ABBREVIATIONS

% : Per cent

@ : At the rate of

°C : Degree Celsius

°C : Centigrade degree

µg : Microgram

µl : Microlitre

A/G : Albumin/Globulin

AChE : Acetyl cholinesterase

ALP : Alkaline phosphatase

ANOVA : Analysis of variance

BALT : Bronchial associated lymphoid tissue

Cf : Cysticercus fasciolaris

CLR’s : Control laboratory rats

Cmm : Cubic millimeter

DLC : Differential leukocyte count

EDTA : Ethylene diamine tetra acetic acid

ESR : Erythrocytic sedimentation rate

g : gram

GSH : Reduced Glutathione

H&E : Haematoxylin and Eosin

Hb : Haemoglobin

Hd : Hymenolepis diminuta

Hm : Hepatozoon muris

hr : hour

Kg : Kilogram

LP : Lipid peroxidase

LR’s : Laboratory rats

m : micro

m : meter

m : mole

MDA : Malondialdehyde

Introduction

1

INTRODUCTION

he diversity of research for which the laboratory rat is used is

greater than that associated with any other laboratory animal.

It is difficult to determine who should be credited for recognition of the

value of using animals to study human diseases, but it is safe to say

that modern medical research began in the late 1700s A.D with the

successful conquest of a number of human diseases by the use of

animals in which diseases comparable to those in humans (models)

were discovered and studied. (Lindsey, 1979)

The genus Rattus emerged from the Murid family about 3.5

million years ago, and Norway rats themselves emerged about 2

million years ago. Wild rat originated in the temperate regions of

Central Asia from Southern USSR through North China. Norway rats

traveled to Europe in human ships in the 16th century and reached

T

Introduction

2

the New World in the 18th century. By immigration along-trade and

military routes the cosmopolitan rat has spread around the world.

Today, Norway rats live in cities, suburbs and agricultural areas in a

human-dependent relationship called commensalisms (Calhom, 1963

and Boice, 1977).

Rats are various medium sized rodents. "True rats" are

members of the genus Rattus, the most important of which to humans

are the black rat, Rattus rattus, and the brown rat, R. norvegicus. Wild

rats live in colonies. Female rats, usually related to each other, live in

little groups of one to six in a little burrow system of their own. Each

female have their own nest chamber, but they may share the burrow

and may raise their young together (called communal nesting).

According to Wikipedia (2007) scientific classification of rats is as

follows:

• Kingdom - Animalia

• Phylum - Chordata

• Class - Mammalia

• Order - Rodentia

• Super family - Muroidea

• Family - Muridae

• Sub-family - Murinae

• Genus - Rattus

• Brown rat - Rattus norvegicus

• Black rat - Rattus rattus

Wild and albino mutants were first used for experimental

purposes in Europe in the mid-1800s and in the United States shortly

Introduction

3

before 1900 A.D. The Wistar Institute in Philadelphia was prominent

in the development of the rats as a laboratory animal and here

originated many of the rat strains now used worldwide. Henry

Donaldson and his colleagues at the Wistar Institute used these early

rat strains for a variety of studies dealing with neuro-anatomy,

nutrition, endocrinology, genetics and behaviour. The history and

evolution of many rat strains used today have been summarized by

earlier workers. Many strains originated by selective breeding viz.,

Sprague-Dawley, Wistar, Long Evans and Charles Foster are used in

specific research (Lindsey, 1979).

About 3-5 million rats are used annually in laboratories all over

the world, which are almost 10-15 % of total number of laboratory

animals used in biomedical research. The laboratory rat is second

only to the mouse in the utilization as a research animal. The rat has

been utilized for a wide range of studies involving practically all fields

of research, including nutritional, genetic and environmental studies.

There are various strains of inbred rats that are available for

spontaneous tumor production, physiologic and anatomical

differences and other unique aspects which should be considered

before and any research study is undertaken utilizing the rat as a

model.

Various random bred strains are used in toxicology, nutritional

and biochemical work. The rat is widely used because of its suitability

Introduction

4

for a wide variety of experimental designs, its docility and ease of

handling and care, its short gestation period, wide dietary preferences,

intelligence and easy availability. The laboratory rat is commercially

available through many sources. Once a particular strain is selected,

a suitable and practical breeder should be identified and maintained

through the entire study.

Wild rats plays a role for transmission and spreading of

diseases related to viruses, bacteria, fungi, rickettsia and parasites.

Here the parasitic diseases occurring in rats and also those have some

zoonotic importance attract our interest. Diseases due to infectious

agents constitute a significant environmental variable that influence

on research data, derived from laboratory rats than the diseases due

to other reasons. Laboratory rats seldom show clinical signs of disease

upon arrival to the laboratory from commercial sources.

A pathogen is a biological agent that causes disease or illness to

its host. However, pathogens can infect unicellular organisms from all

of the biological kingdoms. There are several substrates and pathways

where by pathogens can invade a host; the principal pathways have

different episodic time frames, but soil contamination has the largest

or most persistent potential for harboring a pathogen. However, the

rats may harbour pathogens that are of low to moderate virulence and

are capable of severely compromising the health of animals, when

exposed to various types of experimental stresses. (Wikipedia, 2007)

Introduction

5

Parasitic agents are commonly responsible to show some

clinical signs in rats. Parasites are those organisms that are

metabolically and physiologically dependent on other organisms, their

host for survival and development. They include most of the infectious

agents but for some obscure reason it has been restricted to parasitic

species of protozoa, helminths and arthropods. Most of parasites are

associated with "epidemic diseases" both clinical and sub-clinical

forms and a very few of them are highly infectious. In other words they

do not usually results in epidemics. Their major impact is reflected in

production losses eroding economy of the animal husbandry

(Mamtani, 2005).

The essence of a parasitic relationship is its exploitative nature

as many parasites have a negative impact on host fitness. However, in

general, the level of virulence is determined by strategies that

maximize parasite transmission (Combes, 1997). Thus, if a host that

is rapidly killed by an infection that may not provide the parasite with

resources for long enough for it to complete the current phase of its

life-cycle or to maximize its chances of transmission to the next host.

Parasitic diseases occurring in laboratory rats are very few than

wild rats. If the modes of infection of parasitic diseases are studied,

then frequently an intermediate host is required for spreading of

parasites. Since the laboratory rats remain in better management, so

Introduction

6

the chances of picking parasitic agents are less, in close complex with

better shelter and selective feeds.

Parasitism may be due to ectoparasites or endoparasites.

Although the term ectoparasites can broadly include blood-sucking

arthropods such as mosquitoes (because they are dependent on a

blood meal from a human/animal host for their survival), this term is

generally used more narrowly to refer to organisms such as ticks,

fleas, lice and mites that attach or burrow into the skin and remain

there for relatively long periods of time (e.g., weeks to months).

Arthropods are important in causing diseases in their own right, but

are even more important as vectors, or transmitters, of many different

pathogens that in turn cause tremendous morbidity and mortality

from the diseases they cause.

Endoparasitism is mainly occurs by helminthic and protozoan

parasites. Focus of this study is on helminthic parasites, which can

harm their hosts in number of ways including ingestion of blood,

mechanical blockage, compete for nutrients, tissue damage, disrupt

mechanical functions, pressure atrophy and may produce cancer.

(Anon, 2007)

As stated above parasitic infections include three groups of

agents, i.e. helminthes, protozoan and arthropods. A list of

helminthes, protozoa and arthropods, generally found on rats

(including wild rats) are presented in Table: 1

Introduction

7

Table:1 Helminths, arthropods and protozoans of rats.

