumulation results in decrease in the activity of SOD possibly through feedback inhibition (Sienkiewicz et al., 2004, Aloho-Bekong et al., 2011). Small deviations in the physiological concentrations of these enzymes may have a dramatic effect on the resistance of cellular lipids, proteins and DNA to oxidative damage as these enzymes work together to eliminate ROS (Mates and Sanchez-Jimenez, 1999; Ince et al., 2010). Thus, activities of these enzymes play an important role in the advancement of the disease (Mates et al., 1999).

It was demonstrated by Kono and Fridovich (1982) that superoxide anions which act as a substrate for SOD inhibit the activity of catalase thus, leading to the conclusion that SOD and catalase constitute mutually-acting protective set of enzymes. Studies conducted by Batra et al. (1989) found that in animals infected with Dipetalonema viteae, the activity of SOD decreased in liver and spleen, but increased in the lungs. Ince et al. (2010) observed that the levels of SOD and catalase decreased in the erythrocytes, liver and kidney tissue homogenate of rats infected with Syphacia muris. However, treatment of these rats with levamisole and its combination with vitamin C resulted in elevation of the SOD and catalase levels. Gomathi (2000) analysed the SOD levels in all organs of Mastomys natalensis infected with B. malayi and observed a similar decrease. However, treatment with DEC resulted in an increase in the level of SOD in all the organs except the testes, whereas, lungs showed a decrease in the enzyme level. Aloho-Bekong et al. (2011) reported significantly higher catalase activity in the sera of uninfected, mild ascariasis and trichuriasis than that observed in the serum of moderate S. mansoni infection.

Although catalase and GPx are known to detoxify H2O2 to water, the latter appears to have more potent activity (Halliwell and Gutteridge, 1999; Margis et al., 2008). GPx catalyses the reduction of hydrogen and lipid peroxides in animal tissues and provides protection against oxidative stress and therefore, acts as an indicator of the same (Halliwell & Chirico, 1993; Tüzün et al., 2002). Siwela et al. (2013) observed unaltered GPx activity in hepatic cells during helminth infection in ostriches, while Kolodziejczuk et al. (2006) reported similar effect in rat’s serum infected with F. hepatica. GPx activities in liver, brain, testes, and heart of Mastomys natalensis was

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decreased when infected with B. malayi however, its activity increased in all organs after treatment with DEC (Gomathi, 2000).

Another enzyme, glutathione S-transferase (GST) provides protection against ROS which is achieved by conjugating glutathione on the sulphur atom of cysteine to various electrophiles (Chasseaud, 1979). Sagara et al. (1998) and Pedrosa et al. (2001) studied GST (a phase II enzyme of drug biotransformation) and reported that it has a remarkable scavenging potential. The small and large intestines of mice infected with Trichinella spiralis revealed a significant increase in the GST activity (Wojtkowiak-Giera et al., 2011). The secretory GST from O. volvulus exhibits several features like (i) easy accessibility as it is located directly at the parasite-host interface, (ii) ability to detoxify and/or transport various electrophilic compounds and secondary products of lipid peroxidation and (iii) involvement in synthesis of potential immunomodulators. These features make it an excellent drug target. The structure based design of specific inhibitors is supported by its significant structural differences to the host homologue as observed in xenobiotic binding sites (Sommer et al., 2003; Perbandt et al., 2008; Liebau et al., 2008).

Chemical, physical or biological parameters that are used to measure progress of a disease or the effects of treatment are considered as biomarkers (Loukopoulos et al., 2003). In case of filarial infections, earlier studies have reported elevated levels of liver biomarkers such as alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the sera of infected animals (Kar et al., 1993; Ojiako and Onyeze, 2009). The efficacy of doxycycline during W. bancrofti infection was assessed by Makunde et al. (2006) when administered orally and it was found that the haematological, hepatic, renal and clinical parameters remained unaltered.

In case of wistar rats infected with L. carinii, the activities of AST and ALP were observed to be enhanced while that of ALT remained in the normal range (Nadkarni et al., 1989). Sharma and Joshi (2002) reported a marked increase in the activity of AST which signified necrotic degeneration of liver cells caused by microfilariae in cattle.

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Filarial nematodes are among those human pathogens that enjoy longest lifespans, as individual adult worms may live for 7 years or more while their microfilarial offsprings can remain in the blood stream for atleast a year (Piessens and Partono, 1980; Eberhard, 1986). Mohanty et al. (2000) undertook a study on the prevalence of natural infections of S. digitata and its immune responses in cattle to gain awareness for future investigations on zooprophylaxis as well as zoonotic infections of human populations with the bovine filarial parasite. Antibodies to four different filarial antigen preparations of S. digitata (i.e., adult females, adult males, microfilariae, ES products of adult females) quantified by ELISA in all cattle showed uniformly high titres of anti-filarial antibodies in all groups and there was no significant difference in antibody levels among them. Sundar and D’Souza (2015) carried out studies on survival, activity and release of antigenic excretory secretory products and microfilariae of S. digitata maintained in artificial media.

As immunological pathways are becoming increasingly well defined and the filarial genome project is advancing, it is now possible to search for and characterize individual molecules from these parasites that encode proteins involved in neutralizing or evading the host immune response which may also serve as ideal vaccine or drug targets. Nematode parasites may use the genetic heritage that they share with mammals and adapt conserved regulatory molecules for immune evasion instead of capturing immunomodulatory genes from their hosts. Since nematodes have relatively large genomes (100 million base pairs or more) that encode approximately 20,000 proteins, the scope for expressing direct immune modulation products is immense (Pastrana et al., 1998; Gomez-Escobar et al., 2000). Another strategy adopted by the filarial nematode may be that it can induce the host cells to adopt suppressive functions (Loke et al., 2000) thereby providing the long term immunological tolerance (Maizels and Lawrence, 1991; King et al., 1992).

Malhotra et al. (1986) used certain immunological techniques to study the protein and antigenic patterns of adult (female/ male) and microfilariae of S. cervi. The presence of 9–10 precipitin lines in adults and only four precipitin lines in microfilarial antigenic preparations have been revealed by immunoelectrophoresis while crossed immunoelectrophoresis has resolved these antigenic preparations further and has revealed the presence of about 22–24 and 12–14 antigens in adults and microfilariae, respectively.

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Kaushal et al. (1987) used immunoblotting technique for the identification and characterization of antigenic proteins of S. cervi adults and microfilariae using hyperimmune rabbit sera against S. cervi and B. malayi and found fifteen different antigens common between adult and microfilarial stages of the parasite with a few stage-specific antigens. An immunodominant filarial protein identified as SXP-1 from W. bancrofti and previously exploited for diagnosis of human lymphatic filariasis has been shown to be well conserved across several filarial species, including S. digitata (Sasisekhar et al., 2005). Dalai et al. (1998) reported that S. digitata has shown strong antigenic reactivity with sera of several filarial species.

Zwitterionic glycolipids of all filarial nematode species have been observed to contain the antigenic determinant phosphocholine by the use of an epitope specific monoclonal antibody (Wuhrer et al., 2000). Filariasis is among those parasitic infections that cause immune suppression during the course of infection in both humans and experimental animals. John and Raj (1998) isolated a 29 kDa protein from detergent-soluble antigen of S. digitata that has shown maximum inhibition of the cell-mediated immune response, thereby contributing to the factors responsible for the survival of filarial worms in hosts. Bright and Raj (1990) have demonstrated the presence of cuticle-specific antigens (CSAs) in S. digitata that might prove a useful tool for the detection of filariasis. Treatment with Triton X-100/EDTA extraction was used to isolate the surface antigens of S. digitata whose purification was done by affinity chromatography using Sepharose-bound W. bancrofti antibodies obtained from chronic human filarial sera (Bright and Raj, 1992; Theodore and Kaliraj, 1990). Theodore and Kaliraj (1990) resolved the surface antigen into more than six protein bands in non-SDS PAGE whereas analysis of surface antigens by SDS-PAGE has shown that the proteins with molecular weights 17, 29 and 36 kDa were the three major polypeptides, and different combinations of these gave rise to six native surface proteins. The 29 kDa protein was found to exist as a monomer as well as cross-linked with the 17 and 36 kDa proteins. All surface antigen fractions have shown antigenicity, however, the 29 kDa protein remains as a high avidity surface antigen. Bright and Raj (1990) reported the release of 29 kDa surface protein during in vitro culture of adult parasites, and its cross-reactivity with antiserum specific to surface antigens, indicating towards the possible natural shedding of surface molecules into the host system. The purified and crude antigens have been used in ELISA for the

Historical Review

detection of serum antibodies in bancroftian filariasis (Bright and Raj, 1991; Sharma and Upadhyay, 1993). Filarial Hsp70 is also reported to be highly immunogenic in case of B. malayi and O. volvulus (Rothstein et al. 1989; Selkirk et al. 1989).

Various immunodiagnostic techniques like indirect haemagglutination test (IHAT), indirect fluorescent antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA) were employed to compare their efficacy in the detection of antibodies by using W. bancrofti microfilaria antigens. The utility of human filarial serum immunoglobulin (FSI) in detecting circulating antigen of filariids by counter immunoelectrophoresis (CIEP) and IHAT has been explained by Kaliraj et al. (1981b) whereas, Tandon et al. (1981) studied the use of rodent and bovine filarial antigens for diagnosis of human filariasis by antibody detection through ELISA. Though passive cutaneous anaphylaxis (PCA) test was a simple method for the diagnosis of filariasis, it was highly sensitive (Singhal, 1983). Another highly sensitive, consistent and reproducible method for the diagnosis of filariasis described by Chandrasekhar et al., (1984) was isosmotic percoll (IOP) technique in 0.25 M sucrose in which the recovery of microfilariae was 85-97% and by this technique they isolated pure populations of microfilariae of L. carinii, B. pahangi, B.malayi and D. vitae. Secretory acetylcholinesterases from S. cervi were found to be antigenic and cross- reactive with W. bancrofti infected asymptomatic microfilaraemic human sera when tested by ELISA and immunoblotting and utilized for the diagnosis of early filarial infections (Sharma et al., 1998).

Dissanayake and Chandana (1988) demonstrated the use of immunoperoxidase staining techniques to study the egg surface antigens of S. digitata and found that the soluble egg antigen reacted with the surface antigens of eggs, immature microfilariae of W. bancrofti and Dirofilaria repens. Symptomatic cases of human lymphatic filariasis were distinguished from the asymptomatic cases by Dot-ELISA using B. malayi adult worm (Tandon et al., 1988). Almedia et al., (1990) used S. cervi antigen to diagnose serum antibodies of human filariasis by CIEP, IFAT and ELISA out of which IFAT and ELISA showed higher degree of sensitivity (1:12800 to 1:102400). Microfilaraemic plasma was fractionated by Reddy et al. (1986) by 36-75% ammonium sulphate on ultragel which gave four protein fractions under which CFA-1 and CFA-2 showed antigenic activity. These antigens were further used to analyze different groups of sera for the presence of filarial IgM and IgG antibodies in indirect

Historical Review

ELISA. Zheng et al., (1990) produced monoclonal antibody of IgG subclass against excretory-secretory antigen of adult B. malayi which was used to detect circulating parasite antigens in human lymphatic filariasis by dot- ELISA, while in another study, dot-ELISA was compared with standard sandwich ELISA for detecting parasite antigen in sera of patients suffering from bancroftian filariasis. ELISA and IHAT were used in a study to compare the efficiency of W. bancrofti microfilarial and L. carinii adult antigens in immunodetection of bancroftian filariasis where microfilarial antigen of W. bancrofti was found to be more sensitive (Ganayni, 1992). Urinary filarial antigen was analysed by Chenthamarakshan et al., (1993) using SDS-PAGE and immunoblotting technique. John et al. in 1995 studied the diagnostic application of detergent soluble surface protein of S. digitata and found it very sensitive in antibody and antigen detection tests. Mustafa et al., (1995) identified the active antigen which was similar to filarial circulating antigen by fractionating the E-S product of S. cervi on Sephadex G150 column which showed high reactivity of ES-1 fraction with the monoclonal antibodies for detecting the filarial circulating antigen.

Kaushal and Kaushal (1995) produced monoclonal antibodies against antigenic epitopes common between S.cervi and human filarial parasites. Padigel et al., (1995) developed sandwich ELISA for detection of filarial antigen both in blood and urine by using urinary filarial antigen (UFAC2-DE1) obtained by fractionation of urine on U- Gel column, followed by anion exchange chromatography. Weil et al. (1997) used the immunochromatographic card test (ICT; Binax, Inc., USA) to quickly determine the W. bancrofti antigen within 10 minutes. Robert and Steven (1976) conducted CIEP using microfilarial antigen prepared from D. immitis to detect filarial antibodies in dogs and cats having dirofilariasis, while Robert et al. (1978) conducted CIEP with microfilarial antigen to detect antibodies from cats infected with B. pahangi.

Mishra et al. (2014) examined hydrocoel fluid for diagnosis of its filarial origin. Og4C3 antigen assay was conducted to detect circulating filarial antigen (CFA) in serum and hydrocoel fluids both. On one hand, the levels of IgG, IFN-γ and IL-10 were found to be high in CFA-negative, while IgM and IgE were high in CFA- positive hydrocoel fluid and serum samples. On the other hand, neither CFA-positive nor CFA-negative hydrocoel fluids and serum samples associated with hydrocoel showed any difference in IgG4 level. The synthetic peptides of WbSXP-1 were studied by Pandiarajaa et al. (2010) for the diagnosis of human lymphatic filariasis in

Historical Review

which B-cell epitopic analysis showed 4 potent immunodominant regions (N1, N2, N3 and N4) spanning the whole antigen that were produced and used as a diagnostic candidate to detect anti-SXP antibody and conversely to detect the infected individuals. These chimeric peptides which were constructed based on WbSXP-1 were reactive, specifically with microfilaraemic sera by ELISA. This peptide-based diagnostic method can serve as a standard better tool without cross-reactivity in lymphatic filariasis elimination program. SXP-1, a good diagnostic candidate was explored as an immunodiagnostic candidate in a rapid format, which detects early infections of brugian and bancroftian filariasis (Rao et al., 2000; Lalitha et al., 2002; Baskar et al., 2004; Lammie et al., 2004; Janardhan et al., 2007).

Many studies that utilised S. cervi antigens have shown cross-reactivity between the heterologous parasites which is common in many tropical diseases (Kaliraj et al., 1979; Kaushal et al., 1987; Sharma et al., 1998; Srivastava et al., 2004; Gupta et al., 2005; Singh and Rathaur, 2005; Pokharel and Rathaur, 2008). However, the suitability of such an antigen in detecting the disease by immunological methods is described by the degree of specificity. Hamilton and Scott (1984) and Sugunan et al. (1990) detected circulating parasites in humans using ELISA and western blotting, while Dasgupta et al. (1984) discovered the same in animal filariasis by polyclonal antisera. Srivastava et al. (2010) evaluated the antigenic efficacy of purified ScHSP70 against W. bancrofti infected filarial human sera using ELISA and immunoblotting. ELISA showed a similar pattern of antigenic cross-reactivity with sera of different categories of filarial worms when studied for IgG.

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INTRODUCTION

Every host remains in a constant danger of infection caused by pathogens. Host defense against the pathogens is well regulated through inflammatory response marked by leucocyte migration into the site of infection, inflammation, destruction of the microorganisms and finally, healing and repair of the affected tissue. This interaction between the host and parasite determines the outcome of the infection (Pina-Vazquez et al., 2012). There might be some pathogenic effects caused by mechanical and chemical damages along with host immune response against the parasite. Most of the helminth parasites including filarial worms by virtue of being large in size and invasive in nature provide antigenic stimulus to the host to produce antibodies to counter the infection. Immune-mediated inflammatory changes occur in the skin, liver, lungs, eyes and central nervous system as worms migrate through these organs (Wkelin, 1996). During helminth invasion, granulocytes such as eosinophils, basophils, monocytes and neutrophils play an important role. But after establishment and chronicity, lymphocytes get activated and start secreting antibodies to counteract the infection (Parvathi and Karemungikar, 2011). Depending on the situation, these leucocytes may have pivotal roles in host protection, immunopathology, or facilitation of establishment of infection (Makepeace et al., 2012).

