Post on 20-Aug-2020
transcript
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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33
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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37
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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39
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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40
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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42
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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43
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:
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132
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