Helminths Arthropods Protozoans Echinostoma ilocanum, Polyplex spimulosa , Trypanosoma cruzii, Eschisostoma hortense, Hoplopleura pacifica, Trypanosoma lewisi, Hymenolepis nana, Hemobartonella muris, Tritrichomonas spp, Hymenolepis diminuta, Xenopsylla spp., Tetratrichomonas spp., Taenia taeniaformis, Leptopsylla spp., Pentatrichomas spp., Syphacia obvelata, Ormithonyssus baeti, Trichomitis spp., Syphacia muris, Laelaps echidnimus, Hexamastix spp., Hetrakis spp., Radfordia ensifera, Enteromonas spp., Strongyloides ratti, Demolex spp. Retortamonas spp., Nippostrongylus brasiliensis, Notoedres muris Chilomastia sp., Aspicularis tetraptera, Monocercomonides spp., Trichosomoides crassicauda, Octomitus spp., Trichinella spiralis, Giardia muris, Capillaria hepatica, Spironucleus muris, Gongylonema neoplastium, Hexamita muris, Angiostrongylus cantonensis, Toxoplasma gondii, Trichuris muris Sarcocystis muris,

Frenkelia spp., Encephalitozoon cuniculi, Eimeria miyairii, Eimeria nieschulzi, Eimeria separate, Pneumocystis carinis, Entamoeba muris, Balantidium coli Hepatozoon muris

Clinical examination of the animals has to be carried out

thoroughly and systematically. It is a complex procedure because the

parasite may be found almost in all parts of the body and as such it is

not possible to neglect any location of the body during examination.

Faeces are the most important clinical material for the diagnosis

of parasitic infections as eggs/cysts/oocysts of parasites inhabiting

the host’s body. Some parasitic forms seen in the faeces have

characteristic morphology that is diagnostic for a particular species of

parasites.

Introduction

8

Haematology is a strong conventional tool in diagnosis of

parasitic diseases. Anemia is a characteristic feature in a number of

blood sucking parasitic diseases. Some blood protozoans are

demonstrated in erythrocytes or leucocytes. Young migrating parasitic

larvae induce eosinophilia which is characteristic feature of parasitic

diseases (Jain, 1986).

The concentration of protein, metabolites and enzymes in body

fluids and tissues can serve as useful indications of the metabolite

state of host. These are helpful in clinical diagnosis of certain parasitic

diseases. Enzymatic analysis plays a prominent role in clinical

pathology for the determination of metabolic concentration and

enzymatic activities (Brar et al., 2000).

The serum enzymes used routinely in clinical diagnosis are

present in high concentration in the liver. Liver plays a vital role in the

metabolism of ingested dietary components. Insufficient liver

functions accompanied by clinical signs are evident in animals only

when more than 70-80% of liver tissue gets damaged (Klimes, 1997).

The possibility to monitor liver state using cytology and

histology of liver biopsy specimens seems very practical (Day, 2000).

The microscopic structure and functions of liver in small mammals

are to a greater extent similar to those in other vertebrates. ALT and

AST are localized mainly in the cytoplasm of hepatocytes (Sparkes and

Gruffydd-Jones, 1993). Final stages of liver diseases are due to the

Introduction

9

depletion of hepatocytes accompanied by levels of enzymes that are

within limits or only slightly elevated (Center 1993 and Sparkes and

Gruffydd-Jones, 1993).

Several types of reactive species are generated in body as a

result of metabolic reaction in form of free radicals or non radicals.

These species may be either oxygen or nitrogen derived and are called

peroxidants. They attack macromolecule including protein, DNA, lipid

etc. to counter their effect the body is endowed with another category

called as antioxidants. These are produced either endogenously or

received from exogenous sources and include enzyme SOD, catalase,

reduced glutathione, minerals like Se, Mn, Cu, Zn, vitamin A and

other include bilirubin and uric acid. In healthy body peroxidants and

antioxidants maintain a ratio and shift in this ratio towards

peroxidant and give rise to oxidative stress which may be mild to high.

Each biological system is armed with a sophisticated network

of antioxidants (scavengers) with multiple functions and overlapping

specific activities, which enables the cell or organism also to react, to

a certain limit, to increase in oxidant stress. The effect of antioxidants

can be located intra-and extra-cellularly. Antioxidants can be divided

in lipid and water soluble molecules and can be defined as substances

that: prevent the generation of toxic oxygen species, convert oxidants

into less toxic species, compartmentalise reactive species away from

Introduction

10

vital cellular structures and repair the molecular injury induced by

the toxic oxidant (Verhoef, 1991).

Scanning electron microscope (SEM) is a modern tool used to

find out three-dimensional appearance of organ in which minute

helminthic or other pathogens are easily detected. This technique

reveals alterations in surface structures of mucosa of tubular organs.

Therefore it has been frequently used for investigating pathological

effects of helminthes. Histopathological findings of Hymenolepis in

intestine are supported by similar observation of SEM features (Martin

and Holland, 1984). SEM of C. hepatica eggs revealed barrel shaped

appearance of eggs with presence of depressed and concave polar eggs

(Patel et al., 2004). Somvanshi, (2007) studied SEM features of T.

crassicauda infected urinary bladder in laboratory rats.

Systematic necropsy is open book of pathology. It is a message

of wisdom from dead to alive. This is a necessary procedure for study

of all parasitic diseases particularly in laboratory rats (Sinha, ).

Histopathological technique is a strong conventional tool which

is concerned with the demonstration of minute tissue structures in

diseases. This is very useful in study of pathogenesis and diagnosis of

parasitic diseases (Culling, 1974).

Hymenolepiasis caused by H. diminuta (rat tape worm) and H.

nana (dwarf tapeworm) is not uncommon in wild and laboratory rats.

Both tapeworms are known for their zoonotic significance. The varying

Introduction

11

prevalence of Hymenolepis spp. is reported in brown rats from

Belgium, UK and Iran. (Cotteleer et al., 1982; Webster and Macdonald

1995 and Sadjjadi and Massoud, 1999). Somvanshi (1997) reported

their very high prevalence in laboratory and wild rats from Uttar

Pradesh. Grossly on necropsy no appreciable lesions were seen with

exception of presence of H. diminuta. Histopathologically, pressure

atrophy of mucosal villi, mucin secretion, degeneration and

desquamation of lining epithelial cells and presence of sections of

tapeworms were common findings in small intestine (Somvanshi,

1997).

Rats can serve intermediate host for Taenia taeniformis, the

intestinal cat tape worm. From intestine after hatchering of eggs,

oncospheres migrate to the liver and form strobilocerci or Cysticercus

fasciolaris (Oldham, 1967). Its varying (moderate to high) prevalence

in wild and laboratory rats has been reported in UK (Lewis, 1968,

Webster and Macdonald, 1995) in India (Somvanshi et al., 1994;

Jithendran and Somvanshi, 1999 and Bhelonde and Ghosh, 2002). C.

fasciolaris infection is clinically asymptomatic and considered

harmless in rodents (Oldham, 1967). Histopathological features of C.

fasciolaris have been described earlier (Somvanshi et al., 1994 and

Shivakumar et al., 2003). Occasionally, concurrent lesions of C.

hepatica were also reported along with this intermediate stage

(Bhattacharya et al., 1998).

Introduction

12

High incidence of Capillaria hepatica has been frequently

recorded in wild rats in Thailand (Chaiyabutr, 1979), UK (Webster and

Macdonald, 1995) and in India (Somvanshi et al., 1995 and Patel et

al., 2004). Capillaria may be fatal in mice and probably in rats

(Meehan, 1984). Histopathologically, in severe cases characteristic

multi focal granulamatous inflammation with presence of typical eggs

of worms along with dystrophic calcification of egg shell is typical

findings (Brucinska and Nielsen, 1993 and Somvanshi, et al., 1995).

This parasite was recently recorded in liver and spleen in 102 black

rats of Kolkata (Patel et al., 2004).

Trichosmoides crassicauda is common is some rat colonies but

now it is not generally encountered in commercially raised animals. It

inhabits in urothelium and lumen of urinary bladder. Incidence of T.

crassicauda was reported very high in laboratory and wild rats from

different countries. (Bone and Harr, 1967; Gay et al., 1974; Zubaidy

and Majeed, 1981; George and Iyer, 1981 and Shingatgeri et al.,

2000). This parasite is known to cause focal hyperplastic changes in

mucosa of urinary bladder, dilation of renal pelvis, presence of

sections of T. crassicauda and their eggs (George and Iyer, 1981).