Among parasitic diseases, filariasis in man and livestock is a major health problem in tropical countries. The debilitating effects and economic losses caused by these infections severely affect man and animal power resources in developing countries (Sundar and Ravindaran, 2009). Setaria cervi is a cosmopolitan filariid inhabiting the peritoneal cavity of buffaloes (Bubalus bubalis). This infection is very common in India showing incidence as high as 70-85% (Singhal et al., 1972; Mohanty et al., 2000). The parasites are generally considered to be non pathogenic in their natural hosts, but may result in serious and often fatal neuropathological disorders known by the term “cerebro-spinal nematodiasis” in unnatural host (Pachaury, 1972; Baharsefat et al., 1973; Bazargani et al., 2008). Larvae of S. cervi which normally parasitized the peritoneal and thoracic cavities had been found in the cerebrospinal cavity resulting in lumbar paralysis of the hosts (Shoho, 1958; Blazek et al., 1968; Wang et al., 1991; Tung et al., 2003). Animal models of filariasis have been used widely for understanding the pathogenesis of the disease, host response, drug screening and other

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biochemical aspects (Mohanty et al., 2000). Keeping the above facts in view, microfilarial density, differential leucocyte count and host reaction by the inflammatory cells were observed in the invaded tissues before and after the treatment of DEC, NTZ and NTZ+AgNPs in experimentally infected white rats. Walling up operation of the adult worm, destruction and its piece meal clearance was also observed by the defensive cells in white rats implanted with S. cervi.

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MATERIALS AND METHODS

Establishment of microfilaraemia in white rats and treatment with drugs:

Adult S. cervi worms were collected from the peritoneal cavity of freshly slaughtered buffaloes (B. bubalis) and washed thoroughly with Ringer’s solution at 37 °C to remove any debris. Microfilariae were obtained by dissecting uterus of gravid females and incubated in the same solution at 37 °C before use. 25 laboratory-bred white rats (Rattus norvegicus), weighing 125–150 g were used for this study. Microfilariae were injected in the peritoneal cavity of all the albino rats which were divided into four groups each having 5 rats. Groups 1, 2 and 3 were treated with diethylcarbamazine (DEC), nitazoxanide (NTZ) and nitazoxanide combined with silver nanoparticles (NTZ+AgNPs), respectively 10th day onwards when microfilariae appeared in the peripheral circulation. All the drugs were given orally at a dose of 100 mg/kg/day for 6 days. Group 4 served as untreated infected control. In another group of 5 rats, adult worms were implanted intraperitoneally to see the host reaction. The efficacy of the drugs was observed by recording the longevity of microfilariae in treated and untreated rats every third day until they disappeared. Differential leucocyte count was recorded every third day from the peripheral blood of infected rats to see the changes in the blood picture in treated as well as untreated rats during the course of infection. Peritoneal fluid was aspirated aseptically, smeared and stained with Leishman’s stain to observe the host response against the microfilariae in the peritoneum. Pathological changes were also observed in the tissues of vital organs such as liver, lungs and spleen.

Preparation of Leishman’s stain

Leishman’s stain was prepared by dissolving 0.2 g of powdered Leishman dye in 100 ml of methanol in a conical flask. This mixture was thoroughly mixed at 37 °C for half an hour with occasional shaking. The stain was allowed to cool, filtered and stored in a bottle wrapped with black paper.

Preparation of thin blood film for differential leucocyte count (DLC)

A small drop of blood was placed near one end of a clean glass slide and another slide (spreader) with smooth edges was placed at an angle of 30-45 degree near the drop of

Chapter -1 blood. The spreader was moved backward so that it touches the drop of blood and then moved rapidly forward over the slide to prepare thin blood smear which was air dried before staining.

Procedure for staining blood film for DLC

Few drops of Leishman’s stain were poured and spread on the slide and allowed to stain for 3 minutes followed by its dilution with equal volume of buffered water (pH 6.8), taking caution that the stain does not overflow. Water was mixed with the stain by gentle blowing with a plastic bulb pipette. Then slides were allowed to stain for 12-15 minutes. Stained slides were washed in running water for 1 to 2 minutes, dried in air and examined under the Nikon Eclipse-600 research microscope.

Preparation of smear from peritoneal exudates and its staining

Peritoneal exudate was aspirated from the infected rats with the help of syringe and needle and its smear was prepared on a clean slide which was allowed to dry in air. Dried smears were then flooded with Leishman’s stain and allowed to stain for 3 minutes. Equal volume of buffered water was poured over the slide, mixed with a plastic bulb pipette and allowed to stain for 15 minutes. Finally, the slides were washed in water for 2-3 minutes, air dried and observed under the microscope. Stained slides of peritoneal exudates were examined under the Nikon Eclipse-600 research microscope at x400 to observe the defensive cells around the microfilariae which were present in the peritoneal cavity of experimentally infected albino rats.

Procedure for the preparation of tissue sections for histopathological examination

Tissues such as mesentery, lungs, liver and spleen were collected at necropsy from microfilaraemic rats and fixed in Bouin’s solution for 24 hours. Fixed tissues were washed with distilled water 3-4 times and dehydrated for 30 minutes each in ascending grades of alcohol (30% to 100%). After dehydration, tissues were cleared in a mixture of absolute alcohol and xylene (1:1) for one hour, followed by pure xylene for 30 minutes. After clearing, tissues were left in the mixture of xylene and paraffin wax (1:1) at 60-65 °C and then transferred to pure wax for its impregnation

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for 4 hours. Finally, tissue blocks were prepared in molten wax which were trimmed and sectioned at 5 microns. These sections were stretched on the glass slide coated with egg albumin (egg albumin and glycerol in 1:1 ratio) and stored until used for staining.

Materials required for the preparation of Ehrlich’s haematoxylin

Haematoxylin 2 g

Glacial acetic acid 100 ml

Absolute alcohol 100 ml

Potassium alum 10 mg

Water 100 ml

Preparation of Ehrlich’s haematoxylin

Potassium alum was dissolved by heating it in water. Haematoxylin was dissolved in glacial acetic acid with about 25 ml of absolute alcohol by stirring. When the haematoxylin was dissolved, it was poured into a flask, and glycerine was added gradually by constantly agitating the flask. The warm alum solution was then added to the stain slowly while stirring. The freshly prepared stain was placed in a wide mouthed flask, which was covered with a muslin cloth. It was agitated daily for 3 weeks while placed under the sunlight for ripening. When the stain becomes dark in colour, it was kept in a dark place.

Materials required for the preparation of eosin

Eosin Y 0.1 g

Absolute alcohol 95 ml

Glacial acetic acid 0.5 ml

Preparation of eosin and staining of the sections

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Eosin stain was prepared by dissolving 0.1 g of eosin Y in 95 ml ethanol and 0.5 ml glacial acetic acid. Before staining, sections were dewaxed by putting them in coupling jar containing xylene for 5 minutes. Dewaxed sections were passed through the mixture of xylene and absolute alcohol (1:1), followed by two changes of absolute alcohol for 5 minutes each. Then the sections were rehydrated by passing through descending grades of alcohol followed by a dip in distilled water. These sections were stained in Ehrlich’s haematoxylin for 10 minutes and differentiated in 1% hydrochloric acid. Now sections were dehydrated by passing through alcoholic grades (30-70%) and stained in eosin solution for 2-3 minutes. Eosin stained sections were dehydrated by passing through ascending grades of alcohol, 70% onwards up to absolute alcohol for 2 minutes each, cleared in xylene for 5 minutes and mounted in DPX.

Statistical analysis

Data of the present findings was statistically analyzed using SPSS version 17.0 software. The statistical significance of the data was assessed by ANOVA for comparing means.

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RESULTS

Microfilariae appeared in peripheral circulation after 8±2 days of their intraperitoneal infusion in white rats, which continued to persist for 54 days. Maximum microfilarial density of 20.40/mm3 was recorded on 31st day of infection followed by a declining trend which ultimately led to disappearance of microfilariae on day 55. Effect of DEC, NTZ and NTZ+AgNPs on the longevity of microfilariae in treated and untreated rats is shown in Fig. 1.1. Significant variations were seen in the density as well as longevity of microfilariae when data was compared with that of treated rats. Maximum efficacy was observed in rats treated with NTZ+AgNPs where microfilarial density prior to the treatment was 2.40/mm3 on 10th day, started declining right from the beginning of the treatment and was recorded as 2.00, 1.40, 1.00, 0.60, 0.40 0.20/mm3 after 3, 6, 9, 12, 15 and 18 days, respectively, exhibiting significant difference when compared with DEC where microfilarial density showed an increase which was 2.60/mm3 on 3rd day post-treatment then declined and minimum density of 0.20/mm3 was recorded on 24th day. Rats treated with NTZ also showed an increase in the microfilarial density which was recorded as 2.80/mm3 on 6th day, then declined to 2.20/mm3on 12th day. Density again increased to 4.40 on 21st day and then declined to 0.60 on 33rd day. As for the efficacy of these drugs is concerned, NTZ+AgNPs was found to be most effective which cleared microfilariae within 18 days of infection, followed by DEC which took 24 days, while NTZ was the least effective drug which could clear microfilariae after 33 days.

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Table 1.1. Effect of DEC, NTZ and NTZ+AgNPs on the longevity of the microfilariae of Setaria cervi in untreated and treated rats.

Days Microfilarial density/mm3

after Untreated DEC NTZ NTZ+AgNPs treatment

10 1.80±0.20a 2.00±0.84a 2.00±0.71a 2.40±1.14a

13 2.40±0.89a 2.60±0.89a 2.80±0.84a 2.0±00.71 a

16 5.40±1.14a 2.40±1.14b 2.60±0.55b 1.40±0.55b

19 7.60±1.14a 2.20±0.45bc 2.40±0.55b 1.00±0.00c

22 7.60±1.95a 2.00±0.00b 2.20±0.84b 0.60±0.55b

25 12.80±4.15a 1.80±1.30b 2.40±1.14b 0.40±0.55b

28 16.60±4.10a 1.60±1.14b 3.20±1.84b 0.20±0.45b

31 20.40±2.70a 1.20±0.84c 4.40±0.55b 0.00±0.00c

34 15.20±3.34a 0.20±0.45b 3.40±1.67b 0.00±0.00b

37 12.40±2.07a 0.00±0.00c 2.40±1.14b 0.00±0.00c

40 8.20±2.59a 0.00±0.00b 1.60±0.89b 0.00±0.00b

43 7.00±1.58a 0.00±0.00b 0.60±0.55b 0.00±0.00b

46 3.60±1.14a 0.00±0.00b 0.00±0.00b 0.00±0.00b

50 1.40±0.89a 0.00±0.00b 0.00±0.00b 0.00±0.00b

54 0.80±0.84a 0.00±0.00b 0.00±0.00b 0.00±0.00b

58 0.00±0.00a 0.00±0.00a 0.00±0.00a 0.00±0.00a

Data is expressed as mean ± SD. Mean values followed by a common letter do not differ significantly (Tukey’s test, p<0.05).

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25 DEC NTZ NTZ+AgNPs Untreated 20 3

10 Microfilarial density/mm

0 10 20 30 40 50 60 Number of days

Fig. 1.1. Effect of DEC, NTZ and NTZ+AgNPs on the longevity of the microfilariae of Setaria cervi in untreated and treated and rats.

Results of differential leucocyte count (DLC) are shown in figs. 1.2-1.5. Eosinophils were increased during first two weeks, then almost disappeared and started reappearing 5th week onwards in all the groups of treated as well in untreated rats. In untreated rats, peak for neutrophils was observed on 22nd day, while its peak in DEC treated rats was seen on 19th day (Figs. 1.2 and 1.3). Monocytes were in maximum numbers during the third week, except in DEC, where the highest number was recorded during the fourth week. As for lymphocytes in untreated and DEC treated rats are concerned, its peaks were observed on 40th and 28th days, respectively. In NTZ and NTZ+AgNPs peaks for neutrophils were recorded on 19th and 16th days, respectively, while peaks for lymphocytes were observed on 37th day and 34th day (Figs. 1.4 and 1.5).

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100 25 Monocytes Lymphocytes Neutrophils Eosinophils 80 Basophils 20 Microfilarial density 3

60 15

40 10

20 5 Microfilarial density/mm Leucocyte countLeucocyte in untreated rats

0 0

0 10 20 30 40 50 Days of infection

Fig. 1.2. Differential leucocyte count in white rats infected with Setaria cervi.

100 25 Monocytes Lymphocytes Neutrophils Eosinophils 80 Basophils Microfilarial density 20 3

60 15

40 10

20 5 Microfilarial density/mm Leucocyte countLeucocyte in DEC treated rats 0 0

0 10 20 30 40 50 Days of infection

Fig. 1.3. Differential leucocyte count in microfilaraemic white rats treated with DEC.

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100 25 Monocytes Lymphocytes Neutrophils Eosinophils 80 Basophils 20 Micrpfilarial density 3

60 15

40 10

20 5 Microfilarial density/mm Leucocyte countLeucocyte in treated NTZ rats 0 0

0 10 20 30 40 50 Days of infection

Fig. 1.4. Differential leucocyte count in microfilaraemic white rats treated with NTZ.

100 25 Monocytes Lymphocytes Neutrophils Eosinophils 80 Basophils 20 Microfilarial density 3

60 15

40 10

20 5 Microfilarial density/mm

0 0 Leucocyte countLeucocyte in NTZ+AgNPs treated rats

0 10 20 30 40 50 Days of infection

Fig. 1.5. Differential leucocyte count in microfilaraemic white rats treated with NTZ+AgNPs.

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The stained smears prepared from the peritoneal exudate showed attachment of monocytes, lymphocytes and eosinophils around and on the microfilariae (Fig. 1.6). Microfilariae released in the peritoneum penetrated through venules of mesenteries reached in peripheral circulation and encroached liver, lungs and spleen. Pathological changes were seen around the microfilariae at the sites where they got trapped. In few cases, trapped microfilariae were in the process of destruction due to the inflammatory reactions of defensive cells of the host. Fragments of the microfilariae were visible in the lung tissue with leucocytes (Fig. 1.7 B). Intact microfilaria was found in the section of rat’s liver which was surrounded by defensive inflammatory cells (Fig. 1.7 D). Granuloma of inflammatory cells was also present around it. Similar aggregation of defensive cells was observed in spleen where lymphocytes were the predominant cells around the transverse section of microfilariae (Fig. 1.7 C).

Fig. 1.6. Microfilariae of Setaria cervi in the process of cell adhesion and destruction by the leucocytes in the peritoneal exudates (Yellow arrows denote attached leucocytes where as green arrow denotes ruptured body surface).

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Fig. 1.7. Sections of mesentery (A), lung (B), spleen (C) and liver (D) showing inflammatory cells in the vicinity or around the trapped microfilariae in these viscera (x100).

The infected rats were sacrificed at 5 days interval to observe the condition of the worms. 100% live worms were recovered from the peritoneal cavity after 10 days. Except few live worms, most of them were dead and in the process of degeneration on 15th day. Inflammatory patches were visible in the mesenteries, liver and spleen due to their encroachments. Dead worms were also found embedded in the peritoneal wall.

Haematoxylin and eosin stained tissue sections of mesenteries revealed infiltration of inflammatory cells around the embedded adult worms. These sections showed walling up operation, where leucocyte infiltration was clearly visible around the worm (Fig. 1.8 A). Histological sections revealed aggregation of defensive cells especially monocytes, macrophages, neutrophils and lymphocytes which started sticking to the cuticle of the worm (Fig. 1.8 B). Since parasites were trapped in the tissue permanently, inpocketing by the leucocytes started around the worm (Fig. 1.8 C), 45

Chapter -1 followed by plug formation which migrated within the body of the parasite to destroy and clear it off slowly (Figs. 1.8 D). Worms were encapsulated but not calcified as the study was restricted to 40 days only. There is a possibility of calcification at a later stage as the parasite was within the capsule formed from the host tissue. Since the parasite was trapped in the host tissue for a longer duration, cell infiltration increased. Macrophages, neutrophils and lymphocytes were the predominant cell types surrounding the worm.

Fig. 1.8. Encapsulation and leucocyte infiltration showing aggregation, attachment, inpocketing and plug formation around the worm embedded in the mesenteries.

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DISCUSSION

Longevity of microfilariae during the present study was 54 days with a peak on 31st day in untreated infected control rats which is in agreement with the earlier findings where almost similar peak and longevity were recorded for Setaria cervi in white rats (Singhal et al., 1973). In contrast, much lower survival periods of 23 days for the same parasite was recorded in guinea pigs (Baqui and Khatoon, 1982). Eosinophil count was found to be increased during 2nd and 3rd weeks, along with the monocytes and neutrophils which were more prominent after the 3rd week in both treated and untreated microfilaraemic rats. Early increase of these cells both in treated as well as untreated infected rats showed their active involvement in walling up operation and destruction of the parasite. There appeared a reversal mechanism in neutrophils and lymphocytes. When neutrophils showed a declining trend during 3rd and 4th week, lymphocyte numbers get increased in almost all treated rats that continued for a comparatively longer duration in untreated microfilaraemic rats. It clearly indicated that lymphocyte increase was directly proportional to the longevity of the microfilariae. In untreated rats, microfilariae survived for 54 days and therefore, lymphocytes also persisted for a longer duration as they are responsible for the secretion of antibodies in response to antigenic stimulus provided by the circulating microfilariae. Early increase of lymphocyte in treated rats may be due to the influence of drugs which stimulated lymphocytes for an early multiplication and secretion of antibodies.

In the present study, NTZ+AgNPs was proved to be most effective which cleared microfilariae in rats within 18 days, while DEC took 24 days to clear them. Enhanced efficacy of NTZ+AgNPs was probably due the presence of silver nanoparticles which get attached to the body surface and might have disturbed its permeability and interfered with cellular respiration. The nanoparticles may also penetrate deep inside the cell, thus causing cellular damage by interacting with phosphorus and sulphur containing compounds such as DNA and protein as earlier observed by Melayie and Youngs (2005). DEC treated rats also showed a significant antifilarial effect i.e., it cleared microfilariae from the peripheral circulation on the 24th day after treatment as it harnesses the innate inflammatory system and generates oxidative stress. Similar results were obtained against the microfilariae of Brugia malayi treated with DEC

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(McGarry et al., 2005). Active immunization of microfilaraemic animals with methyl piperazine carboxylic acid and bovine serum albumin showed rapid clearance of microfilariae when the sub-curative dose of DEC was administered in Mastomys coucha infected with Setaria digitata, indicating opsonization of the drug which enhanced its efficacy (Mukhopadhyay and Ravindran, 1997). These observations are indicative of the fact that host immune system played a key role in DEC mediated parasite elimination. A slow and steady decline was also observed in the microfilarial density of Brugia malayi and Wuchereria bancrofti after treatment with DEC alone, but the decline was immediate after the treatment with DEC-albendazole combination as it acts as a synergist (Ramaiah et al., 2011; Husain et al., 2014). NTZ suppressed survival of microfilariae to some extent which reappeared when medication was over, showing its inefficacy when compared with DEC and NTZ+AgNPs. NTZ is known to interfere with energy metabolism in anaerobic organisms and inhibits transcription/replication in infected cells and most importantly the secretion of proinflammatory cytokines, which in turn play an important role in generating reactive oxygen species (ROS) which slowed down the clearance of microfilariae from the peripheral circulation as earlier indicated by Rossignol et al. (2005). Rao et al. (2009) could not get any significant effect of NTZ against microfilariae of B. malayi in experimentally infected gerbils which seem true in our case also, where this drug failed to clear microfilariae from peripheral circulation of S. cervi in white rats.

Establishment of a parasite in a host invokes its defense tactics to resist reactions of the host that may be deleterious. The parasite in the host body sensitizes the host tissue, as a result of which a struggle is initiated between the parasite and the host in which the host generally succeeds and the parasite is expelled and/or killed at the expense of tissue damage. S. cervi which is freely available in the peritoneum of buffaloes in our study area was occasionally found embedded in the mesenteries and peritoneal walls. In the white rats too when it was implanted in the peritoneum, few worms were found embedded in the mesenteries. Similar penetrations and encapsulations by S. cervi were observed in patches of inflammatory tissue of the mesenteries, liver and peritoneum of rabbit (Khatoon et al., 1983; Sundar and Ravindran, 2009). Almost identical type of host-parasite relationship exists in rat-S. cervi model, where the worms were found embedded in the mesenteries and revealed infiltration of inflammatory cells around them. Similar reactions from the host were

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noticed around the microfilariae which were infused in the peritoneum. The eosinophils and monocytes were the first to register their increase around the worm as well as microfilariae and at a later stage they started sticking to the body surface so that they damage that particular part of the parasite by their secretions and could clear it off in due course of time. At a later stage when the stay of the parasite was prolonged, neutrophils too became active in the process of destruction along with macrophages and lymphocytes. During the last phase of infection, lymphocyte dominated as they got sensitized by the parasite’s antigen at an early stage, multiplied, converted into plasma cells and started secreting antibodies, which were ultimately involved in destruction of the parasite. Eosinophils, lymphocytes and macrophages were the predominant cell type surrounding the worm and microfilariae. Similar sequences of destruction of S. cervi, S. digitata, S. labiatopapillosa and S. tundra were recorded in buffaloes, reindeer calves and experimentally infected rats and rabbits by earlier workers in Italy, Finland and India (Chauhan and Pande, 1980; Khatoon et al., 1983; Bregoli et al., 2006; Laaksonen et al., 2007; Wajihullah, 2011; Maxie, 2016). Similar aggregation of inflammatory cells was also reported around Litomosoides carinii which were embedded in the tissues of experimentally infected white rats, cotton rats and multimammate rats (Bertram, 1966; Mohan, 1973; Weiner and Soulsby, 1976). For the long-term chronic helminth infection, modified Th2 response appears to be an adaptive phenomenon which limits the parasite burden as well as pathology (Metenou et al., 2011; Fairfax et al., 2012; Ferreira et al., 2013).

During the present study when haematoxylin-eosin stained sections of mesentery, liver, lungs and spleen were examine; few microfilariae were observed as transverse or oblique sections. There was moderate to intense infiltration of defensive cells in these organs except the mesenteries, where it was less pronounced. Probable reasons for this may be that the microfilariae find their way to the venules/ lymphatics quickly in the mesenteries and reach the portal circulation, while in lungs they stay for a brief period and perforate the endothelial wall to reach the lung alveoli, which result in lung inflammation. Finally, these microfilariae, if dead are dumped in spleen where plenty of lymphocytes surround and destroy them. The demonstration of microfilariae in the lungs of infected rats in the present study indicated that this filarial infection is a cause of tropical pulmonary eosinophilia (TPE) which was evidenced by nasal discharge, is in agreement with the findings of earlier workers who observed TPE

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along with emphysematous, degenerative, necrotic and infiltrative changes and exudation in response to S. cervi infection in rabbits (Kumar et al., 1991). Presence of only few microfilariae in the section of lungs may be due to the reason that most of them might have passed through and a few which were trapped, were subjected to the destruction by cell-mediated cytotoxicity involving eosinophils, basophils and macrophages along with the antibodies produced by lymphocytes. The degenerating microfilariae release somatic allergens that bind to specific cell-bound IgE and thereby trigger the release of vasoactive and inflammatory molecules by lung basophils and mast cells. Almost similar observations were recorded by the earlier workers in filarial and other helminthic infections (Ottesen and Nutman, 1992; Hoerauf et al., 2005; Elliot et al., 2007; Gupta, 2009).

In the present study, damage in liver and spleen was observed by the microfilariae which were manifested as dilated liver sinusoids along with increased kupffer cells, and heavily congested spleen. Destruction of microfilariae in liver and spleen was observed by the aggregation of inflammatory cells which restricted and began to destroy them as earlier observed against the microfilariae of bancroftian filariid where microfilariae were trapped and destroyed by leucocytes (Gupta et al., 2009; Figueredo-Silva et al., 2010; Pandey et al., 2015). The demonstration of microfilariae in liver and spleen lesions indicates that filarial infection is a possible cause of hepatomegaly and splenomegaly with eosinophilia. Histopathological examination of spleen of man and monkey showed similar eosinophilic tissue reactions in the form of granulomas around the microfilariae of W. bancrofti (Naramak et al., 1985; Bakhshi et al., 2012). Infiltration of mononuclear cells and polymorphs around the trapped microfilariae showed moderate host reaction in the rat-S. cervi system. Microfilarial granulomas containing eosinophils with a few lymphocytes and plasma cells around the microfilariae in the tissue section of spleen were also observed earlier in wild- caught cynomolgus monkeys (Naramak et al., 1985). Eosinophils, monocytes, macrophages and neutrophils play a significant role in early defense in the host against the parasites. In microfilaraemic rats, we observed elevation in the count of eosinophils and monocytes earliest, followed by the neutrophils during the early phase. These cells started declining from the peripheral circulation afterwards, indicating their migration and infiltration which can be attributed towards the destruction and elimination of the parasites in the organs where microfilariae and/or

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adult worms were trapped. In the deeper invaded tissues, these defensive cells might have been associated with piece meal clearance of the parasite as earlier observed in bancroftian filariasis where large numbers of eosinophils were found associated with DEC treated worms (Figuerdo-Silva et al., 2010). The cationic proteins of the eosinophils damage target cells through membrane interaction. During the present study, it was observed that eosinophils, monocytes and lymphocytes eventually targeted the microfilariae whether it was in the peritoneum or trapped in the viscera. Similar attack by these defensive cells was observed against the microfilariae of Onchocerca and Brugia by earlier workers (Mackenzie, 1980; Greene et al., 1981; Choong and Mak, 1991). Increase in lymphocytes clearly indicated towards the immunological defense which was evidenced by increased number of lymphocytes in the peripheral circulation in later phase of infection suggesting the formation of antibodies to neutralize the microfilariae. In the DEC treated microfilaraemic rats peaks for neutrophils and lymphocytes occured little earlier, if compared with experimental control, that might be effect of opsonization of these defensive cells and their involvement in early destruction and clearance of microfilariae. Almost similar sequence of events was observed in Trichinella and Onchocerca infections (Larsh, 1967; William et al., 1987). Increase in lymphocyte count in treated rats might have been due to the transformation of lymphocytes in plasma cells which secrete antibodies to eliminate the parasite from infected rats. Similar increase in lymphocyte proliferation was recorded in DEC treated mice infected with Nocardia braziliensis (Garcia-Hernández, 2014).

NTZ+AgNPs was proved to be the most effective formulation, as it cleared

microfilariae within 18 days of infection followed by DEC which took 24 days, while NTZ was least effective which could clear microfilariae after 33 days. During early phase of infection, there was an increase in the numbers of eosinophils, basophils, monocytes and neutrophils which invaded the tissues and enveloped the trapped microfilariae as well as adult worms for a piece meal destruction. During the late phase, there was an increase in lymphocyte count which was directly proportional to the longevity of the microfilariae. In response to the circulating microfilariae lymphocytes multiplied quickly, transformed into plasma cells and secreted antibodies to restrict and destroy them. Microfilariae were in the process of destruction where they got trapped. Immune mediated pathology was also observed around the

Chapter -1 microfilariae which were spotted in the tissue sections of lungs, spleen and liver. Nodular lesions were observed in the spleen. It may, therefore, be surmised that this model invokes immunopathological response against both adult as well as microfilariae in the tissues where they got trapped. But since the microfilariae survived for 54 days in the peripheral circulation of white rats, it will prove to be a good model to study various aspects of filariasis.

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INTRODUCTION

Although biochemical studies on TCA cycle enzymes have been studied in detail but their localization and physiological role inside the parasite after drug treatment has not been explored extensively. Parasite components like enzymes have certain biological function that makes them essential for their survival. Alterations in the output of these enzymes may play an important role in host parasite interactions and disease process (Bhandary, 2006). Fragmentary reports are available regarding the effects of anthelmintics on the survival and enzyme activities of the adult worms and microfilariae of different species of Setaria in vitro and in vivo (Ahmad and Srivastava, 2007; Singh and Rathaur, 2010; El-Shahawi, 2010; Srinivasan et al., 2011). Most of the findings related to pharmacology based on the biochemical and physiological aspects of total worm homogenates, as the isolation of the different organs and organ systems is not possible. The results thus obtained may not provide possible clue to the biological significance of a particular drug in relation to specific drug sites. To overcome this difficulty, histochemical studies are preferred. The effect of anthelmintics on a number of enzymes of glycolytic and oxidative pathways have been assessed in the parasites living in the peritoneum, gastrointestinal (GI) tract and tissues of hosts (Anwar et al., 1977; Walter, 1979; Agarwal et al., 1990; Hussain et al., 1990; Ahmad and Srivastava, 2007; Srinivasan et al., 2011; Khan et al., 2012). Widespread anthelmintic resistance has been reported among nematodes of livestock as a consequence of frequent administration of the same class of compounds over long periods (Wolstenholme et al., 2004, Von Samson-Himmelstjerna and Blackhall, 2005) and therefore, combination chemotherapy was considered as a powerful strategy to slow it down (Barnes et al., 1995; Nyunt and Plowe, 2007). Nitazoxanide, a broad-spectrum thiazolide compound possesses anthelmintic, antiprotozoal and antiviral properties (Rossignol and Stachulski, 1999; Hemphill et al., 2006). Its therapeutic efficacy has been observed against Ascaris lumbricoides and Trichuris trichiura when multiple doses were administered (Fox and Saravolatz, 2005; Anderson and Curran, 2007; Van den Enden, 2009). Recently, nanomedicine has been successfully tried against microorganisms (Dubey, 2006; Patel et al., 2011; Brigger et al., 2002; Merisko-Liversidge et al., 2003; Gherbawy et al., 2013). Singh et al. (2012) observed marked ultrastructural damage by SEM in the microfilarial sheath after incubation in nanoparticles of silver, whereas no such changes were observed when

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microfilariae exposed to AuNPs. AgNPs formulation demonstrated significant antileishmanial effects by inhibiting the proliferation and metabolic activity of promastigotes (Allahverdiyev et al., 2011). SEM images revealed cuticular damage in nematode parasites against benzimidazole class of drugs (Hanser et al., 2003; Matadamas-Martínez et al., 2013). Plant extracts and nitazoxanide has also showed antiparasitic action against Trichuris muris (Stepek et al., 2006; Tritten et al., 2012). Keeping the above findings in view, the present study was conducted to assess the efficacy of diethylcarbamazine (DEC), nitazoxanide (NTZ) and nanocomposite of nitazoxanide and silver nanoparticles (NTZ+AgNPs) against the microfilariae and adult Setaria cervi. Histochemical localization of TCA cycle enzymes such as succinate, malate and isocitrate dehydrogenases in the control and treated microfilariae and adult worms were also observed. In vitro effect of these drugs was also observed on the microfilariae of S. cervi by scanning electron microscopy.

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MATERIALS AND METHODS

Synthesis of NTZ+AgNPs

The AgNPs were prepared by mixing 10 ml of 1 mM AgNO3 solution with 100 µl of rhamnolipids extract under vigorous stirring for 1 hour at 30 °C. Subsequently, 2

ml of 5 mM NaBH4 solution was added under constant stirring to obtain a red coloured solution. 1 ml aliquot of 1mM AgNPs was mixed with 4 ml of 25.0 mg/ml of NTZ. The resulting mixture was sonicated in an ultrasonic bath for 2 minutes, and continuously stirred overnight at ambient temperature (30 °C). Adsorbed preparations were centrifuged at 5000 rpm for 30 minutes, washed thrice with Milli Q water and finally stored at 4 °C for further use.