Diagnosis is made by demonstration of thick shelled, oval shaped,

bipolar plugged, larvaenated eggs in urine samples (Gounalan et al.,

1999).

Introduction

13

Two surveys of parasitic infections and infestations were

conducted in 18 and 67 rat colonies in USA and UK, respectively

(Lindsey et al., 1986 and Sparrow, 1976). It concealed that prevalence

of Entamoeba muris, Spironucleus muris, pin worms, Tritrichomonas

muris, Giardia muris and mites were fairly common. However,

Hymenolepis spp. and Eimeria spp. were not diagnosed. Authors

concluded that even after use of modern technology to prevent

infection by commercial and accredited breeders only very modest

success has been achieved in prevention of parasitic infections and

infestations of contemporary rats.

In principles of rodent disease prevention, modern terms used

in defining rodent microbial status vary greatly. Four terms (germfree,

gnotobiotic, defined flora and conventional), representing the extremes

of microbial status, have clear definitions that are generally accepted

and understood by scientists and technical personnel (NRC, 1991).

However, there is major confusion about the definition and use of

terms representing the middle ground of pathogen status. Pathogen

free, specific pathogen free, virus antibody free and clean conventional

terms are relative terms that require explicit definition every time they

are used. The definition should include the background of the rodent

sub-population in question (e.g., cesarean derived, isolator and barrier

maintained), details of current housing (e.g., isolator and barrier) and

data from laboratory tests for pathogens (the specific tests done, the

Introduction

14

number of tests, the frequency of testing and the results. (Lindsey et

al., 1986)

Traditionally, rodent diagnostic laboratories have tended to give

highest priority to the investigation of clinical illness and necropsy

evaluations of dead animals. That approach is no longer acceptable.

While those investigations are certainly necessary, the needs of

modern research and the principles of scientific method demand that

diagnostic laboratories give greater priority to disease prevention.

Most of the pathogen infections and pathogen-induced diseases of

laboratory rodents including those of parasitic are preventable.

Overview of literature revealed that haemato-biochemical tools

are infrequently used in study of helminthic infection in laboratory

and wild rats. Most of studies remained confined on epidemiology,

parasitology and pathology of helminthiasis. With the above

knowledge present study is proposed with following objectives:

Objectives:

1. To study prevalence of certain helminthic (Cysticercus

fasciolaris, Hymenolepiasis, Hepatic capillariasis and

Trichosomoidiasis), in wild and laboratory rats of different

ages and both sexes.

2. To determine clinical, haematological and biochemical

alterations of highly prevalent helminthic parasites in these

rodents.

Review of literature

15

REVIEW OF LITERATURE

arasites are those organisms that are metabolically and

physiologically dependent on other organisms, their host for

survival and development. Parasitic agents are commonly responsible

to show some clinical signs in rats. They include most of the

infectious agents but for some obscure reason it has been restricted to

parasitic species of protozoa, helminths and arthropods.

Historically, fear of the wild rat as a carrier of disease is

embedded within culture and immortalized within literature. It is

surprising therefore, to discover that the scientific literature relating

to rat-borne infection is scanty. Webster and Macdonald, (1995)

described results of survey of parasites of wild brown rats in 11 rural

farmsteads in UK. These rats contained several zoonotic and non-

P

Review of literature

16

zoonotic parasites. The study suggested that wild brown rats are

serving as vectors of disease and represent a serious risk to the health

of humans and domestic animals.

Nematododiasis

Syphacea muris: It is a common oxyurid (pin worm) of the caecum

and colon. Embryonated eggs are passed in the faeces or deposited on

the anus. Rats are infected by ingestion of eggs or by larval migration

into the colon from the anus. S. muris closely resembles with S.

obvelata, the mouse pin worm. Both species of Syphacia can patently

infect rats or mice, but preference seems to be shown for the

homologous host. Another common oxyurid of rats and mice is

Aspicularis tetraoptera, which is also found in the caecum and colon.

Aspicularis eggs are non-embryonated and are not deposited near the

anus.

Nippostrongylus brasiliensis: This strongyloid parasite is also not a

zoonotic of humans or domestic livestock, although, it is often used as

a model of human hookworm disease and the study of helminthiasis

immunity. N. brasiliensis causes inflammation of the rat's skin, lungs

(larval stages) and intestine (adult stages) and can be fatal in heavy

infections (Owen, 1992). Transmission of N. brasiliensis is mainly by

penetration of the larvae through the skin.

Capillaria hepatica: Since first report of Capillaria hepatica in 1850

in a rat liver, it had been recorded in rodents from time to time and

Review of literature

17

the prevalence rate varied from 0.7-85% (Palmer et al., 1998). These

liver worms favour rats as their host. Transmission to man from rat is

rare. Infection in domestic animals has only been recorded in dogs,

cats and pigs (Meehan, 1984). Hepatic capillariasis is an

anthropozoonoses caused by C. hepatica responsible for hepatic

damage in rodents and human beings (Soulsby, 1969). With only 11

human cases of capillariasis being recorded most of them were fatal

(Meehan, 1984).

C. hepatica have been frequently found in wild rats in Thailand

as high as 50% (Chaiyabutr, 1979), UK 23% (Webester and

Macdonald, 1995), India 27.3% (Somvanshi et al., 1995) and 88.33%

(Patel et al., 2004). Capillaria can be fatal in mice and probably also in

rats (Meehan, 1984).

From India, besides record on first human case (Parija and

Bhatnagar, 1990) the disease has been well documented in wild

rodents from time to time (Somvanshi et al., 1995; Chahota et al.,

1997; Bhattacharya et al., 1998 and Patel et al., 2004).

In India, the infection has been recorded in hill regions viz.,

Mukteswar, Kumaon, Uttranchal (Somvanshi et al., 1995); Palampur,

HP (Chahota et al., 1997) as well as from plains viz. (Raut et al., 2003)

and Kolkata, West Bengal (Patel et al., 2004). Since black and Norway

rats serve as the primary host of parasite (Singleton et al., 1991) this

may be possible reason for high rate of infection. C. hepatica

Review of literature

18

commonly invades liver and occasionally spleen as well (Bhattacharya

et al., 1999).

In animals and man clinicopathologically its infection causes

splenomegaly, pneumonitis, abdominal distension, fever, ascites,

epigastric pain and moderate to high lecocytosis during the course of

ailment.

Grossly, in this infection, whitish, irregular patches are seen on

the liver. Sub-capsular whitish granular deposits and sometimes-

irregular patchy cracks are also observed. Examination of smear

reveals numerous whitish, oval, thick-shelled, double layered eggs

with unsegmented yolk-measuring 48.43±7.28 (mean 37.50)

(Somvanshi et al., 1995). Scanning electron microscopy of C. hepatica

eggs revealed barrel shaped appearance of eggs with the presence of

depressed and concave polar plug (Patel et al., 2004).

Histopathologically, in severe cases there was characteristic multifocal

granulamatous inflammation, which contained eggs of worms along

with dystrophic calcification of the egg-shell (Brucinska and Nielsen,

1993; Somvanshi et al., 1995). Detailed pathology of this infection is

reported by a number of earlier workers (Brucinska et al., 1997; Choe

et al., 1993 and Patel et al., 2004). Further, besides high prevalence

(88.23%), Patel et al., (2004) also reported hepatic and splenic lesions

in 102 black rats of Kolkata. Number of lesions was positively

correlated with number of parasitic eggs per gram of liver and spleen

Review of literature

19

tissues. Histopathological changes in liver and spleen revealed loss of

architectural details with 1+ to 5+ lesions besides presence of varying

sizes of nodules.

Trichosomoides crassicauda (Bellingham, 1845). This is also known

as bladder threadworm. It is a nematode belonging to the family

Trichinellidae. First of all Bellingham in 1845 found it in bladder of

wild rats. Yokogawa (1920) recorded T. crassicauda in wild rats

Epimys norvegicus in Baltimore, USA. It caused swelling and

congestion of mucosa of urinary bladder resulting in catarrhal cystitis

depending on number of the parasites present.