Characterization of NTZ+AgNPs

Nanocomposite of NTZ+AgNPs was characterized by UV-Visible spectral analysis, Fourier transform infrared spectroscopy, scanning and transmission electron microscopy as described below:

UV–Visible spectral analysis

The spectra of the surface plasmon resonance of AgNPs and NTZ+AgNPs in the supernatants were recorded in wavelength range of 200 and 800 nm. The interaction of AgNPs and NTZ+AgNPs was monitored both by visual inspection and absorbance measurements using double beam UV–Vis spectrophotometer (Perkin Elmer lambda 36, U.S.A).

Transmission electron microscopy (TEM)

For TEM analysis, specimens of AgNPs alone and NTZ+AgNPs were prepared by dropping 10 μl of aqueous suspensions of AgNPs and NTZ+AgNPs complex on a TEM grid. Excess of solution was removed by soft filter paper and samples were allowed to dry in air overnight before imaging. TEM images were captured at 200 kV and analysed at x40000 using TEM (JOEL-2100, Tokyo, Japan).

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Scanning electron microscopy (SEM)

SEM analysis was carried out for AgNPs alone and NTZ+AgNPs using JSM 6510LV scanning electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV. The elemental analysis of pure AgNPs and NTZ+AgNPs nanocomposite was performed using Oxford Instruments INCAx-sight EDAX spectrometer equipped with SEM.

Fourier transform infrared spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) was performed for examining the functional groups of the pure AgNPs and NTZ+AgNPs nanocomposite, using Perkin Elmer FT-IR spectrometer Spectrum Two (Perkin Elmer Life and Analytical Sciences, CT, USA). The separated samples of AgNPs and NTZ+AgNPs powder were mixed with spectroscopic grade potassium bromide (KBr) in the ratio of 1:100 and the spectra were recorded in the range of 400–4000 wave number (cm−1) in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets.

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RESULTS

The UV-Vis absorption spectra exhibited the maximum absorbance at ~420 nm. The shift of maximum peak position was recorded due to the complex formation between AgNPs and NTZ (Fig. 2.1.1). FTIR analysis showed the N-H stretching vibration at 3357 cm-1 in NTZ, while new band appeared at a lower frequency in the spectra of the NTZ+AgNPs complex (Fig. 2.1.2). TEM images confirmed NPs which were spherical with size ranging from 10 to 30 nm. The TEM images obtained for AgNPs alone and NTZ+AgNPs film deposited on a carbon-coated copper grid is shown in Fig. 2.1.3. The image showed individual as well as capped silver nanoparticles with NTZ. Scanning electron microscope images of NTZ and NTZ capped AgNPs are shown in Figs. 2.1.4 a&b. EDAX spectrum of NTZ+AgNPs is shown in Figs. 2.1.5, where a silver signal in the EDAX spectrum exhibited the presence of elemental silver along with the signals of C, O and S as the components in the reaction medium.

1.0

0.8 AgNPs NTZ + AgNPs

0.6

0.4 Absorbance

0.2

0.0 300 400 500 600

Wavelength (nm)

Fig. 2.1.1. UV–Vis absorption spectra showing surface plasmon resonance (SPR) of AgNPs and NTZ+AgNPs.

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Fig. 2.1.2. FTIR spectra depicting the vibration of NTZ alone and NTZ+AgNPs.

Fig. 2.1.3. TEM images of AgNPs alone and NTZ+AgNPs at x40000.

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Fig. 2.1.4. SEM images showing NTZ alone and NTZ+AgNPs, marked by arrows (x1000).

Fig. 2.1.5. EDX-ray spectrum of NTZ+AgNPs.

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DISCUSSION

Nanocomposite of NTZ+AgNPs was prepared and characterized to evaluate its efficacy against the microfilariae of S. cervi. Synthesis of NTZ+AgNPs was also validated by the UV-Vis spectrum. The characteristic peak of AgNPs at 420 nm was due to the surface plasmon resonance. The shift of maximum peak position was due to the complex formation between AgNPs and NTZ. NTZ band at 436 nm could be assigned to intermolecular charge transfer (ICT) from the 2-amino to 5-nitro group via thiazol ring as earlier observed by Sarwade et al. (2015). SEM images showed the binding of NTZ with AgNPs. The FTIR spectra of AgNPs alone and NTZ+AgNPs also validate the interaction of NTZ with AgNPs. The feeble signal was possibly due to the biomolecules which were bound to the surface of AgNPs as earlier indicated by Ali et al. (2015). The N-H stretching vibration was recorded at 3357 cm-1 in NTZ along with the band of lower frequency in the spectra of the NTZ+AgNPs complex which was probably due to metal-nitrogen. An intense absorption band observed at -1 1370 cm corresponds to NO3 stretching of free nitrate anions as earlier elucidated by Tavman (2010). FTIR spectrum of NTZ+AgNPs also showed intense absorption bands at 2925, 2855, 1736, 1661, 1459, 1378, 1124 and 1046 cm-1. The absorption band observed at 1046 cm-1 represents the C-O-H bending vibrations as earlier observed by Dwivedi et al. (2015).

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MATERIALS AND METHODS

Collection of the adult and microfilariae of S. cervi

Adult S. cervi worms were collected from freshly slaughtered buffaloes and brought to the laboratory. The worms were washed in normal saline and adult females were dissected to recover the microfilariae from the gravid segment of the uterus for in vitro study.

Screening of DEC, NTZ and NTZ+AgNPs against the adult worm of S. cervi in vitro

4 concentrations of each drug i.e., 25, 50, 100 and 200 µg/ml were prepared in Ringer’s solution and adult worms were incubated in each concentration at 37 oC. The effect of these drugs was assessed by motility inhibition assay till the worms were dead. Control contained only Ringer’s solution to compare the results. After every 6 hours, solutions were changed.

Screening of DEC, NTZ and NTZ+AgNPs against the microfilariae of S. cervi in vitro

10 concentrations of DEC, NTZ and NTZ+AgNPs ranging from 10 µg/ml to 100 µg/ml were prepared in Ringer’s solution, to see their effect against the microfilariae in vitro. The effect of these drugs was assessed by observing the motility of the microfilariae. Motility inhibition assay was carried out by keeping microfilariae in different drug concentrations prepared in 200 µl of Ringer’s solution at 37 oC in 96- well titer plate. Control was run by incubating microfilariae in Ringer’s solution to compare the results. The inhibition of motility or mortality of the microfilariae in the above treatments was used as the criterion for anthelmintic activity. The motility inhibition was observed at hourly intervals until microfilariae died.

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RESULTS

Motility inhibition in vitro

NTZ+AgNPs proved to be the most effective drug against adult and microfilariae of S. cervi. Activity of adult worms was completely inhibited at 100 µg/ml concentration after 5 hours of incubation which was further reduced to 4 hours when the dose was doubled. However, at low concentrations of 50 and 25 µg/ml, 100% mortality was recorded after 10 and 18 hours, respectively. 30 µg/ml concentrations of NTZ+AgNPs caused 100% mortality of microfilariae within 1 hour, while 20 and 10 µg/ml of this formulation immobilized them completely within 2 and 3 hours at 37 ºC, respectively.

NTZ alone was less effective as it caused complete inhibition of adult motility after 10 hours of incubation in 100 µg/ml which was reduced to 8 hours when the dose was doubled. However, at low concentrations of 50 and 25 µg/ml, 100% mortality was recorded after 14 and 24 hours, respectively. Maximum mortality of microfilariae was recorded at 100 µg/ml concentration of NTZ alone in 1 hour, while it took 6 hours to kill them at the concentration of 10 µg/ml. DEC treated adult and microfilariae showed no significant effect at all the concentrations, when compared with untreated adult and microfilariae which were incubated in Ringer’s solution.

Table 2.1. Antifilarial activity of DEC, NTZ and NTZ+AgNPs against adult Setaria cervi in vitro.

Drug Concentrations Time taken for 100% mortality (µg/ml) (hours)

NTZ NTZ+AgNPs Control and DEC 25 24 18 - 50 14 10 - 100 10 5 - 200 8 4 -

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Table 2.2. Antifilarial activity of DEC, NTZ and NTZ+AgNPs against microfilariae of Setaria cervi in vitro.

NTZ NTZ+AgNPs Incubation Microfilarial Microfilarialmortality Concentration time(hrs) (Control and DEC) Concentration mortality (µg/ml) (µg/ml) (%)

10 µg/ml - 6 100 0

20 µg/ml - 5 100 0

30 µg/ml - 4 100 0

40 to 60 µg/ml 10 µg/ml 3 100 0

70 to 90 µg/ml 20 µg/ml 2 100 0

100 µg/ml 30 µg/ml 1 100 0

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DISCUSSION

During the present study efficacies of DEC, NTZ and NTZ+AgNPs were observed in vitro. NTZ+AgNPs was the most effective drug which killed the microfilariae at the concentration of 30 µg/ml within 1 hour of incubation which might be due to the induction of mitochondrial apoptotic pathway by increased production of reactive oxygen species (ROS). The increased level of ROS creates redox imbalance and promotes apoptosis through upregulating pro-apoptotic genes as earlier observed by green silver nanoparticles from Acacia auriculiformis at a very low dose (Saini et al., 2015). No significant effect was observed on the microfilariae treated with DEC in vitro. The probable reason might be lack of body factors especially immune bodies which opsonize DEC to increase its efficacy as earlier indicated by Misra et al. (1990). Similar in vitro results in DEC treated Brugia malayi was obtained by Murthy and Chatterjee (1999). NTZ was effective against the microfilariae in the present study where marked motility was noticed. Similar mortality was observed in the larvae and adult worms of Trichuris muris and Ancylostoma ceylanicum using the motility assay (Stepek et al., 2006; Kopp et al., 2008; Silbereisen et al., 2011). Earlier workers indicated that NTZ targets energy metabolism by inhibition of pyruvate ferredoxin oxidoreductase (PFOR) and protein disulfide isomerase (PDI) enzyme activities to attenuate and kill the parasite (Hemphill et al., 2006).

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MATERIALS AND METHODS

The effect of DEC, NTZ and NTZ+AgNPs was observed on the microfilariae of Setaria cervi by scanning electron microscope (SEM). After 6 hours of incubation in 100 µg/ml concentration of the drugs, microfilariae were fixed in 2.5% glutaraldehyde in PBS (pH 7.4) for approximately 24 hours at room temperature. After fixation the microfilariae were washed three times in PBS and stored in it at 4 ºC until used. Before SEM examination, the samples were dehydrated stepwise for 10 minutes each in ascending grades of ethanol (30%, 50%, 70%, 80%, 90% and 100%) at room temperature and kept in 96% ethanol at 4 ºC as described by Manneck et al. (2010). Finally, the microfilariae were dried to critical point, fixed on stubs and sputter coated with gold particles. SEM images were taken with a high- resolution scanning electron microscope (JEOL-JSM-6510LV).

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RESULTS

No significant effect was observed in SEM images of microfilariae treated with 100 µg/ml of DEC and NTZ except shrinkage of sheath, when compared with control. (Figs. 2.2.1-2.2.3). NTZ+AgNPs treated microfilariae showed eroded sheath at few places along with nanoparticles which were sticking to their body surface (Fig. 2.2.4).

Fig. 2.2.1. SEM images of Setaria cervi microfilariae incubated in Ringer’s solution (control) for 6 hours (x4000).

Fig. 2.2.2. SEM images of Setaria cervi microfilariae incubated for 6 hours in Ringer’s solution containing 100 µg/ml DEC (x4000).

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Fig. 2.2.3. SEM images of Setaria cervi microfilariae incubated for 6 hours in Ringer’s solution containing 100 µg/ml NTZ (x4000).

Fig. 2.2.4. SEM images of Setaria cervi microfilariae incubated for 6 hours in Ringer’s solution containing 100 µg/ml NTZ+AgNPs (x4000).

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DISCUSSION

SEM did not show any marked effect of DEC and NTZ on the microfilariae of Setaria cervi. But destruction of sheath and attachment of nanoparticles on the body surface of microfilariae was visible in NTZ+AgNPs treated microfilariae. Nanoparticles which were attached with the body surface of microfilariae would have hampered the permeability and interfered with the metabolism and those which penetrated deep might have caused cellular damage by interacting with phosphorus and sulphur containing compounds, such as proteins and nucleic acids as earlier observed in microbes (Melayie and Youngs, 2005). On contrary to this Peixoto et al. (2003) reported sheath loss and wrinkled appearance of the microfilariae of Wuchereria bancrofti, when treated with DEC. Chandrashekar et al. (1984) observed changes on the surface of microfilariae of Littomosoides sigmodontis and Brugia pahangi and concluded that these changes help in exposing the main body of the microfilariae to the antigenic determinants which in turn trigger secondary immunological damage. Therefore, DEC seems to have different pharmacological mechanism of action for different filarial species and various developmental stages of the same species as earlier indicated by Ottesen (1984). Recent studies using SEM in the third stage of sub periodic B. malayi showed no morphological changes with DEC or ALB alone or in combination (Tippawangkosol et al., 2004). Effect of NTZ and TZ was also reported in B. malayi, Echinococcus granulosus and Hymenolepis diminuta where these drugs caused some structural and functional changes in mitochondria (Walker et al., 2004; Hemphill et al., 2006; Rao et al., 2009).

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MATERIALS AND METHODS

Localization of succinate, malate and isocitrate dehydrogenases

For histochemical localization, adult and microfilariae of Setaria cervi were incubated in 100 µg/ml of DEC, NTZ and NTZ+AgNPs for 24 and 6 hours, respectively, at 37 oC and were processed for the localization of TCA cycle enzymes (SDH, MDH and ICDH). The control and drug treated microfilariae and adult worms were incubated for 1-2 hours at 37 oC in incubating medium consisting of 18 ml incubating solution (0.2 M Tris buffer, pH 7.4; nitro BT; MgCl2; water) and 2 ml substrate of 2.5 M disodium succinate for the localization of succinate dehydrogenase (SDH), 1 M malic acid for malate dehydrogenase (MDH) and 1 M trisodium isocitrate for isocitrate dehydogenase (ICDH). 2-4 mg coenzyme NAD was added for MDH and NADP for ICDH enzyme activities. After incubation, counter staining of microfilariae was done by 2% methyl green for 15 minutes, mounted in glycerol and sealed by paraffin wax to observe the sites of localization under the microscope as earlier described by Bancroft and Gamble (2002).

After enzyme localization, adult worms were dehydrated for 10 minutes each in ascending ethanol concentrations (30%, 50%, 70%, 80%, 90% and 100%), cleared in xylene and embedded in paraffin wax. Sections were cut at 5 microns, stretched on the slide and dewaxed in xylene. After rehydration in descending grades of alcohol, sections were counterstained in 2% methylene green for 15 minutes. Sections were dehydrated again, cleared in xylene, mounted in DPX and observed under the Nikon Eclipse 600 microscope.

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RESULTS

Localization of TCA cycle enzymes is shown in Tables 2.2.3-2.2.5. Succinate dehydrogenase (SDH) activity was moderate throughout the body in DEC, mild in NTZ and negligible in NTZ+AgNPs treated microfilariae when compared with untreated control where intense activity of this enzyme was observed. In DEC and NTZ treated microfilariae, SDH activity in excretory pore and anal pore was moderate to intense, while it was mild in NTZ+AgNPs treated microfilariae (Fig. 2.2.5). Malate dehydrogenase activity was moderate throughout the body except cephalic cells, excretory pore and anal pore where the localization was intense in untreated microfilariae. In DEC treated microfilariae, enzyme activity was mild throughout the body except in excretory and anal pores where activity was high. Low activity of MDH was observed in anal pore which was very feeble on rest of the body of microfilariae treated with NTZ (Fig. 2.2.6). In NTZ+AgNPs treated microfilariae, no enzyme activity was seen. Isocitrate dehydrogenase activity was mild throughout the body in untreated as well as DEC treated microfilariae, while it was mild and very feeble in the microfilariae treated with the combination of NTZ+AgNPs. In untreated microfilariae, intense activity of ICDH was observed in the cephalic cells, excretory and anal pore which was moderate in excretory and anal pore of DEC and NTZ treated microfilariae. However no activity of this enzyme was observed in the microfilariae treated with NTZ+AgNPs (Fig. 2.2.7).

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Table 2.3. Succinate dehydrogenase localization in control and treated microfilariae of Setaria cervi.

Body parts Control DEC NTZ NTZ+AgNPs

Nerve ring +++ ++ + -

Cephalic cells +++ ++ + -

Body column +++ ++ + ±

Excretory pore +++ ++ ++ +

Anal pore +++ +++ ++ +

(Strong) +++, (Moderate) ++, (weak) +, (Slight) ±, (Absent) –

Fig. 2.2.5. Localization of succinate dehydrogenase in control (A), DEC (B), NTZ (C) and NTZ+AgNPs (D) treated microfilariae of Setaria cervi (x400).

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Table 2.4. Malate dehydrogenase localization in control and treated microfilariae of Setaria cervi

Body parts Control DEC NTZ NTZ+AgNPs

Nerve ring ++ - - -

Cephalic cells +++ ± - -

Body column ++ ++ - -

Excretory pore +++ ++ - -

Anal pore +++ +++ + -

(Strong) +++, (Moderate) ++, (weak) +, (Slight) ±, (Absent)–

Fig. 2.2.6. Localization of malate dehydrogenase in control (A), DEC (B), NTZ (C) and NTZ+AgNPs (D) treated microfilariae of Setaria cervi (x400).

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Table 2.5. Isocitrate dehydrogenase localization in control and treated microfilariae of Setaria cervi

Body parts Control DEC NTZ NTZ+AgNPs

Nerve ring - - - -

Cephalic cells ++ + - -

Body column + + ± -

Excretory pore +++ ++ + -

Anal pore +++ ++ + -

(Strong) +++, (Moderate) ++, (weak) +, (Slight) ±, (Absent) –

Fig. 2.2.7. Localization of isocitrate dehydrogenase in control (A), DEC (B), NTZ (C) and NTZ+AgNPs (D) treated microfilariae of Setaria cervi (x400).

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Histochemical localization of TCA cycle enzymes in control and treated adult Setaria cervi worms is given in Tables 2.2.6-2.2.8. In control worms, strong activity of succinate and malate dehydrogenase was observed in the hypodermis, fibrillar region of muscle cells, uterine wall and GI tract, while isocitrate dehydrogenase activity was intense in hypodermis and moderate in rest of the body parts.

In DEC and NTZ treated worms, moderate activity of SDH was observed in musculature, GI tract and uterine epithelium, but was absent from cuticle and hypodermis. NTZ+AgNPs treated worms showed moderate SDH activity in the hypodermis, while rest of the body parts exhibited slight activity. However, no enzyme activity was seen in the cuticle (Fig. 2.2.6).

MDH activity was moderate throughout the body of the worm treated with DEC with the exception of cuticle which was devoid of this enzyme. As for NTZ treated worms are concerned, they exhibited moderate activity of MDH in cuticle and hypodermis, while rest of the body parts showed slight activity. Worms which were treated with nanocomposite of NTZ+AgNPs showed slight activity only in cuticle and hypodermis, while no activity was observed in other body parts (Fig. 2.2.7).

DEC treated worms showed moderate localization of ICDH in hypodermis, which was feeble in rest of the body parts. In cuticle, activity of the enzyme was totally absent. In NTZ treated worms, slight activity of ICDH was observed throughout the body except hypodermis which showed moderate activity. NTZ+AgNPs treated worms showed no enzyme activity throughout the body except hypodermis which exhibited slight reaction (Fig. 2.2.8)

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Table 2.6. Distribution of succinate dehydrogenase activity in the adult Setaria cervi

Status Cuticle Hypodermis Muscle GI tract Uterine

epithelium

Control - +++ +++ +++ +++

DEC - - ++ ++ ++

NTZ - - ++ ++ ++

NTZ+AgNPs - ++ + + +

(Strong) +++, (Moderate) ++, (weak) +, (Slight) ±, (Absent) –

Fig. 2.2.8. Localization of succinate dehydrogenase in control (A), DEC (B), NTZ (C) and NTZ+AgNPs (D) treated Setaria cervi (T.S.) (x100).

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Table 2.7. Distribution of malate dehydrogenase activity in the adult Setaria cervi.

Status Cuticle Hypodermis Muscle GI tract Uterine

epithelium

Control + +++ +++ +++ +++

DEC - ++ ++ ++ ++

NTZ ++ ++ ± ± ±

NTZ+AgNPs ± ± - - -

(Strong) +++, (Moderate) ++, (weak) +, (Slight) ±, (Absent) –

Fig. 2.2.9. Localization of malate dehydrogenase in control (A), DEC (B), NTZ (C) and NTZ+AgNPs (D) treated Setaria cervi (T.S.) (x100).

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Table 2.8. Distribution of isocitrate dehydrogenase activity in the adult Setaria cervi

Status Cuticle Hypodermis Muscle GI tract Uterine

epithelium

Control + +++ ++ ++ ++

DEC - ++ + + +

NTZ ± ++ ± ± ±

NTZ+AgNPs - ± - - -

(Strong) +++, (Moderate) ++, (weak) +, (Slight) ±, (Absent) –

Fig. 2.2.10. Localization of isocitrate dehydrogenase in control (A), DEC (B), NTZ (C) and NTZ+AgNPs (D) treated S. cervi (T.S.) (x100).

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DISCUSSION

Glycolytic and tricarboxylic acid (TCA) cycle enzymes have been observed in adult filarial worms, but only a few fragmentary reports are available regarding their localization and distribution in microfilariae. Anthelmintics are known to inhibit a variety of enzymes in adult nematodes. We observed NTZ+AgNPs as the most potent inhibitor of succinate, malate and isocitrate dehydrogenases as compared to DEC and NTZ. Localization of these enzymes almost on the entire body of microfilariae and adult worms indicates the presence of PEP-succinate pathway in this parasite. Moderate to strong activities of SDH, MDH and ICDH in the microfilariae and adult worms indicate their role in catalysis of different steps of TCA cycle to generate energy for the worm. Similar intense localization of this enzyme was reported by earlier workers in S. digitata and Brugia malayi (Banu et al., 1991; Bhandary et al., 2006). SDH converts succinate to fumarate while MDH oxidizes malate to oxaloacetate and vice versa. It has been observed that the oxaloacetate reduction reaction was more active than its respective opposite reaction in S. digitata, suggesting that the pathway proceeds in the direction of malate formation which seems true in our study also where localization of MDH was intense throughout the body of microfilariae and adult worms. It has been reported by many workers that malate which formed as a result of cytosolic MDH activity enters into the mitochondria for further catabolic processes, affecting the parasite’s survival adversely (Barrett, 1981; Saz, 1981; Ward, 1982). Thus, decrease in SDH and MDH activities in treated microfilariae and adult worms points towards the blockage of PEP-succinate pathway and a shift towards homolactate fermentation as earlier reported by Rathaur et al (1982). In this study, low activity of ICDH enzyme was observed throughout the body except cephalic cells and anal pore where activity was more pronounced indicating its active involvement in the parts related to secretion and excretion. Localization of complete sequence of the TCA cycle enzymes was reported in many filarial nematodes including, Onchocerca volvulus, O. gibsoni, O. gutturosa, O. lienalis, B. pahangi and Dirofilaria immitis. Though the enzymes involved in the initial steps of the cycle showed low activity in most of the species of Onchocerca except in O. faciata which showed strong ICDH activity pointing towards an additional pathway (pyruvate-succinate) of glucose metabolism via a reverse

Chapter -2

sequence of TCA cycle in the parasite (Middleton and Saz, 1979, Omar et al., 1996, Walter and Albiez, 1986, Dunn et al., 1988).

In the present study, we observed NTZ+AgNPs as most effective inhibitor of TCA cycle enzymes as it slowed down SDH to a large extent and completely inhibited MDH and ICDH activities in treated microfilariae and adult worms. This indicates that nanocomposite of NTZ+AgNPs adversely affected the PEP-succinate pathway due to the synergistic effect of AgNPs with nitazoxanide in which NTZ inhibited the energy metabolism and AgNPs caused mitochondrial apoptosis in the microfilariae as earlier indicated by Hemphill et al. (2006) and Saini et al. (2016). DEC was less effective as moderate SDH and comparatively low MDH and ICDH activities were observed in the microfilariae as well as adult worms after treatment, indicating its action as inhibitor of glucose uptake and glycogen synthesis in S. cervi which is in agreement with the earlier findings where similar effects were observed in the microfilariae of S. cervi and Litomosoides carinii (Anwar et al., 1978; Rathaur et al., 1980; Zakai and Khan, 2015). Thus, decrease in SDH, MDH and ICDH in DEC treated microfilariae pointed towards the blockage of PEP-succinate pathway and a shift towards homolactate fermentation as earlier observed by Rathaur et al. (1982).

NTZ+AgNPs was the most effective synergistic combination against the TCA cycle enzymes which blocked the ICDH and MDH completely and SDH to a large extent in the microfilariae and adult worms of S. cervi. AgNPs ruptured the sheath which made NTZ accessible to the main body of the microfilariae and produced maximum effect by penetrating through the body wall and acting on the TCA cycle enzymes, which plays a vital role in the energy metabolism and survival of microfilariae. This formulation may be used to control the transmission of bovine and other filariids in tropical countries.

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INTRODUCTION

During any infection, chemical mediators elaborated by the inflammatory cells may generate oxidative stress and affect the clinical outcome (Kumar et al., 2004; Pal et al., 2006). Parasites including filarial worms cause inflammation when invade the host tissue, as a result of which reactive oxygen species (ROS) are generated and cause oxidative damage which prove deleterious and may lead to senescence and cell death (Sies, 1991; Bello et al., 2000; Egwunyenga et al., 2004; Pal et al., 2006; Siwela et al., 2010). Since the redox system plays indispensable role for parasite survival within their host, drugs that either promote ROS generation or inhibit cellular antioxidant systems will lead to redox imbalance by pushing ROS levels above a certain threshold level that ultimately cause parasite death (Müller et al., 2003; Massimine et al., 2006). Highly reactive oxygen species which are produced during normal cellular metabolism especially by the activated phagocytes and by some antifilarial drugs acts as a powerful effector mechanism against invading parasites (Oliveira and Cecchini, 2000). Hydrogen peroxide, for example, is produced against the parasite by macrophages that freely crosses the membrane and produces the reactive hydroxyl radical, when reacts with haeme proteins in accordance with the Fenton reaction (Oliveira and Cecchini, 2000). Whilst there have been several studies on antioxidant enzymes as a protective mechanism of parasites from ROS, arising from host immune response (LoVerde et al., 2004).

Oxidative stress has been reported against the parasites causing malaria, leishmaniasis and schistosomiasis (Erel et al., 1997; Biswas et al., 1997; Gharib et al., 1999). Evidences suggest that diethylcarbamazine (DEC) is responsible for the modification of the host’s immune response which is more rapid in vivo than in vitro suggesting the involvement of host factors (Fidelus and Tsan, 1986; Maizels and Denham, 1992; Baeuerle and Henkel, 1994). The action of DEC may be mediated through enhanced immune mechanisms of the host. A purported mechanism of action is that the DEC may attach to and alter the surface of microfilariae, thereby enhancing the immune response (Srivastava et al., 1984). Nitazoxanide’s (NTZ) activity against parasites was found to be associated with energy metabolism and inhibition of pyruvate ferredoxin oxidoreductase (PFOR) and protein disulfide isomerase (PDI) enzyme activities (Hemphill et al., 2006; Aslam and Musher, 2007; Pal and Bandyopadhyay,

Chapter -3

2012; Abdel-Hafeez et al., 2015). Metal oxide nanoparticles (NPs) possess effective antimicrobial activities as they possess large surface area (Azam et al., 2012). Recently silver nanoparticles (AgNPs) which have very distinct physicochemical and antibacterial properties have been integrated in biomedical research, especially in the context of nanomedicine. In vitro and in vivo effects of NTZ and tizoxanide (TZ) have been established against Brugia malayi in experimentally infected gerbils (Rao et al., 2009). Different forms of NPs have been used to control the parasites inhabiting lymphatic and circulatory system (Kirthi et al., 2011; Ramyadevi et al., 2011; Ali et al., 2014). The chemical reactivity of NPs makes them capable of generating ROS to kill the infectious agents. Among various metal NPs, silver is of a particular interest as it posses excellent biocompatibility and exert inhibitory effects at a concentration that is below their cytotoxic limits, and hence, used as antimicrobial agents (Pillai et al., 2012; Mohamed et al., 2014). Thus, the development of effective alternatives may be explored for nanoparticle based drug formulations.

Liver is the most sensitive predictor of chemical induced toxicity due to its involvement in metabolism, detoxification and storage of drugs and their metabolites. Liver is an important target for drug induced injury in mammals in which pathological changes may be seen in the affected hepatocytes along with the fluctuations in the liver enzymes (Sturgill and Lambert, 1997; Abd El-Rahman et al., 1999; Lin et al., 2003; Qureshi, 2013; Idowu et al., 2015). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are the biochemical markers of hepatocellular necrosis and are considered sensitive indicators of hepatic drug induced injuries (Choi et al., 2008; Timbrell, 2009). It has also been suggested by the earlier workers that the administered drugs and/or their metabolites cause lipid peroxidative damage to the hepatocytes which increase cell membrane permeability to the cellular enzymes (Idowu et al., 2015). By now, it is quite clear that AST, ALT and alkaline phosphatase (ALP) get elevated in acute or mild hepato-cellular injury (Peters, 1989; Ojiako and Onyeze, 2009). Significant increase in the activity of AST and ALT was observed from the necrosed and degenerated liver cells of microfilaraemic cattle, buffaloes, horses, dog and man (Rudolph et al., 1957; Dalsanto, 1959; Malherbe, 1960; Sharma and Pachauri, 1982; Sharma and Joshi, 2002; Murugan and Jeeva, 2016).

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Keeping above findings in view, activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase, liver enzymes namely aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase were observed in the sera of microfilaraemic rats and those treated with DEC, NTZ and NTZ+AgNPs during the course of infection

Chapter -3

MATERIALS AND METHODS

Establishment of microfilariae in white rats and drug treatment:

Microfilariae which were recovered from uterus of gravid females were injected in the peritoneal cavity of all the albino rats which were divided into four groups, each having 5 rats. Groups 1, 2 and 3 were treated with DEC, NTZ and nanocomposite NTZ+AgNPs, respectively, 10th day onwards when microfilariae appeared in the peripheral circulation. All the drugs were given orally at a dose of 100 mg/kg/day for 6 days. Group 4 served as untreated infected control.

Collection of sera from infected untreated and treated rats:

To study enzyme activity, blood samples were collected from the tail of rats from 0 to 40th day of infection at 10 days interval. Blood was kept at room temperature for 30 minutes and then transferred to the refrigerator at 4 °C for 2 hours, so that serum could be squeezed out from the clotted blood. Then it was transferred to 1.5 ml vials and centrifuged at 1000×g at 4 °C for 5 minutes. Serum was collected and stored at - 80 °C until used.