It is a member of the order Enoplida and is therefore

phylogenetically related to Trichuris sp., Trichinella spiralis and

Calodium hepaticum. The small male worms, 1.3 to 3.5 mm long, live

permanently in the uterus or vagina of the adult female worm. The

latter is 9 to 10 mm long. The eggs are oval, brown and thick-shelled

with an operculum at each end, measuring 55 to 70 by 30 to 35 µm.

The life cycle of T. crassicauda is direct. Infection is by ingestion of

embryonated eggs passed in the urine. Infection most often occurs

shortly after birth, from adult females to offspring (Weisbroth and

Scher, 1971). Ingested eggs hatch in the stomach. Newly hatched

larvae penetrate the stomach wall to migrate via the blood stream to

the lungs, kidneys and ureters, by which, they arrive in the urinary

bladder where they mature into the adult worms. The prepatent

Review of literature

20

period is 50-60 days. The infections with T. crassicauda are

asymptomatic.

It is common in some rat colonies but is not generally

encountered in commercially raised animals. It inhabits the

transitional epithelium and lumen of the urinary tract, preferably

bladder. The anterior end of the adult female embeds within the

tunnels burrowed into the transitional epithelium of the urinary

bladder. Migratory larvae can incite eosinophilia and formation of

granulomatas, particularly in the lungs. Diagnosis depends on

demonstrations of characteristics eggs in urine or female worms in

bladder.

Incidence of T. crassicauda is reported in laboratory and wild

rats from different continents (Bone and Harr, 1967; Gray et al., 1974;

Nemeseri and Szakall, 1975; Erturk et al., 1978; Zubaidy and Majeed,

1981 and Ebisui et al., 1992) and India (Ramanujachari and Alwar,

1954; George and Iyer, 1981; Gounalan et al., 1999 and Shingatgeri et

al., 2000).

In India, T. crassicauda was recorded in bandicoots in Madras

(Ramanujachari and Alwar, 1954), laboratory rats in Izatnagar (George

and Iyer, 1981 and Gounalan et al., 1999) and Mumbai (Shingatgeri et

al., 2000). Gounalan et al. (1999) recorded its high incidence as 31.7%

while Shingatgeri et al. (2000) reported 16.68% prevalence in

experimental laboratory rats. Clinically, microscopic examination of

Review of literature

21

urine of positive cases showed numerous oval, brown eggs with thick

wall and bipolar plugs. They had coil larvae inside (George and Iyer,

1981 and Gounalan et al., 1999). Only a few female worms were

detected in urine. Males were parasitic in uterus of females. The

females were 1.8 to 2 cm long. Uterus was found heavily packed with

embryonated eggs (Gounalan et al., 1999).

This parasite is known to cause hyperplastic changes and

dilatation of renal pelvis (George and Iyer, 1981), pyelonephritis and

granulamatous lesions in lungs (Gray et al., 1974; Zubaidy and

Majeed, 1981), hyperplasia and papilloma in urinary bladder (Erturk

et al., 1978) and associated conditions, like focal hyperplasia of

urothelium and presence of sections of T. crassicauda and its eggs.

Gounalan et al. (1999) and Shingatgeri et al. (2000) described

pathology of this parasitic disease. In addition to bladder lesions,

occasionally the lining epithelium of pelvis of kidneys showed sections

of T. crassicauda along with hyperplasia and dilatations (Gounalan et

al., 1999). Shingatgeri et al. (2000) reported that besides other

microscopic lesions described, mild granular degeneration of tubular

epithelium in kidneys, degeneration of lining epithelium and oedema

of urinary bladder with occasional mononuclear cellular aggregation

below epithelium was observed. Cut sections were seen in lumen or

attached to the mucosa. Earlier, Smith (1946) studied precipitation

reaction of rat serum to eggs of T. crassicauda. The source of infection

Review of literature

22

could not be traced out but due to improved management conditions

and strict hygienic measures subsequent infection in batches of rats

were prevented.

Other nematodes: Certain other nematodes are common in wild rats

but rare in laboratory rats. Trichinella spiralis adults occur in the

intestine and larvae migrate extensively encysting in muscles.

Transmission is affected by ingestion of contaminated meat.

Angiostrongylus cartonensis, which requires the ingestion of molluscs

intermediate host, infects the brain and lungs. The intestinal

nematodes Heterakis spumosa, Strongyloides ratti and Trichuris muris

lack intermediate hosts, but rarely occur naturally in laboratory rats.

None is known to be highly pathogenic.

Cestodiasis

Cysticercus fasciolaris: Rats can serve as the intermediate host for

Taenia taeniformis (Batsch, 1786) (Syn. Hydatigera taeniformis and T.

crassicollis), the intestinal cat tapeworm. Following ingestion of ova by

rodent its eggs are hatched in the small intestine and oncospheres

migrate to the liver and form strobilocerci or bladder worm stage, C.

fasciolaris (Oldham, 1967). Host connective tissue capsules can give

rise to sarcomas. This parasite has been used as a model of parasite-

induced oncogenesis in the rat. C. fasciolaris is generally encountered

in laboratory rodents with contaminated food or bedding. Surveys of

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23

C. fasciolaris in wild rats in UK revealed its incidence ranged from11

to 41 percent (Lewis, 1968 and Webster and Macdonald, 1995). The

later authors observed its peak incidence in subadults. Comparatively

low prevalence (3.1 percent) of C. fasciolaris was reported in

Khuzestan province of South-West Iran (Sadjjadi and Massoud, 1999).

In India, relatively higher incidence of C. fasciolaris was recorded in

wild rats at Mukteswar, UT 48% (Somvanshi et al., 1994) in laboratory

rats, 38.9% at Himachal Pradesh (Jithendran and Somvanshi, 1999);

33.88% in laboratory rats at Anjora, Durg, Chattisgarh (Bhelonde and

Ghosh, 2002) and a case of very high infection in laboratory rats at

Thrissur, Kerala (Sivakumar et al., 2003).

C. fasciolaris infection is clinically asymptomatic and considered

harmless in rodents (Oldham, 1967). However, it could lead

misinterpretation of results for biological experiments (Tirina, 1953)

and host connective tissue capsule can give rise to sarcomas (Hanes,

1995). Jithendran and Somvanshi (1999) traced out source of

infection to contamination of Taenia taeniformis eggs in feed/bedding

materials by cats. Embryonated eggs, when ingested by rats, hatch in

small intestine and embryos pass to liver where they develop into

strobilocercus larva. The large scolex of C. fasciolaris was not

invaginated, with prominent suckers and had a conspicuous

segmented neck region at anterior end segmented and with strobila

ending in a small bladder at the posterior end resembling a small tape

Review of literature

24

worm (Jithendran and Somvanshi, 1999).

Grossly, rats of either sex and different age groups showed pea-

size, whitish, raised single to multiple parasitic cysts. Generally, livers

had 1 to 3 or 4 cysts (Somvanshi et al., 1994). Rarely more number of

cysts (up to 8) was seen. Sivakumar et al. (2003) reported that a

laboratory rat had numerous cysts. The surface of the liver was

diffusely studded with a total number of 206 cysts. The diameter of

cysts varied from 4 to 8 mm and their weight ranged from 75 to 120

mg. Microscopic examination of the cyst revealed a scolex followed by

segmented strobila measured 3-6 cm (Somvanshi et al., 1994) or 12-

20 cms (Bhelonde and Ghosh, 2002) or 12-19 cms (Jithendran and

Somvanshi, 1999) and 20 cm (Cheng, 1991). However, transformation

of the hepatic cells into larvae is about 30 days (Wardle and McLeod,

1952).