Protein estimation

Detection and quantification of total protein was done by the method of Bradford (1976), modified by Spector in 1978, using Coomassie Brilliant Blue G-250 (CBBG- 250) dye. The dye reagent consisted of 0.01% (w/v) CBBG-250, 4.75% (v/v) absolute ethanol and 8.5% (v/v) orthophosphoric acid. 4 µl of each serum was taken in separate test tubes in triplets and diluted with 96 µl of distilled water and allowed to conjugate with 2 ml of CBBG-250 dye. The optical density was recorded at 595 nm by JASCO spectrophotometer. The protein concentration of each sample was determined with the help of standard calibration curve using bovine serum albumin.

Lipid Peroxidation

The level of malondialdehyde (MDA), a marker for lipid peroxidation was determined by the procedure described by Buege and Aust (1978). 2 ml of TCA-TBA-HCl was added in serum samples and mixed thoroughly. This solution was heated for 15 minutes and centrifuged at 1000×g for 10 minutes. Absorbance of the sample was

Chapter -3

determined at 535 nm against blank. The malondialdehyde concentration of the serum was calculated using an extinction coefficient of 1.56×105 M-1cm-1.

Superoxide dismutase

Superoxide dismutase (SOD) was assayed by following the inhibition of autooxidation of pyrogallol at 420 nm wavelength as earlier described by Marklund and Marklund (1974). The assay system contained 1 mM DTPA, 1 mM EDTA, 50 mM air-equilibrated triscacodylate buffer (pH 8.5) and serum in a final volume of 3 ml. The reaction was initiated by the addition of 100 μl of freshly prepared 0.13 mM pyrogallol solution in the assay mixture which was transferred to a cuvette and rate of increase in the absorbance at 420 nm was recorded for every thirty seconds in a double beam spectrophotometer with recorder. The increase in the absorbance at 420 nm after addition of pyrogallol was inhibited by the presence of SOD. One unit of SOD is described as the amount of enzyme required to cause 50% inhibition of pyrogallol autoxidation per 3 ml of assay mixture. Results have been expressed in units per mg protein.

Catalase

Catalase activity was measured as per the method of Aebi (1984). 3 ml reaction

mixture contained 2 ml serum and 1ml H2O2 at 20 °C against a blank containing 1ml phosphate buffer and 2ml serum solution. The reaction started by addition of H2O2. Decrease in optical density was measured at 240 nm for about 30 seconds in spectrophotometer. The molar extinction coefficient of 43.6 M-1cm-1 was used to determine the CAT activity. One unit of activity is equal to the number of moles of

H2O2 reduced/mg protein/min.

Glutathione peroxidase

Glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler (1984). Reaction mixture contained 500 µl 0.1 M PB, serum sample, 100 µl glutathione reductase, and 100 µl of 10 mM glutathione (GSH), which was incubated at 37 0C for 10 minutes to which 100 µl NADPH solution was added. The overall

reaction was started by adding 100 µl of H2O2 solution. Sodium azide was used in the reaction to suppress CAT activity. The linear decrease in NADPH absorption was

Chapter -3

recorded at 340 nm. Amount of NADPH oxidized was calculated using molar extinction 6.22 × 103 M−1 cm−1. Specific activity of GPx was expressed as micro moles of NADPH oxidized/min/mg protein at room temperature.

Glutathione S-transferase

Glutathione S-transferase (GST) activity was assayed by the conventional method of Habig et al. (1974). The reaction mixture of 3 ml consisted of l ml 0.1 M potassium phosphate buffer (pH 6.5), serum, 1 mM CDNB and 1 mM GSH and its total volume was made up to 3 ml with distilled water. The increase in absorbance was read at 340 nm and quantification was done using 9.6 ×103 M-1cm-1 as the extinction coefficient. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of one micromole of 2,4-dinitro benzene GSH adduct per minute. Specific activity was expressed as micromoles of GSH conjugate formed per milligram of protein.

Alkaline phosphatase: This enzyme was assessed by following the protocol of Kind and King’s kit (Kind and King, 1954).

Assay principle

Alkaline phosphatase from serum converts phenyl phosphate to inorganic phosphate and phenol at pH 10.0. Phenol so formed reacts in alkaline medium with 4- aminoantipyrine in the presence of oxidizing agent potassium ferricyanide and forms an orange-red coloured complex, which can be measured colorimetrically. The colour intensity was proportional to the enzyme activity.

The reaction steps can be represented as follows:

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Reagents (Supplied in the kit)

Reagent 1: Buffered substrate, pH 10.0

Reagent 2: Chromogen reagent

Reagent 3: Phenol standard, 10 mg%

Preparation of working solution

Reconstitute one vial of reagent 1, buffered substrate with 2.2 ml of purified water, reagent 2 and 3 were ready to use.

Procedure

Reagents Blank Standard Control Test

Volume in ml

Working 0.5 0.5 0.5 0.5 buffered substrate

Purified water 1.5 1.5 1.5 1.5

Mixed well and incubated at 37 °C for 3 minutes

Serum - - - 0.05

Reagent 3 - 0.05 - -

Mixed well and incubated at 37 °C for 15 minutes

Reagent 2 1.0 1.0 1.0 1.0

Serum - - 0.05 -

O.D. was measured at 510 nm.

Calculations

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Aspartate aminotransferase (AST): 2,4-DNPH (Reitman and Frankel, 1957)

Assay principle

Aspartate aminotransferase (AST) catalyses the transamination of L-Aspartate and α- Ketoglutarate (α -KG) to form oxaloacetate and L-glutamate. Oxaloacetate so formed is coupled with 2,4-Dinitrophenyl hydrazine (2,4-DNPH) to form a corresponding hydrazone, a brown coloured complex in alkaline medium and this can be measured spectrophotometrically.

ɑ - KG+L-Aspartate Oxaloacetate + L- Glutamate

Oxaloacetate + 2,4-DNPH Corresponding Hydrazone (brown colour)

Reagents (Supplied in the kit)

Reagent 1: Buffered Aspartate-α-KG substrate, pH 7.4

Reagent 2: 2,4-DNPH colour reagent

Reagent 3: Sodium hydroxide, 4 N

Reagent 4: Working pyruvate standard, 6 mM (114 IU/L)

Working reagent preparation

Reagent 1, 2 and 4 were ready to use.

Solution I: Dilute 1 ml of reagent 3 to 10 ml with purified water.

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Procedure

Reagents Blank Standard Test Control Volume in ml Reagent 1 0.25 0.25 0.25 0.25 Serum - - 0.05 - Standard - 0.05 - - Mixed well and incubated at 37 °C for 60 minutes Reagent 2 0.25 0.25 0.25 0.25 Deionised 0.05 - - - water Serum - - - 0.05 Mixed well and allowed to stand at room temperature (+15 °C to +30 °C) Solution I 2.5 2.5 2.5 2.5 Mixed well and read the O.D. against purified water in a spectrophotometer at 505 nm, within 15 minutes.

Calculations

Alanine aminotransferase (ALT) : 2,4-DNPH (Reitman and Frankel, 1957)

Assay principle

Alanine aminotransferase (ALT) catalyses the transamination of L-Alanine and α- Ketoglutarate (α-KG) to form Pyruvate and L-glutamate. Pyruvate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH) to form a corresponding hydrazone, a brown coloured complex in alkaline medium and this can be measured spectrophotometrically.

ɑ - KG+L-Alanine Pyruvate + L- Glutamate

Pyruvate + 2,4-DNPH Corresponding Hydrazone (brown colour)

Chapter -3

Reagents (supplied in the kit)

Reagent 1: Buffered Alanine-α-KG Substrate, pH 7.4

Reagent 2: 2,4-DNPH colour reagent

Reagent 3: Sodium hydroxide, 4 N

Reagent 4: Working pyruvate standard, 8 mM (150 IU/L)

Working reagent preparation

Reagent 1, 2 and 4 are ready to use.

Solution I: Dilute 1 ml of reagent 3 to 10 ml with purified water.

Procedure

Reagents Blank Standard Test Control

Volume in ml

Reagent 1 0.25 0.25 0.25 0.25

Serum - - 0.05 -

Standard - 0.05 - -

Mixed well and incubated at 37 °C for 60 minutes

Reagent 2 0.25 0.25 0.25 0.25

Deionised 0.05 - - - water

Serum - - - 0.05

Mixed well and allowed to stand at room temperature (+15 °C to +30 °C)

Solution I 2.5 2.5 2.5 2.5

Mixed well and the O.D. was read against purified water in a spectrophotometer at 505 nm, within 15 minutes.

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Calculation

RESULTS

MDA is an end product of lipid peroxidation process which showed progressive increase in microfilaraemic rats till day 10. On 20th day when medication was over, there was a further significant (p<0.05) elevation in MDA level, which was recorded as 13.00 nm/mg and 9.62 nm/mg in the sera of rats treated with NTZ+AgNPs and DEC, respectively, whereas decline of 7.39 nm/mg was observed in NTZ treated rat serum. Decline in MDA was observed in all treated groups and its level was noted as 3.08, 3.68 and 3.05 nm/mg on the 40th day for NTZ+AgNPs, DEC and NTZ, respectively. However, the untreated group showed a significant increase in MDA level till 30th day and then declined at a slow pace (Fig. 3.1).

All the groups of microfilaraemic rats showed a significant decrease in SOD activity on the 10th day compared to day 0, i.e. before infection. Elevated SOD activity was observed in all the groups except those treated with NTZ. NTZ+AgNPs showed significant (p<0.05) elevated activity (17.14 U/mg) on the 20th day of infection and then it declined to 8.96 and 6.91 U/mg on 30th and 40th day. But in DEC treated rats, SOD activity was elevated to 10.92 U/mg on 20th day which further increased to 11.78 U/mg on 30th day, then declined to 5.82 U/mg on the 40th day of infection. NTZ showed decrease in SOD activity (3.96-3.40 U/mg) between 20th and 40th days of infection. In the untreated control group, there was a constant decline in SOD activity (8.20-5.23 U/mg) throughout the infection (Fig. 3.2).

Significant increase in catalase activity was observed in the sera of all infected groups of rats on the 10th day. The level of catalase was elevated to 1838.99 U/mg in rats treated with NTZ+AgNPs on 20th day compared to 511.37 U/mg of day 0 (Fig. 3.3). After this peak, it declined to 1463.40 and 803.11 U/mg on 30th and 40th day of infection, whereas in DEC treated rats catalase activity was increased to 1763.06 and 1801.35 U/mg on 20th and 30th day of infection and then the level decreased to 1427.26 U/mg on the 40th day. In NTZ treated rats, activity of this enzyme was significantly decreased to the level of 635.14, 600.92 and 580.56 U/mg on 20th, 30th

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and 40th day of infection, respectively. In untreated control, enzyme activity showed its peak on 20th day when its concentration was 1670.94 U/mg which declined to 991.41 U/mg on 40th day.

In microfilaraemic rats, increase in GPx activity was observed in the sera of all groups of rats up to 10th day. Rats treated with DEC showed significant (p<0.05) increase in GPx activity, i.e. 156.50 U/mg on the 20th day and then decreased to 143.18 and 124.82 U/mg on 30th and 40th day, respectively. A constant decrease was noticed in NTZ and NTZ+AgNPs treated microfilaraemic rats which accounted for 54.02 and 50.58 U/mg and 45.10 and 33.52 U/mg on 20th and 40th days of infection. DEC treated microfilaraemic rats showed a decline in GPx activity to143.18 and 124.82 U/mg on 30th and 40th day of infection (Fig. 3.4). In untreated rats, enzyme level showed slow progressive increase right from the beginning to 40th day of infection and rose from 60.19 U/mg to 95.90 U/mg.

There was a marked increase in GST activity in the sera of all infected rats on the 10th day. Increase in the level of GST was recorded in DEC treated rats (177.30 U/mg), which decreased in the sera of rats treated with NTZ and NTZ+AgNPs and recorded as 85.12 and 72.05 U/mg, as compared to 148.70 U/mg of untreated control on the 20th day of infection. GST level significantly (p<0.05) decreased in the NTZ+AgNPs treated rats (61.78-56.59 U/mg) followed by NTZ (76.37-70.46 U/mg) and DEC (87.89-81.11 U/mg) from 30-40 day of infection (Fig. 3.5).

The concentration of liver enzymes in treated and untreated rats are shown in Figs. 3.6-3.8. Liver function tests (LFT) performed from the sera collected from all the microfilaraemic rats showed an increased level of all the enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) on 10th day. From 20th day onwards levels of these enzymes get decreased substantially which continues till 40th day of infection in DEC, NTZ and NTZ+AgNPs treated rats. However in untreated rats, AST and ALT showed increase right from the beginning and continue till 40th day, while ALP level increased till 30th day then started decline.

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Table 3.1. Malondialdehyde levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNPs

of days (nmoles/mg (nmoles/mg (nmoles/mg (nmoles/mg protein)

protein) protein) protein)

a a a a 0 2.92±0.63 3.70±0.14 3.60±1.04 3.06±0.65

a a a a 10 7.34±0.81 8.01±0.43 7.45±1.05 7.84±0.99

b b b a 20 7.81±0.52 9.62±1.09 7.39±1.01 13.00±1.91

bc ab c a 30 8.16±0.72 10.16±1.25 5.27±0.66 12.66±1.58

a b b b 40 8.05±1.00 3.68±0.96 3.05±0.74 3.08±0.60

Data is expressed as mean ± SD. Mean values followed by a common letter do not differ significantly (Tukey’s test, p<0.05).

Fig. 3.1. Malondialdehyde levesl in the sera of untreated and treated microfilaraemic rats.

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Table 3.2 Superoxide dismutase levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNPs

of days (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein)

a a a a 0 12.61±1.15 12.09±0.97 12.44±0.70 12.56±0.99

a a a a 10 8.20±.17 8.45±0.75 7.92±0.52 9.00±0.70

c b d a 20 7.02±0.61 10.92±0.88 3.96±0.94 17.14±0.75

c a d b 30 6.76±0.95 11.78±0.74 3.43±0.56 8.96±0.63

b a b a 40 5.23±0.71 5.82±0.86 3.40±0.53 6.91±0.57

Data is expressed as mean ± SD. Mean values shown with common letters do not show significant difference (Tukey’s test, p<0.05).

20 Untreated DEC 18 a NTZ NTZ+AgNPs 16

14 a a a a a 12 b

10 a b a a a 8 c c a a 6 a Serum SOD level (U/mg) d 4 d b

0 0 10 20 30 40 Days of infection Fig. 3.2. Superoxide dismutase levels in the sera of untreated and treated microfilaraemic rats.

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Table 3.3. Catalase levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNP s

of days (U/mg protein) (U/mg protein) (U/mg protein) (U/mg protein)

0 500.00±30.30a 448.90±25.67a 438.42±23.72a 511.37±30.57a

10 1568.00±77.50a 1553.72±45.28a 1532.87±42.56a 1624.97±48.35a

20 1670.94±45.14b 1763.06±47.72ab 635.14±36.61c 1838.99±52.78a

30 1600.43±55.83b 1801.35±49.09a 600.92±31.41d 1463.40±49.90c

40 991.41±35.65b 1427.26±37.43a 580.56±53.25d 803.11±49.78c

Data is expressed as mean ± SD. Mean values shown with common letters do not show significant difference (Tukey’s test, p<0.05).

Fig. 3.3. Catalase levels in the sera of untreated and treated microfilaraemic rats.

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Table 3.4. Glutathione peroxidase levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNP s

of days (U/mg protein) (U/mg protein) (U/mgprotein) (U/mg protein)

0 51.86±5.81a 47.36±6.94a 47.95±8.44a 53.29±11.17a

10 60.19±2.51a 60.98±2.51a 58.03±10.13a 62.06±14.99a

20 64.89±4.29b 156.50±11.69a 54.02±6.15bc 45.10±9.21c

30 70.19±7.66c 143.18±14.33a 52.90±4.72c 33.75±10.76b

40 95.90±8.14ab 124.82±11.38a 50.58±8.06b 33.52±16.03b

Data is expressed as mean ± SD. Mean values shown with common letters do not show significant difference (Tukey’s test, p<0.05).

180 Untreated a DEC 160 NTZ a NTZ+AgNPs

140 a

120

b 100

80 b a a b a a a c 60 a c c a a Serum GPx level (U/mg) c

40 c c

0 0 10 20 30 40 Days of infection Fig. 3.4. Glutathione peroxidase levels in the sera of untreated and treated microfilaraemic rats.

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Table 3.5. Glutathione S-transferase levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNPs of days (U/mg protein) (U/mg protein) (U/mgprotein) (U/mg protein)

a a a a 0 65.81±10.44 62.93±8.69 55.09±11.77 57.66±11.86

a a a a 10 119.79±19.79 112.50±20.00 100.49±27.88 103.48±10.71

b a b b 20 148.70±8.10 177.30±25.86 85.12±12.32 72.05±13.17

a b b b 30 269.12±21.70 87.89±13.07 76.37±9.59 61.78±17.58

a b a b 40 150.49±14.48 81.11±6.61 70.46±9.68 56.59±8.49

Data is expressed as mean ± SD. Mean values shown with common letters do not show significant difference (Tukey’s test, p<0.05)

300 a Untreated DEC NTZ 250 NTZ+AgNPs

200 a

a a 150 a a a a 100 b b b Serum level GST (U/mg) b b b a a b a a b 50

0 0 10 20 30 40 Days of infection Fig. 3.5. Glutathione S-transferase levels in the sera of untreated and treated microfilaraemic rats.

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Table 3.6. Alkaline phosphatase levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNP s

of days (KA unit) (KA unit) (KA unit) (KA unit)

0 8.04±0.66a 7.61±0.46a 7.51±0.45a 8.40±0.62a

10 11.30±1.22a 10.30±1.01a 11.27±1.10a 10.27±0.76a

20 12.19±0.84bc 10.99±1.21c 14.30±1.13ab 16.63±0.90a

30 13.51±1.00a 9.47±0.89b 13.62±0.82a 11.51±0.50ab

40 8.93±0.74a 8.34±0.97a 9.28±0.45a 8.74±0.45a

Data is expressed as mean ± SD. Mean values shown with common letters do not show significant difference (Tukey’s test, p<0.05).

20 Untreated DEC 18 NTZ a NTZ+AgNPs 16 ab a a 14 bc a a 12 c ab a a b a 10 a a a a a a 8 a a

Serum ALP level (KA) 6

0 0 10 20 30 40 Days of infection

Fig. 3.6. Alkaline phosphatase levels in the sera of untreated and treated microfilaraemic rats.

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Table 3.7. Aspartate aminotransferase levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNPs

of days (IU/L) (IU/L) (IU/L) (IU/L)

0 75.13±7.99a 72.80±3.89a 80.65±2.15a 73.77±4.96a

10 114.45±14.68a 112.62±11.06a 120.16±6.73a 110.00±7.16a

20 136.21±8.60a 119.65±9.27a 125.27±9.07a 126.40±6.65a

30 147.41±9.29a 90.11±5.10c 122.65±4.19b 118.72±3.12b

40 160.00±10.00a 75.10±2.68c 116.33±2.89b 112.56±2.69b

Data is expressed as mean ± SD. Mean values shown with common letters do not show significant difference (Tukey’s test, p<0.05).

180 Untreated a DEC 160 NTZ a NTZ+AgNPs a 140 a a a a b a a a b b 120 b

100 c

a a 80 a a c

60 Serum AST level (IU/L)

0 0 10 20 30 40 Days of infection

Fig. 3.7. Aspartate aminotransferase levels in the sera of untreated and treated microfilaraemic rats.

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Table 3.8. Alanine aminotransferase levels in the sera of untreated and treated microfilaraemic rats.

Number Untreated DEC NTZ NTZ+AgNPs

of days (IU/L) (IU/L) (IU/L) (IU/L)

0 24.48±1.98a 22.44±0.94a 24.72±1.44a 22.97±1.3a

10 48.26±1.39a 45.76±0.92a 47.54±1.09a 47.32±0.95a

20 53.80±1.04a 46.58±2.91c 48.29±1.58bc 52.35±1.51ab

30 55.03±0.84a 31.12±1.06d 42.60±0.87c 48.18±1.04b

40 57.25±1.10a 27.92±0.99d 38.04±0.44c 43.96±1.06b

Data are expressed as mean ± SD. Mean values shown with common letters do not show significant difference (Tukey’s test, p<0.05).

70 Untreated DEC NTZ 60 NTZ+AgNPs a a a ab a bc 50 a a c b a b c

40 c

d 30 d a a a a

Serum level ALT (IU/L) 20

0 0 10 20 30 40 Days of infection Fig. 3.8. Alanine aminotransferase levels in the sera of untreated and treated microfilaraemic rats.

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DISCUSSION

Parasites cause inflammation when they get embedded or penetrated under the mucosa. Inflammatory reactions are accompanied by generation of reactive oxygen species (ROS) which may lead to deterioration of vital functions and cell death (Sies, 1991; Bello et al., 2000; Egwunyenga et al., 2004; Siwela et al., 2010). Lipid peroxidation is ongoing physiological processes which also play its role in the pathogenesis of several parasitic diseases (Bagchi et al., 1993). ROS induce the oxidation of polyunsaturated fatty acids in the biological systems and lead to the formation of lipid peroxidation products. Malondialdehyde (MDA) is one of the most frequently used ROS biomarkers to determine the overall lipid peroxidation level (Serarslan et al., 2005; Siwela, 2013). MDA level was increased in the serum of microfilaraemic rats on 10th day compared to the serum of uninfected rats on day 0. Oxidative stress increased with the increase in microfilarial count indicating its putative role in infected rats. Similar correlation was observed between the degree of parasitaemia and lipid peroxidation as earlier observed in Mastomys natalensis tissues infected with Brugia malayi (Gomathi, 2000). In the present study, MDA level in DEC and NTZ+AgNPs treated microfilaraemic rats was found to be increased which is in agreement with earlier studies from different countries indicating similar rise in MDA level in the blood and tissues of infected organisms (Bagchi et al., Basu, 1993; Kilic, 2003; Rezaei and Dalir-Naghadeh, 2006; Ince et al., 2010; Siwela et al., 2010; França et al., 2012; Siwela, 2013; Dkhil, 2015). MDA level showed a decrease in rats treated with NTZ when compared with untreated control, which showed a slight increase in serum MDA up to 30th day of infection. MDA level was much higher in NTZ+AgNPs treated rats reflecting inflammation and oxidative stress. NPs have been reported to influence intracellular calcium concentrations, activate transcription factors, and modulate cytokine production via generation of free radicals (Huang et al., 2010; Li et al., 2010). Cellular internalization of NPs has been shown to activate immune cells, including macrophages and neutrophils, contributing to ROS/RNS (Knaapen et al., 2004; Risom et al., 2005). This process usually involves the activation of NADPH oxidase enzymes (Manke et al., 2013). MDA level was increased significantly in Schistosome infected mice as well as in those treated with the combination of gold nanoparticles (AuNPs) and praziquantel (PZQ). However, the

Chapter -3

level of MDA gets reduced if infected mice were treated with AuNPs alone or PZQ (Dkhil et al., 2015). Similar observations were recorded in the present study where MDA level was much higher when microfilaraemic rats were treated with the combination of NTZ+AgNPs.

In the present study, MDA level in DEC treated rats showed an increase in rats infected with S. cervi as it harnesses the innate inflammatory system in vivo to combat the infection and the resultant effect was probably induced through possible oxidative mechanism. Similar results were obtained against microfilariae of B. malayi treated with DEC (McGarry et al., 2005). With these evidential proofs coupled with our experimental results, the direct antifilarial effect might be envisaged as a function of oxidative rationale as earlier indicated by Mahajan et al. (2010). NTZ treatment in rats showed a progressive decrease in MDA level, and therefore, suppression of microfilariae was much less compared to DEC and NTZ+AgNPs. This difference in efficacy might be due the reason that it inhibits transcription/replication in infected cells and most importantly it inhibits the secretion of proinflammatory cytokines which play most important role in generating ROS as described by Rossignol et al. (2005). Similar low level of MDA was observed when mice infected with blastocystis were treated with NTZ (Abdel-Hafeez et al., 2015). It clearly indicates that the NTZ alone is not effective antifilarial agent.

Enzymatic antioxidants, including superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GPx) represents the main form of intracellular antioxidant defense system. Plasma SOD catalyses the dismutation of superoxide to hydrogen

peroxide (H2O2) and it is considered as the first defense against pro-oxidants. SOD protects the tissue to a certain degree from the harmful effects of superoxide radicals (Halliwell and Chirico, 1993; Fridovich, 1995; Celi, 2010; Ince et al., 2010). When SOD dismutes superoxide free radicals, another ROS such as hydrogen peroxide is formed, which is the substrate for CAT (Nelson and Kiesow, 1972; Aebi, 1984). Both CAT and GPx detoxify hydrogen peroxide to water, although the latter appears to have more potent activity (Halliwell and Gutteridge, 1999; Margis et al., 2008). These enzymes work together to eliminate active oxygen species and small deviations in physiological concentrations may have a dramatic effect on the resistance of cellular lipids, proteins and DNA to oxidative damage (Mates and Sanchez-Jimenez, 1999;

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Ince et al., 2010). GPx activity contributes to the oxidative defense of animal tissues by catalyzing the reduction of hydrogen and lipid peroxides, and is also considered an indicator of oxidative stress (Flohe et al., 1973; Halliwell and Chirico, 1993; Tüzün et al., 2002). The glutathione-S-transferases (GSTs) are a family of proteins that catalyze the conjugation of glutathione on the sulfur atom of cysteine to various electrophiles and protecting cells against ROS (Chasseaud, 1979). GST which is a phase II enzyme responsible for drug biotransformation, also presents remarkable scavenging potential (Sagara et al., 1998; Pedrosa et al., 2001). The antioxidant enzymes SOD and CAT limit the effects of antioxidant molecules on tissues and provide defense against oxidative cell injury by acting as free radical scavengers (Kyle et al., 1987). The activity of these enzymes plays an important role in suppressing the pathological injury inflicted by the parasite (Mates et al., 1999b).

In the present study, the level of serum SOD was decreased in infected rats on the

10th day as a fraction of it might have been utilized in production of H2O2 during oxidative metabolism through feedback inhibition as earlier observed by Sienkiewicz et al. (2004). In untreated microfilaraemic rats, a significant increase in CAT, GPx and GST activities was observed on the 10th day compared to day 0. CAT activity increases in response to increase in hydrogen peroxide as it acts as a substrate for CAT. Similar results were obtained in rats infected with Syphacia muris (Ince et al., 2010). Dogs infected with Rangelia vitalii, showed an increased CAT activity at day 20 post infection (PI), while SOD was activated at days 10 and 20 post infection (França et al., 2012). Highest increase in SOD and CAT activity was observed in NTZ+AgNPs treated rats, followed by DEC, whereas NTZ showed a declining activity of these enzymes in infected rats on the 20th day of infection during the present study. Earlier it was reported that SOD and CAT levels were inconsistently elevated after exposure to the NPs. SOD is an inducible enzyme and its elevated levels may indicate the presence of reactive species (Lovric et al., 2005; Adeyemi et al., 2012). NTZ+AgNPs and NTZ treated rats showed a constant decline in SOD activity from 30th-40th day as compared to those treated with DEC where decline was seen on 40th day. In untreated rats, SOD showed a constant declining trend from 10th- 40th day of infection, whereas the CAT levels were increased between 10th -20th day and then decline started from 30th day and continued till 40th day of infection. Similar

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observations were made regarding the activity of these enzymes in animals infected with B. malayi, Ascaris lumbricoides and Trichuris trichura (Gomathi, 2000; Aloho- Bekong et al., 2011).

In untreated microfilaraemic rats, significant increase in GPx and GST activity was observed on the 10th day in the present study. The increased level of GPx in rats might

be due to the reason that this enzyme is responsible for conversion of H2O2 and/or lipid peroxides, while GST has scavenging potential (Oliveira and Cecchini, 2000; Pedrosa et al., 2001). Similar increase was observed in GST in rats infected with Trichinella spiralis (Wojtkowiak-Giera et al., 2011).

During the present study, an increase in GPx and GST activity was observed in rats against DEC on 20th day. A similar increase in GPx and GST activity was observed in most of the organs of B. malayi infected animal treated with DEC (Gomathi, 2000). Decrease in GPx and GST activity was observed in NTZ+AgNPs and NTZ treated rat’s sera in the present study. Thiazolide hampers the enzymatic activity of GST1 by inhibiting the coupling of GSTP1 to glutathione which reduces the chemoresistance by this enzyme (Tew, 1994). When GST1 combines with thiazolides, it transform later into a toxic derivative that triggers cell death by affecting the interaction of GSTP1 and apoptosis-related JNK (Adler et al., 1999; Turella et al., 2005). Similar observations were recorded earlier in cancer chemotherapy by Muller et al. (2008).

NTZ+AgNPs caused inconsistent reductions in the levels of GST and GPx in sera collected after 20th-40th day of infection. GST reduction may be due to its role in detoxification of endogenous and exogenous substances (Litwack et al., 1971; Habig

et al., 1974), while GPx was probably being utilized for reduction of H2O2 and alkyl hydroperoxides (Mannervik, 1985). Since silver nanoparticles have affinity with thiol groups of glutathione (GSH), they deactivate this enzyme (Ravindran et al., 2012; Dakal et al., 2016). Similar reductions of these enzymes were observed when silver nanoparticles were used against multidrug resistant bacteria (Salma et al., 2011; Srivastava et al., 2011; Adeyemi et al., 2014). Level of GPx showed a constant increase in untreated rats from 0th-40th day, whereas GST showed increased activity from 0th-30th day which declined afterwards. The probable reason for the elevation in

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these enzymes might be the increasing microfilarial density which exerts oxidative stress, in response to which secretion gets accelerated to neutralize the infection.

In all the treated microfilaraemic rats, level of ALP, AST and ALT was low as compared to untreated rats which were proportionate to the microfilarial density in infected rats. Since there was a progressive decrease in the microfilarial density in the rats treated with aforesaid drugs and level of liver markers too decreased accordingly. It is a known fact that DEC interferes with the metabolism of arachidonic acid whose product plays a significant role in liver injury, DEC blocks the production of prostaglandins, resulting in capillary vasoconstriction and infringement in the passage of microfilariae as described earlier in CCl4-induced liver damage in rats (Gonzàlez et al., 1994). In the present study, increase in ALP, AST and ALT enzymes was recorded in untreated and treated rats when microfilarial density was high and was possibly due to the encroachment and degeneration of liver cells caused by microfilariae and obstruction of bile ducts. Similar significant increase in the activity of AST and ALT was observed in the experimentally infected microfilaraemic man, cattle, buffaloes, horses and dogs, which were infected with Wuchereria bancrofti, Dirofilaria immitis and Dipetalonema reconditum in which hepatic parenchyma was damaged (Sharma and Pachauri, 1982; Sharma and Joshi, 2002; Hashem and Badawy, 2007; Tabrizi, 2012; Murugan and Jeeva, 2016).

In the light of results obtained during the present study, it may be concluded that the combination of nitazoxanide and silver nanoparticles possesses the potent antifilarial activity as AgNPs has the capacity to increase antioxidant defense systems in the host, particularly by upregulating antioxidant enzymes such as SOD and CAT. AgNPs also promotes increase in oxidative stress that exerts pressure on the parasite, while NTZ interfere with the energy metabolism of microfilariae. This suggests that NTZ+AgNPs acts as a synergist that offers a balance of effects with respect to its capacity to promote protection in the host. The differences between the parasite and host in terms of the biochemical systems that afford the antioxidant defenses of the two organisms, could provide exploitable targets for the development of an effective chemotherapeutic agent for filariasis. Future investigations could also elucidate the mechanism of action of these compounds, to ascertain their widespread use.