Somvanshi et al. (1994) described histopathology of this

parasitic condition. The cyst wall was made up of fibrous tissue lined

by flattened epithelial cells and inside luminas C. fasciolaris larva was

present. Generally, it caused pressure atrophy, engorged blood vessel,

and mononuclear cellular infiltration with presence of lymphocytes

and plasma cells. In some case thick zone or cuffing by mononuclear

cells was seen. Few larger cells with bluish granules (mast cells) were

also observed. Occasionally, concurrent lesions of C. hepatica were

also seen (Bhattacharya et al., 1998).

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25

According to Soulsby (1969) C. fasciolaris appears to be fairly

harmless in rats even when it occurs in large numbers. However,

Sivakumar et al. (2003) observed that highly infected C. fasciolaris

liver showed granulamatous changes and fibrous tissue proliferation

in the liver parenchyma. Section of liver revealed cysts of varying

stages of development. Some of cyst contained sections of larvae with

fibrous tissue encapsulation. The adjacent hepatic cells were

compressed. Neoplastic cells could not be observed. Presence of

infiltrating mononuclear inflammatory cells in portal areas was seen

along with moderate fibrous connective tissue proliferation.

Dissociation of the hepatic cells with inflammatory oedema was also

observed.

Hymenolepis diminuta and H. nana (von Siebold, 1852): Although

rats are the principal host of these two cestodes but both can infect

humans, particularly children. H. nana (the dwarf tape worm) has

been found in man, rats and mice while H. diminuta (the rat

tapeworm) occurs in rats, mice and has been recorded in man.

However, location of the adult as well as the large size of the eggs

compared to H. nana make differential diagnosis relatively simple.

There are, however, no reports of direct-cross contamination

between humans and rats (Owen, 1992). In humans, Hymenolepis

spp. can cause diarrhea and abdominal pain in heavy infections. In

rodents, infection can be associated with slow growth and pot-bellied

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26

syndrome (Owen, 1992). H. diminuta infection is usually obtained via

ingestion of infected intermediate host insects. H. nana is usually

contracted by eating contaminated faeces, although it's life-cycle can

be completed within one host. Diagnosis of Hymenolepiasis is made by

finding ova containing hexacanth embryos in the faeces of adults in

the lumen of the bowel.

The rat tapeworm, H. diminuta, has a two-host life cycle and is

transmitted, as an embryonated egg, from the definitive host to one of

a range of insects, including the beetle Tenebrio molitor (Arai, 1980).

Metacestodes mature within the haemocoel, where they remain as

long as the insect survives (Arai, 1980). The parasite’s life- cycle is

completed when the beetle is predated by one of several suitable

mammalian hosts, including Rattus rattus and R. norvegicus (Arai

1980 and Webster and Macdonald, 1995).

Macroscopic and microscopic tapeworm examinations were

suggestive of H. diminuta proglottids. The parasitological examination

of concentrated stool samples reveals spherical eggs, 70 m in

diameter, with a striated outer membrane and a thin inner membrane

and containing six central hooklets but no polar filaments, they were

identified as H. diminuta eggs and differentiated from H. nana eggs,

which have a similar appearance but are smaller and have two evident

polar thickenings, from each of which arise four to eight polar

filaments (Zechini, et. al., 2003).

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27

H. diminuta occurs in rats, mice and has been recorded in man.

It is 2-6 cm long or longer and the scolex has no hooks. Its

intermediate hosts are the larvae, nymphs and adults of various

species of moths, earwigs, cockroaches, fleas, beetles and millipedes

(Soulsby, 1969).

Fine structural studies (SEM) of the cestodes have demonstrated

that the surface cytoplasm is extended as microtriches, consisting of

cylindrical cytoplasmic bases capped by dense structures termed

“shafts”. Functionally, these microtriches have been suggested to:

(1) increase the surface area for absorption and secretion (2) aid in

maintaining parasitism in host and (3) agitate the microhabitat. Such

functions are in part supported by observations, which suggest that

the density of microtriches changes throughout the strobila (Ubelaker

et al., 1973).

Berger and Mettrick (1971) reported polymorphism in the

microtriches from H. diminuta, H. microstoma and H. nana by SEM

examination. They suggested that the microtriches played a role in

locomotion of these organisms within the host’s gut. Ubelaker et al.

(1973) described SEM of the surface of H. diminuta particularly the

scolex, which indicated that dense populations of microtriches occur

on the rostellum, suckers and scolex proper. Voge (1980) described

SEM of the surface topography of the eggshell of H. diminuta.

The prevalence of H. diminuta and H. nana is detected in brown

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28

rat as 7 and 0 per cent in Belgium (Cotteleer et al., 1982) and in UK

11 and 22 per cent (Webster and Macdonald, 1995), respectively. In

India, Somvanshi (1997) reported incidence of H. diminuta as 39.6 in

laboratory rats and 27% in wild rats. Earlier, Sriram et al. (1980)

reported spontaneous pathological conditions in 50 bandicoot rats

from Tirupati (AP) including haemorrhagic enteritis associated with H

diminuta and other species of worms. Similarly, Sadjjadi and Massoud

(1999) recorded high incidence of H. nana and H. diminuta as 31.3%

and 12.5%, respectively in wild rodents in Khuzestan Province, South-

West Iran.

Grossly, on necropsy no gross lesions of pathological

significance were observed in visceral organs. However, the presence

of H. diminuta was noted in intestines. The number of tapeworm

varied from 1-3 in individual rats, but occasionally the number was as

high as upto 5 to 7. Histopathologically, the presence of mucinous

content in intestinal lumen and rarely the embedding scolex of worm

in the mucosal layer of small intestine, atrophy of intestinal villi and

mononuclear cellular infiltration were major pathological findings

(Somvanshi, 1997). These findings supported the scanning electron

microscopic features reported earlier (Martin and Holland, 1984).

H. nana is a small tapeworm found in frequently contemporary

rodents including rats. It is mildly pathogenic but has importance as

zoonotic agent for humans. The adult is a slender, white worm, 25-40

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29

mm long and less than 1 mm wide. It has a scolex with 4 suckers and

an armed, refractable rostellum with a row of 20-27 hooks. Mature

proglottids are trapezoidal in shape. The eggs are oval, thin-shelled

and colourless and had prominent polar filaments. They contain an

oncosphere with 3 pairs of hooklets enclosed in an inner envelope.

They measure 36-56 x 44 x 62 µm and are unable to survive long

outside the host. The life cycle includes adult, egg with embryo or

oncosphere and larval (cercocystis) stage. The life cycle can be direct

(14-16 days) or indirect (20-30 days). Weaning and young adult

rodents are most frequently infected.

Most infections are subclinical. However, severe infections have

been reported to cause retarded growth and weight loss. The presence

of adult worms in the small intestine is usually associated with mild

enteritis. The finding of typical cercocystis with an armed rostellum in

the intestinal villi of a rodent is diagnostic of H. nana. Larval stages

occasionally reach mesenteric lymph nodes, liver or lungs where they

incite a granulamatous inflammatory response (Anon, 1991).

Trematoda infections

There are very few reports about the trematoda infection. The

trematoda, Echinostoma ilocanum and E. hoeertense are found in rats.

But as there is the need of an intermediate host for this infection, so

generally the trematoda infections are very rare in rats.

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30

Protozoal infections

Cryptosporidiosis: Cryptosporidium can cause enteritis and

enterocolitis in human and several other mammals and in a

particularly debilitating condition in immunosuppressed patients. It is

transmitted through contact with oocysts. C. parvum and C. muris

have been detected in wild brown rats in Japan. The high prevalence

of C. parvum in rats could represent a significant risk to human

health through the carriage and transmission of this zoonotic

protozoan.

Toxoplasmosis: Comparatively higher incidence (35%) of Toxoplasma

gondii in 7/7 rural farms in UK was observed (Jackson et al., 1986

and Webster and Macdonald, 1995). Toxoplasma has been detected in

wild rats in the UK, upto 10% which represent an important

intermediate host reservoir for T. gondii (Webster, 1994). These

authors detected it by serology, which was performed by indirect latex

agglutinations test (ILAT) and IgG ELISA. The titres of ≥ 1:6 were

considered positive. Parasitology was performed by microscopical

detection of brain cysts (Webster, 1994). In India, Anandan and

Deveda (1995) studied epidemiology of toxoplasmosis in Madras and

diagnosed T. gondii tissue cysts in 5/24 domestic, 8/20 wild rats and

3/10 pigeons indicating the significance of rodents and birds as

source of infection to rats. Somvanshi and Singh (1998) reported 3/54

cases (5.67%) of T. gondii infection in laboratory rats. Microscopically,

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31

rats showed presence of T. gondii cysts along with gliosis and

granuloma formation in brain. Each cyst contained numerous

bradyzoites. Cat faeces contaminated ration and water for these

laboratory rats were suspected for source of infection to the laboratory

rats. Webster et al. (1994) studied the effect of T. gondii on neophobic

behaviour (the avoidance of novel stimuli) in wild rats with natural

infection. The results show that neophobia was significantly

associated with positive Toxoplasma titres in most of groups. It was

concluded that differences between infected and uninfected wild rats

arises from pathological changes caused by Toxoplasma cysts in the

brains of infected rats. Such behavioral changes may be selectively

advantageous for the parasites, as Toxoplasma infected rats are more

susceptible to predation by domestic cats, the definite host of

Toxoplasma and as a side effect more susceptible to trapping and

poisoning during pest control programmes.

T. gondii in humans and other mammals, particularly sheep,

can cause spontaneous abortion or birth defects. Infection may also

reactivate in immunosuppressed patients and can produce

neurological disorders. Transmission occurs through environmental

(via ingestion of contaminated meat or vegetation) or congenital routes

(Beverely, 1976).

Recently, T. gondii was for the first time isolated from free range

desi (local or non descript) chickens from Izatnagar (UP) and Chennai

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32

(TN) region of India. At Izatnagar 46 (21.70 percent) desi chicken were

found seropositive by IFAT. The cats fed with hearts and brains of

these birds excreted oocysts of T. gondii in their faeces ranging from 8

× 106 to 1.5 × 106. The identity of the oocyts was established as

T. gondii by passing them twice successively in Swiss mice (Sreekumar

et al., 2001).

It is well known that marked difference exist between T. gondii

strains especially in their pathogenicity to experimental animals. With

application of recent molecular genetics tools 3 genetically distinct

c1onal lineage (I, ll and III), have been described. Lineage I was found

to comprise of all mouse virulent isolates while most human cases

were associated with type II lineage (Mondragon et al., 1998 and Owen

and Trees, 1999). Researchers believe that knowledge about the

genetic variability among T. gondii will be useful in formulating

measures of effective diagnosis, treatment and control of

toxoplasmosis in human and animals.

Trypanosoma lewisi: This trypanosome exhibits a considerable

degree of host specificity and does not infect humans or domestic

animals. High levels of infection can, however, cause anaemia in rats.

The rat-flea is the vector and infection is a result of contaminated by

T. lewisi.

Enteric flagellates: Tritrichomonas spp., Tetratrichomonas spp.,

Pentatrichomonas spp., Trichomitis spp., Hexamastin spp.,

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Enteromonas sp., Retortomonas sp., Chilomostin sp., Monocerco-

monoides spp. and Octomitus spp. are very common but non-

pathogenic. Giardia muris and Spironucleus (Hexamita) muris are also

common and considered potentially pathogenic, causing untrhiness,

infection, diarrhea, and intestinal lesions in severely affected animals.

Eimeria spp.: Members of the Eimeria genus are apparently the most

common protozoan parasites of mammals. They exhibit marked host

specificity and thus were not zoonosis of human or domestic livestock.

Eimeria usually live in the intestine or in associated parts of the host's

body where they cause considerable damage. Resistant oocysts are

secreted with the host's faeces and new infections are initiated when

oocysts are swallowed. E. seperata has been identified in rats in UK.

Babesia spp.: These are pathogens of humans and domestic animals,

which occur in RBCs and are transmitted by the tick Ixodes

trianguliceps. Zoonotic species of Babesia rodhaini and B. microti have

been reported in virtually all small rodents in the UK, with the apparent

exception of rats (Cox, 1980). The lack of detection of Babesia spp.

within the blood smears examined is consistent with this finding,

together with the lack of ticks carried by rats.

Sarcocystis: This parasite has wide host range, which includes

human and rodents. Infection is characterized by presence of thick

walled cysts in muscles. A total of 93 species of Sarcocystis has been

recorded from a range of rodents including rats in the UK (Levine and

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34

Tadros, 1980). Some Sarcocystis spp. are non-pathogenic but they

may cause serious symptoms in many mammals, which include

anorexia, fever, lameness, abortion and sometimes death (Schmidt

and Roberts, 1989). Little is known about the effects of Sarcocystis in

rodents. Sarcocystis has a potential significance in the cat-rat

predator-prey life cycle. The potential carrier status of this parasite in

wild rats cannot be ruled out without the aid of more complex

serological diagnostic tests.

Ectoparasites

Ectoparasites carried by rats do not directly cause illness in

humans or domestic animals, although as vectors they are

responsible for transmission of serious diseases of humans, such as

typhus and the plague (via Xenopsylla cheopis, rat-flea). Although, the

high prevalence and intensity of fleas, mites and lice detected could

provide a reservoir for such infections, ticks, on the other hand, can

be a vector for Borrelia and Babesia.

Fleas: With regard to species-specific parasites fleas (Nosopsyllus

fasciatus) are the intermediate host vector of the protozoan

Trypanosoma lewisi and the helminths Hymenolepis nana and H.

diminuta. Heavy infestations of lice (Polyplex spinulosa) in rats can

cause irritation and anaemia, and are also the most likely vector for

the rat rickettsian parasite Haemobartonella muris. Finally the mite

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35

(Notoedros muris) can spread to the rat legs and genitalia in severe

infections and can result in erosion of the pinna (Owen, 1992).

Mites: Mesostigmate mites can be encountered on occasion in

laboratory rats. They are rapid bloodsuckers and have a non-selective

host range. They are on the host only during feeding and spending

much of their life cycle in the environment. Diagnosis must be

attained by finding engorged mites on bedding, cages and crevices.

The most important mesostigmate is Omithonyssus bacoti (tropical rat

mite). Heavily infected colonies contain debilitated, anaemic rats with

reduced reproductive efficiency and occasional deaths.

Prostigmate mites have a more selective host range and spend

their life cycle in the fur (or follicles) of the host. Radfordia ensifera,

the rat fur mite, can be common in some rat colonies. This mite

produces few ill effects, but heavy infestation can reduce self-inflicted

trauma. Demodex sp. is also encountered, but its prevalence is

unknown, since it lives deep within their follicles and sebaceous

glands where it produces minimal lesions.

Haematology in Helminthic Infections

Complete blood count (CBC) is a set of test done on blood and

plasma for screening of blood abnormalities. It includes RBC, MCV,

MCH, MCHC, Hb, PCV, PP, TLC, DLC and Platelet count. When CBC

examination reveals decrease in Hb, PCV and RBCs it indicates

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36

anaemia. Haemorrhagic and haemolytic anaemia are seen in certain

haemoprotozoan and helminthic infections or ectoparasitism. In

hemorrhagic anaemia total plasma protein are also decreased while in

haemolytic anaemia haemoglobinurea, red discolouration of plasma,

increased MCHC, most often hyperbilirubinemia and increased

fragmentation of RBCs are seen (Brar et al., 2000).

Red blood cell (RBC): It contains haemoglobin in their cytoplasm and

Hb has great affinity for respiratory gases. Therefore, RBCs perform

the important functions like oxygen transport from lungs to tissue,

carbondioxide transport from tissues to lungs, maintenance of blood

pH as the Hb acts as buffer system and these maintain the viscosity of

blood (Verma et al., 2000).

Haemoglobin (Hb): The function of the Hb is to transport oxygen from

the lungs to the tissues and carbondioxide from tissues to lungs. Thus

Hb tests measure the oxygen carrying ability of erythrocytes.