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INTRODUCTION

Immunodiagnostic tests are of paramount importance for highly prevalent infections, especially those causing severe morbidity. Enzyme-linked immunosorbent assay (ELISA) is a simple and sensitive diagnostic technique for determining the seroprevalence of filarial infections which detect low concentrations of circulating antibodies, excretory-secretory (ES) products or their markers in body fluids of the host (Kaliraj et al., 1981; de Savigny and Speiser, 1985). Immunodiagnosis of human filariasis by the antigen of Setaria digitata was successfully done by ELISA (Dissanayake and Ismail, 1980). ELISA using different antigens of Setaria spp. has been reported to be sensitive in diagnosis of bovine as well as bancroftian filariasis (Theodore and Kaliraj, 1990; Mohanty et al., 2000; Jayalakshmi, 2008). Native PAGE is another technique used for separating biologically active proteins where mobility depends on both size and charge. The immunological profile of Setaria cervi was studied by Singhal (1983) in order to explore the possibility of its use in the diagnosis of filariasis and found antigenic profile of S. cervi simple than other filariids such as Litomosoides carinii and Dirofilaria immitis.

Counter current immunoelectrophoresis (CIEP) and immunodiffusion (ID) were also tried for detection of antigen and antibody; using S. cervi and immune complex antigens in which sixty percent positivity was noted in microfilaraemic cases, against both the homologous and heterologous antibodies (Gupta et al., 1990). Reddy et al. (1984) indicated the utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen in bancroftian filariosis by sandwich ELISA and showed an apparent positive correlation between the microfilariae density and antigen titer. Theodore and Kaliraj (1990) claimed to distinguish stage specific infection in filariasis based on the immunoreactivity of the sera with crude and purified S. digitata antigens. It has been reported earlier that microfilarial antigen is more potent than whole worm in serodiagnosis of bovine filariasis (Sundar and D’Souza, 2015).

Recent studies on immunity to filarial infections revealed the presence of extremely elevated levels of immunoglobulins in patients having high microfilarial density which was observed in Brugia malayi infection, indicating predominance of antimicrofilarial immunity (Lawrence, 2001). S. cervi antigens have been evaluated

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for the immunodiagnosis of human filariasis (Almeida et al., 1990). Immunoelectrophoresis has revealed the presence of 9–10 precipitin lines in adults and only four precipitin lines in microfilarial antigenic preparations. Many antigens of S. cervi adults and microfilariae have also been recognized by rabbit anti-B. malayi serum, showing the existence of common antigenic determinants between the bovine and human filarial parasites (Kaushal et al., 1987). An enzyme-linked immunosorbent assay (ELISA) has been developed using cross-reacting antigens of L. carinii and S. cervi to detect antibodies in filarial patients (Tandon et al., 1983).

The antifilarial drug DEC citrate is known to mediate in vivo microfilaricidal activity in conjunction with the host immune system. Active immunization of microfilaraemic animals with methyl piperazine carboxylic acid (MPCA) coupled with bovine serum albumin (BSA), followed by administration of subcurative doses of DEC, has been reported to result in rapid clearance of microfilaraemia in both S. digitata and B. malayi infected Mastomys coucha, indicating the synergistic activity of DEC and the antibodies to the drug which may be true with other drugs. Since some of the filarial antibodies are known to react with DEC, it is proposed that such antibodies may potentiate the microfilaricidal activity of the drug in vivo (Mukhopadhyay & Ravindran, 1997).

Keeping above facts in view, qualitative effect in serum protein of untreated rats and those treated with DEC, NTZ and nanocomposite of NTZ+AgNPs was observed by native PAGE. Antibody titer was also quantitated from the sera obtained every tenth day from infected untreated and treated rats to see its correlation with efficacy and the microfilarial density during the course of infection.

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MATERIALS AND METHODS

Establishment of microfilaraemia in white rats and their treatment with drugs:

Microfilariae obtained from uterus of gravid female were injected in the peritoneal cavity of 20 albino rats which were divided into four groups. Groups 1, 2 and 3 were treated with diethylcarbamizine (DEC), nitazoxanide (NTZ) and nitazoxanide combined with silver nanoparticles (NTZ+AgNPs), respectively from the 10th day onwards when microfilariae appeared in the peripheral circulation. All the drugs were given orally at a dose of 100 mg/kg/day for 6 days. Group 4 served as untreated infected control.

Collection of sera from infected rats:

Blood was collected from the tail of rats from 0th to 40th day of infection at 10 days interval. Blood was kept at room temperature for 30 minutes and then transferred to the refrigerator at 4 °C for 2 hours so that serum could separate from the clotted blood. Serum was picked and transferred to 1.5 ml vials and centrifuged at 1000 xg for 5 minutes in a cooling centrifuge. The sera was collected and stored at -80 °C until used.

Native-PAGE: Electrophoretic mobility of proteins was observed by the Native-PAGE in sera collected from untreated and treated microfilaraemic white rats as described by Ornstein and Davis (1964). 10% separating gel was prepared by adding 29.2 g/100 ml acrylamide with 0.8 g/100 ml bis-acrylamide and 1.5 M Tris buffer (pH 8.8) in distilled water. 5% stacking gel was prepared by adding 29.2 g/100 ml acrylamide with bis-acrylamide 0.8 g/100 ml and 0.5 M Tris buffer (pH 6.8) in distilled water. Working solutions were prepared by adding TEMED and 10% freshly prepared APS. These solutions were carefully overlaid into the glass plate’s mould and left for polymerization. Loading samples were prepared by dissolving 1:2 volume of serum in sample buffer (1.5 M Tris buffer pH 8.8, glycerol, and 0.05% aqueous bromophenol blue and distilled water). Prepared samples were loaded in the wells of casted gel. The electrophoresis was carried out at a constant voltage of 100 V in vertical electrophoresis unit. Thereafter, the gel was stained with 0.25% Coomassie Brilliant

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Blue-R250 (CBBR-250) dye. After clearing and differentiation, the images were taken on Gel Doc™ XR+. Enzyme linked immunosorbent assay (ELISA) Antigen specific antibody was measured in sera of untreated and treated rats by direct binding ELISA as described by Voller et al. (1976). Ninety six wells of microtitre plates were coated with 50 µl of antigen and left overnight at 4 °C. These plates were washed 3 times with phosphate buffer saline containing 0.1% PBST. The unoccupied sites were saturated by incubation with 1.5% milk in 0.1% PBST for 5-6 hours at room temperature. Plates were washed thrice with 0.1% PBST. The test and control wells were loaded with 100 µl of serially diluted anti-microfilarial serum. These plates were incubated for 2 hours at room temperature and then overnight at 4 °C. 100 µl of appropriate conjugate of anti-rat IgG conjugated with horseradish peroxidase (1:3000) was coated in each well and kept for 2 hours at room temperature. After regular washing with PBST and distilled water, 100 µl of substrate TMB (3,3′,5,5′-Tetramethylbenzidine) was added in each well and incubated for 30- 45 minutes. The reaction was stopped by addition of 100 µl of 3 M NaOH in each well. The absorbance of each well was monitored at 450 nm on a Qualigens ELISA reader.

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RESULTS Protein profile of the sera in untreated and DEC, NTZ and NTZ+AgNPS treated microfilaraemic white rats is given in Fig. 4.1. Serum protein profile of the untreated microfilaraemic rats showed faint protein bands on 10th day which become more prominent 20th day onwards and continue to increase till 40th day. In DEC treated rats, increase in the intensity of protein bands was observed till 30th day which declined afterwards. Serum treated with NTZ showed progressive increase in the intensity of protein bands 10th day onwards showing similarity with untreated serum. Rats treated with NTZ+AgNPs showed highest increase in the intensity of protein bands in the serum on 20th day which declined afterwards and became very feeble on 40th day. The protein bands in the serum of NTZ+AgNPs treated rats were more pronounced if compared with the sera of normal, DEC and NTZ treated rats. The antibody titer on 10th day increased in the sera of all the microfilaraemic rats which were detected at dilution 1:1600-1:3200. In untreated rats, the antibody was detected at the dilution 1:6400 on 20th day which was 1:25600 and 1:51200 on 30th and 40th day, respectively (Fig. 4.2). In DEC treated rats, antibody titer increased from 20th-30th day and was detected at dilutions 1:25600- 1:51200 followed by a decline on 40th day where antibodies were detected at dilution 1: 25600 (Fig. 4.3). In NTZ treated rat’s serum, antibody titer showed a progressive increase in almost similar manner as in untreated rats with positive reaction at dilutions 1:3200, 1:12800 and 1:25600 on 20th, 30th and 40th day, respectively (Fig. 4.4). Rats treated with NTZ+AgNPs showed the highest antibody titer on 20th day as it was positive at the dilution 1:51200 on 20th day followed by a decline on 30th day onwards which continued till 40th day, showing antibodies at dilutions 1:25600 and 1:6400, respectively (Fig. 4.5)

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Fig. 4.1. Protein profile in the sera of untreated and DEC, NTZ and NTZ+AgNPs treated microfilaraemic white rats.

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0th day 10th day 0.30 20th day 30th day 40th day 0.25 Negative control

0.20

0.15

0.10 sorbance at 450nm

Ab 0.05

0.00

2.0 2.5 3.0 3.5 4.0 4.5 5.0 Log serial dilution of serum of untreated rat

Fig. 4.2. Antibody titer in the serum of untreated microfilaraemic rats.

0th day 10th day 20th day 0.30 30th day 40th day 0.25 Negative control

0.20

0.15

0.10 bsorbance at 450nm

A 0.05

0.00

2.0 2.5 3.0 3.5 4.0 4.5 5.0 Log serial dilution of serum of rat treated with DEC

Fig. 4.3. Antibody titer in the serum of microfilaraemic rats treated with DEC.

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0th day 10th day 20th day 0.30 30th day 40th day 0.25 Negative control

0.20

0.15

0.10 bsorbance at 450nm

A 0.05

0.00

2.0 2.5 3.0 3.5 4.0 4.5 5.0 Log serial dilution of serum of rat treated with NTZ

Fig. 4.4. Antibody titer of serum of microfilaraemic rats treated with NTZ.

0th day 10th day 20th day 0.30 30th day 40th day 0.25 Negative control

0.20

0.15

0.10 sorbance at 450nm

Ab 0.05

0.00

2.0 2.5 3.0 3.5 4.0 4.5 5.0 Log serial dilution of serum of rat treated with NTZ+AgNPs

Fig. 4.5. Antibody titer in the serum of microfilaraemic rats treated with NTZ+AgNPs.

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DISCUSSION

Native gel, also known as non-denaturing gel, analyzes proteins that are still in their folded state. Thus, the electrophoretic mobility depends not only on the charge-to- mass ratio, but also on the physical shape and size of the protein. Protein profile of the sera of untreated and treated rats showed some protein bands which differ in their intensities which was supported by antibody titer estimation through ELISA. In this study prominence of protein bands as well as increase in antibody titer was observed in untreated as well as treated rats 20th day onwards which was directly proportional to the microfilarial density. Probable reason of increase in antibody titer after establishment of infection might be the presence of phosphocholine, a ubiquitous and immunodominant molecule on the microfilarial surface, as presence of these molecules are documented in many organisms in nature that evokes antibody response (Brown et al., 1984; Briles et al., 1987). Another reason for increase in antibody titer may be that IgG4 levels rise in response to regulatory cytokines which reflect a dominant regulatory environment as observed earlier in human filariasis (Adjobimey and Hoerauf, 2010). The subsequent decrease in microfilariae after DEC treatment was observed which was probably due to increased IgM and IgG levels as earlier observed in the serum of patient suffering from human filariasis which gradually decreased due to decline in parasitaemia (Ramaprasad et al., 1988; Lal and Ottesen, 1989; Kueniawan-Atmadja et al., 1995).

It has been observed earlier that DEC induced a rapid decrease in the number of microfilariae circulating in the blood of patients infected with W. bancrofti or B. malayi and numerous attempts have been made to identify immunological criteria suitable for use in parallel with or as substitution for parasitological parameters to evaluate the efficacy of antifilarial agents (Ottesen, 1985). Though detection of parasite antigen is thought to have the greatest potential to provide direct evidence of drug efficacy, informations are scanty regarding antibody titer to monitor drug efficacy in humans (Malhotra & Harinath, 1984).

In the present study antibody titer was measured in the sera of microfilaraemic rats before and after treatment with DEC, NTZ and NTZ+AgNPs. It was noted that antibody titer progressively increased till 40th day in untreated rats and those treated with NTZ and was proportionate to the density and longevity of the circulating

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microfilariae. NTZ did not exert much effect on microfilariae as a result of which they declined in number for few days and reappeared. But in DEC treated rats antibody titer started increasing after the treatment which pointed towards antigenic stimulus and activation of helper T-cells which help in initiating multiplication of lymphocytes and secretion of antibody by plasma cells. Increase in antibody had also been reported in both bancroftian and brugian filariasis after DEC treatment (Grove, 1981; Piessens et al., 1981; Lal and Ottesen, 1989). It is possible that such increased antibody levels subsequently played a role in suppression of any recurrent microfilaraemia, as it had been shown experimentally that immune human or mouse serum injected into mice reduce circulating microfilariae (Grove et al., 1979). Similar but higher increase of antibody titer was noticed in white rats treated with NTZ+AgNPs which attained a peak on 20th day followed by a decrease which was again proportionate to the microfilarial density. Since nanocomposite of NTZ+AgNPs has two components which acts synergistically, its efficacy was enhanced and microfilariae were cleared in shortest period due to high antibody titer which showed its peak on 20th day and declined thereafter. The probable reason for its maximum efficacy may be the cumulative effect in which NTZ targets the metabolism of the microfilariae and silver nanoparticles which activate immune response and destroy the cell by targeting protein and DNA of the parasite. The efficacy of this formulation was evident from decrease in number of microfilaraemia right from the beginning of the treatment and decline in antibody titer. Similar antigenic stimulation and destruction of parasites by activating immune response by the nanoparticles of nanomedicines was observed by earlier workers as well (Knaapen et al., 2004; Risom et al., 2005). The prominence in the protein bands of sera of the rats treated with NTZ+AgNPs may be due to the presence of nanoparticles which are known to stimulate lymphocytes responsible for secreting antibodies. This was confirmed by the ELISA results which showed highest antibody titer on the 20th day in rats treated with nanocomposite of NTZ+AgNPs.

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Summary

Drugs used during this study were DEC, NTZ and NTZ+AgNPs. AgNPs and NTZ+AgNPs were prepared and characterized by UV-Visible spectral analysis, Fourier transform infrared spectroscopy, scanning and transmission electron microscopy. Ten concentrations ranging from 10µg/ml to 100µg/ml of DEC, NTZ and NTZ+AgNPs were prepared in 200 µl of Ringer’s solution and tried against the microfilariae in vitro at 37 oC in 96-well titer plate. Controls were run by incubating microfilariae in Ringer’s solution to compare the results. Images of treated and untreated microfilariae were taken by scanning electron microscopy to see the possible effect on their body surface. TCA cycle enzymes such as succinate, malate and isocitric dehydrogenase were localized in adult and microfilariae of S. cervi by incubating them for 24 and 6 hours, respectively at 37 oC in 100 µg/ml of DEC, NTZ and NTZ+AgNPs by the method as described in Theory and Practice of Histological Techniques by Bancroft and Gamble (2002).

Summary

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Summary

remained elevated for a comparatively longer duration. In the untreated control group there was a constant decline in SOD activity throughout the infection. The increase in the level of SOD was inversely proportional to the microfilarial density. Since SOD catalyzes the dismutation of superoxide to H2O2 and protects tissue from harmful effects of superoxide radicals, it gets elevated when microfilariae were more in blood circulation, indicating its protective role.

Summary

markers too decreased accordingly. Increased ALP, AST and ALT in untreated rats indicated degeneration of liver cells caused by microfilariae.

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