Packed cell volume (PCV): The word hemocrit is derived from Greek

words Haima (blood) and Krinein (to seperate) i.e., to separate the

blood. Depending upon the specific gravity of the blood components,

high speed centrifugation separates blood into different components.

PCV is the most accurate, simple and inexpensive method for the

detection of degree of anemia.

Erythrocyte sedimentation rate (ESR): The distance to which

erythrocytes fall during a given period of time when a tube containing

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anticoagulated blood is placed in the upright position is known as

erythrocyte sedimentation rate. The test should be conducted

immediately or within one or two hours after collection of blood. ESR

is increased in general and local conditions like inflammation,

neoplasia, radiation injury, viral diseases etc. Occasionally, it

increases in certain chronic parasitic diseases.

Leukogram: It includes total and differential leucocyte count (TLC and

DLC) and morphological evaluation of blood leucocytes. Leucopenia

and leucocytosis occur in a number of bacterial or viral infections.

These changes may also occur in shock, toxicity or certain

physiological conditions. Eosinophilia is associated with certain

helminthic infections (Brar et al., 2000).

Blood glucose: The blood glucose levels reflect balance between blood

insulin and glucagon. With increase in insulin levels, glucose

utilization increases resulting in decreased blood glucose levels.

Glucagon prevents lowering of blood glucose too low. In diabetes

mellitus, insulin levels decreases resulting in decreased utilization of

glucose and hence increased blood glucose levels. Other agents, which

raise blood glucose levels, are thyroxine, growth hormones,

glucocorticoids and epinephrine. Insulin is the only hormone, which

reduces blood glucose levels.

Hypoglycemia can be observed in beta cell tumor,

hypothyroidism, starvation, hepatic diseases, adrenocortical

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38

insufficiency and ketosis (Brar et al., 2000).

Biochemical profile in Helminthic Infections

It is reviewed in great detail in context with clinical pathology

and parasitology (Brar et al., 2000).

Plasma Proteins (PP): These are mainly produced in liver and

immune system. Protein’s important functions are to maintain

osmotic pressure, to serve as enzymes for various reactions, to act as

buffer in acid-base balance, to provide structural matrix for cells and

tissues, to serve as hormones, to protect the body from pathogens and

to serve as carrier molecules in the plasma. Measurement of total

plasma proteins includes fibrinogen while total serum proteins lack

fibrinogen. Many factors influence the total protein concentration.

These include dehydration or over-hydration, altered protein

breakdown and distribution and altered synthesis from liver.

Albumin: It is important protein, which is 35-50% of the total proteins

in serum or plasma. It is major transport protein in blood, which

maintains osmotic pressure of plasma. Hepatocytes synthesize

albumin.

Globulins: There are three types of globulins- alpha, beta and gamma.

Alpha globulins include lipoproteins, antitrypsin, glycoprotein,

haptoglobin and ceruplasmin. Alpha globulins increase in acute

inflammatory processes, tissue destruction, renal failure, fever and

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neoplastic conditions. Beta globulins include lipoproteins,

complement, transferring and ferritin. Beta globulins are increased in

hepatic diseases and many other pathological conditions. Gamma

globulin are synthesized by plasma cells include IgG, IgD, IgE, IgA and

IgM immunoglobulins. Increase in gamma globulins reflects response

of reticulo-endothelial system to antigens. IgG consist of viral,

bacterial and toxins antibodies. IgA is secreted in genital, urinary,

respiratory and gastrointestinal tracts. IgE is involved in anaphylactic

and allergic reactions.

A:G ratio: It is estimated by dividing the albumin concentration by

the globulin concentration. The ratio is altered in liver diseases and

many types of infections and certain blood protozoan and trematodal

diseases.

Ceruplasmin: The plasma proteins ceruplasmin and transferrin may

also have a great impact in protection, both compounds being strong

inhibitors of degraded OH formation and lipid peroxidation.

Ceruplasmin, a glycoprotein with MW 134.000 is described as one of

the most potent serum antioxidants, binds copper thereby preventing

its participation in degradation of OH generation. It is also catalyses

by ferroxidase activity i.e. oxidation of Fe2+ to the less toxic Fe3+.

Transferrin (MW 80.000), the iron transport protein of plasma has two

high affinity iron binding sites. Once iron is bound to transferrin,

which 'locks in' the metal, it looses its catalytic activity. Thus

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ceruplasmin and transferrin in serum may act coordinately in the

extracellular defense against iron catalysed molecular disruption and

cellular and tissue injury (Verhoef, 1991).

Liver function tests

These may be classified as - (a) tests based upon hepatic

secretions and excretions. These include bile pigments and clearance

of foreign substances, (b) tests based upon serum enzyme activity and

(c) tests based upon the biochemical activity in liver. Liver function

tests include protein, carbohydrate and lipid metabolism tests. Here,

the clinical significance of enzymes estimated in present study will be

briefly reviewed (Brar et al., 2000).

Alanine transaminase (ALT): This enzyme is present in large

quantities in hepatocytes, cardiac and skeletal muscles, pancreas and

renal cells. Damage to these tissues results in higher ALT values.

Aspartate Transaminase (AST): It is present in hepatocytes but also

present in all the tissues of body. It is not organ specific enzyme.

Cardiac and skeletal muscles have high concentration of this enzyme

as such it helps in confirming muscular damage. Thus causes of

increased blood AST values are indicative of hepatic and muscle

damage.

Alkaline Phosphatase (ALP): There are multiple sources of ALP. In

older animals ALP mostly comes from hepato-biliary system. In certain

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41

species of large animals ALP is estimated in cholestasis. This enzyme

may increase in biliary obstruction or metabolic defect in hepatic cells.

Protein metabolism: These tests are based upon biochemical activity

of liver and include total protein, albumin, globulins, coagulative

factors and urea or urea nitrogen. Although liver plays vital role in

protein metabolism yet, total protein concentration is of little value in

assessment of liver functions. Disturbance in normal synthesis of

albumin by liver results in hypoalbuminemia. It is significant in

chronic liver disease. In liver diseases (cirrhosis and hepatitis) there

may be increase in alpha-2, beta and gamma globulins. Urea is

produced in liver from the catabolism of protein. Urea is not sensitive

indicator of hepatic damage. In advance liver damage blood urea

nitrogen may be low.

Lipid metabolism

Cholesterol: It is esterified in the liver and is used in the synthesis of

bile acids and steroid hormones. Esterification is depressed in liver

diseases. Ratio of esterified cholesterol to free cholesterol has been

reported abnormal in animals with liver diseases. Normal ratio ranges

between 0.64 and 0.80. Hypercholesterolemia is seen in animals with

bile duct occlusions. Hypocholesterolemia is seen in animals with

severe hepatic insufficiency and stage of cirrhosis and intestinal

lymphangiectasia.

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Kidney function test

The kidneys play a major role in regulating the internal

environment of the body. The functions of kidney include: (1) retention

of water and electrolytes in a negative body balance, (2) elimination of

water and electrolytes in a positive body balance, (3) excretion or

retention of hydrogen ions to maintain blood pH within permissible

limits, (4) retention of certain substances such as amino acids,

hormones, vitamins, plasma proteins and glucose, (5) removal of

certain end products such as urea, creatinine and allantoin, (6)

elimination of foreign toxic substances, (7) production of rennin and

prostaglandin and (8) help in activation of vitamin D.

Blood urea nitrogen (BUN): This test is used to evaluate the ability of

the kidneys to remove nitrogenous waste from the blood. But this test

is not sensitive as 75% of the kidneys should be nonfunctional for

BUN elevation.

Increased value of BUN is seen in pre-renal causes (shock,

congestive heart failure, dehydration and adrenocortical insufficiency),

renal causes (diseases causing damage to 75% nephrons) and post-

renal causes (obstruction in the urinary passage). BUN is decreased in

hepatic insufficiency, dietary protein restriction, late pregnancy and

overhydration and persistently elevated values are significant.

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Tissue Enzymology

Enzymes are the substances that induce chemical changes in

other substances but are not changed them selves. (Substrate +

Enzyme → Product + Enzyme).

Enzyme can be assayed by two methods. In the end point

method, the final colour intensity reflects the concentration of enzyme

in sample. In kinetic method, rate of product formation is proportional

to the amount of enzymes present. Temperature influence enzyme

activity greatly. Most assays are performed at fixed temperature,

which can either of 25, 30 and 37°C. Enzyme activity is measured in

International Unit (IU).

Liver cells are actively involved in synthesizing many proteins

including an extensive array of enzymes involved in metabolism. It

also play major role in detoxification of foreign and toxic compounds,

by their chemical transformation, which are generally catalysed by

enzymes (GSH, SDH etc.) and endogenous substrate (GSH) many of

which occur in liver. In relation to the important role that liver plays,

it is readily vulnerable to toxic agents, which may alter its

physiological function.

When the oxidant stress is overwhelming the endogenous

antioxidant protection can be provided by exogenous administration of

antioxidants, induction of antioxidants synthesis or a specific therapy,

e.g. iron chelation (Weiss, 1989).

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The determination of organ or tissue specific enzyme pattern

allows easy detection of the pathological condition in acute stage. The

enzyme superoxide dismutase (SOD) catalyses the dismutation of

superoxide anion to hydrogen peroxide.

Cells are armored with a very effective system to degrade O2H2.

First, by the action of catalase, located primarily in peroxisomes

(Halliwell, 1990). Hydrogen peroxide can also be eliminated by the

glutathione redox cycle, distributed throughout the cytosol.

Lipid peroxidase (LP): It refers to the oxidative degradation of lipids. It

is the process whereby free radicals "steal" electrons from the lipids in

cell membranes, resulting in cell damage. This process proceeds by a

free radical chain reaction mechanism. It most often affects

polyunsaturated fatty acids, because they contain multiple double

bonds in between which lie methylene -CH2- groups that possess

especially reactive hydrogens (Wikipedia, 2007). Jimoh et al. (2005)

studied the status of lipid peroxidation as assessed by

malondialdehyde levels in the rat tissues. Authors concluded that

increased tissue lipid peroxidation in rats kept on low protein diet was

indicative of oxidative stress and altered the activity of antioxidant

enzymes.

Catalase: It is the intracellular antioxidant defense mechanism to

cope up with increased oxidant stress. Increased catalase activity may

Review of literature

45

be due to damage of cell membrane by superoxide and hydrogen

peroxide, which had some latent period (Kellog and Fridovich, 1977).

Increase in catalase activity may be due to greater production of

reactive oxygen species due to lipid peroxidation.

Acetylcholinesterase (AChE): It is a membrane bound glycoprotein,

catalyses the hydrolysis of acetylcholine, a neuro-translation may be

due to action of parasite antigen/toxin or due modulation of

membrane fluidity and physio-chemical changes in biological

membranes, which alters the affinity of the enzymes for its substrate

(Mohini, 1991).

Superoxide dismutase (SOD): It is the primary intracellular

antioxidant defense mechanism to cope with increased oxidant stress

(Peng et al., 2000). Significant increase of SOD in different tissues

indicates that these organs were protected from oxidative damage.

Increased activity of SOD eliminates O2 and hydroperoxides that may

oxidize cellular substrates and prevent free radical chain reactions

(Halliwell and Cutteridge, 1985).

Reduced Glutathione (GSH): It is important in protecting cell

damage and important as a sink for free radicals and reactive oxygen

intermediates. Alterations in GSH levels affect these functions and

activities of GSH dependent enzymes. Decrease in GSH might be due

to either decreased synthesis or increased efflux from altered cell

membrane.

Review of literature

46

Sorbitol dehydrogenase (SDH): It is cellular and organ specific

enzymes of liver. Its level increases due to liver dysfunction.

Jimoh et al. (2005) studied the effect of low-protein diet on the

activity of antioxidant enzymes (Catalase and Superoxide dismutase)

and the status of lipid peroxidation as assessed by malondialdehyde

levels in the brain, liver, kidneys, lungs and heart of rats. Male

weaning rats were maintained on low protein diets (2% of protein in

diet instead of 25%) for a period of four weeks. Malondialdehyde

contents (levels) of tissues of animals fed low-protein diet was

significantly increased (P<0.05) when compared with the control. The

heart recorded the highest level of malondialdehyde when compared

with other tissues. The activities of superoxide dismutase and catalase

were significantly increased in the brain and liver of rats fed low

protein diet while a significant reduction was observed in the kidneys

and lungs. Authors concluded that the ingestion of low-protein diet

might led to increased tissue lipid peroxidation (oxidative stress) and

altered the activity of antioxidant enzymes.

Control of parasitism

Control of ectoparasites is gained by preventing the introduction

of infected animals (including wild rodents). Sanitation and treatment

of rats and in the cases of mesostigmatis and fleas, treatment of the

environment is necessary. Treatment seldom completely eliminates

Review of literature

47

infestation and can have an adverse effect on the usefulness of the

treated rats for research. Repopulation or caesarean re-derivation and

subsequent prevention by proper management are the best approach.

Laboratory rats are host to far fewer parasites than wild rats,

since husbandry practices interrupt complex life-cycles and caesarean

re-derivation has simply eliminated many agents altogether. Outline of

selective treatment regimens for control of these agents in infected

colonies is also best achieved by these means. One must beware that

these drugs may render the treated rat useless for experimental work.

Decision to treat rather than eliminate infected rodents must be made

with care, in concert with the needs of the scientific investigator.

Review of literature clearly indicated that scanty or no

information is available on haematology and biochemical aspects of

spontaneously occurring parasitic diseases in wild and laboratory

rats.

Materials and Methods

62

1 EU= mg of protein required to inhibit auto oxidation of pyragallol

by 50%.

Reduced Glutathione (GSH): It was assayed by spectrophotometric

method.

Reagents:

(a) 50 ml of TCA (50%) was added to 100 ml of distilled water.

(b) 12.1 g of Tris buffer was dissolved in 80 ml of distilled water

and the volume was made up to 100 ml.

(c) 0.099g of DTNB was dissolved in 25 ml of methanol.

Procedure: Aliquots of 1ml of 10 % homogenate in NSS were mixed with

4 ml of distilled water and 1 ml of 50% TCA. The tubes were shaken

intermediately for 10-15 minutes and centrifuged for 15 minutes at 3000

rpm. Two ml of clear supernatant was mixed with 4ml of tris buffer pH

8.9 and 0.1 ml DTNB was added to the sample.

Calculations: The calculation was done by using the molar extinction

coefficient at 412 nm.

OD Total volume 1 = -------------------------- × --------------------- × ------------------------------ Extinction coefficient Volume taken mg protein (use dilution factor).

Sorbitol dehydrogenase (SDH): It was estimated in tissue homogenates

by spectrophotometric assay as described by Ulrich and Hiby (1974) with

slight modifications.

Materials and Methods

64

round worms. After this samples were examined qualitatively by direct

smear method.

Direct smear method: A small quantity of faecal matter was taken with

a drop of water or saline on a clean slide. Attempt was made that faeces

should be free from debris and coarse fibres. The slide with a cover slip

placed over it was examined under microscope. This procedure was

repeated 3 times if the sample was found negative. Similarly urine

samples were collected and a drop was examined under microscope for

eggs, larvae or parasites.

Pathological studies

Detailed gross, SEM and histopathological studies of rat tissues

were conducted.

Necropsy examination: Dead/sacrificed animals were weighed prior to

necropsy examination. Brain and visceral organs of all humanely

sacrificed animals were systematically examined. Weight of carcass,

brain, lungs, heart, liver, spleen, kidneys and genitalia (testes/uterus)

and visible patho-anatomical lesions were recorded.

Relative Weight (RW): RW of different organs was derived using

following formula:

References

132

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