GENERAL INTRODUCTION
Rodents pose a threaten towards crops in fields and
stores. In addition, they may attack people and their domestic
animals spreading many infectious diseases via their endo-
and ectoparasites. The control of Norway rat (Rattus
norvegicus Berk.), the most prevailing species lives close to
man, depends mainly on rodenticides such as metal
phosphides, fluoroacetamide, hypercalcemics and the
worldwide commonly used coumarin-derived anticoagulants.
Constituting over 40% of all mammal species, Rodents
are the largest and most successful group of mammals
worldwide. They have a high rate of reproduction and a good
ability to adapt to a wide variety of habitats (Parshad 1999)
Although rodents are often only associated with
infrastructural damages, crop attacking and eating or spoiling
of stored food and products, the veterinary and zoonotic risks
of rodents are frequently underestimated. Wild rodents can be
reservoirs and vectors of a number of agents that cause serious
diseases for human and domestic animal; there are more than
20 transmissible diseases that are known to be directly
transmitted by rodents to humans, by the assistance of blood-
sucking parasites like fleas, ticks and mites (Khatoon et al.
2004). Wild rodents act as definitive and/or intermediate hosts
of many parasites, which are common to domestic animals,
and humans. Some rodent parasites are epidemiologically
2
important as they are prevalent parasites of humans and their
domestic animals. The eggs of parasites are passed out in
rodent droppings in fields, grain stores and amongst foodstuffs
in houses, and are responsible for disease spread (Khatoon et
al. 2004). As rodents live in a close proximity with human and
their animals and expose to the blood-sucking arthropods, the
possibility for transmission of parasites increases.
Controlling of rodents and their endo- and ectoparasites
has been done mainly using anticoagulant rodenticides. The
repeated use and application of such anticoagulant
rodenticides for long periods may result in the rapid
development of resistance to these compounds in wild rodent
species.
Resistance to anticoagulants can develop in a population
after 5-10 years sustained use of anticoagulant rodenticides.
No enough data exist on the baseline susceptibility of rodent
populations in Egypt to anticoagulants or their changing
patterns of susceptibility in areas of sustained use. Monitoring
systems for rodent populations and changes to poisoning
methods will assist Egypt rodent control groups in avoiding
the resistance-induced control problems now seen outside
Egypt. Sustained control of rodents is likely to be
substantially dependent on toxicants, and anticoagulant
poisons in particular, for the foreseeable future .
3
The aim of this work
This study was carried out to determine what the major
Norway rat parasites are, and to monitor its resistance to
warfarin anticoagulant rodenticide at some governorates of
Egypt. Therefore, the scope of the present work was to cover
the following points:
1- To study the Norway rat species population structure at
four different governorates.
2- To identify Norway rat helminthic parasites and to
determine their incidence and distribution at four
different governorates.
3- To identify Norway rat ectoparasites, and to determine
their prevalence and general indices that is useful to
understand the role of arthropod vectors as well as
mammalian reservoirs in the maintenance of various
diseases in the study areas.
4- To monitor the Norway rat resistance to warfarin (First
generation anticoagulant rodenticide) at four different
governorates by using the conventional method, non-
choice feeding test.
5- To monitor the Norway rat resistance to anticoagulants
rodenticides (warfarin) at four different governorates
through VKORC1 analysis using Polymerase Chain
Reaction (PCR) technique.
4
5
Part I: Endo and Ectoparasites of Rattus
norvegicus
INTRODUCTION
Norway rat, Rattus norvegicus (Berk. 1769), is a
cosmopolitan rodent species with a wide distribution in urban
and suburban-rural habitats. It is commonly found living near
sources of food and water, such as garbage and drainage
ditches, streams or sewers. Because of its high ability to
harbor many zoonotic agents, wild Norway rats play a
significant role as definitive and/or intermediate hosts for
vector-borne animal and human diseases (Easterbrook et al.,
2007).
Zoonotic disease or zoonosis are the diseases that can be
transmitted from either wild or domesticated animals to
humans. About 60% of all infectious disease agents affecting
humans are zoonotic in origin and most of the zoonotic
reservoir species are rodents (Taylor et al., 2001). Viral,
bacterial and protozoan pathogens responsible for zoonotic
diseases are excreted by rodent hosts or are transferred via the
bite of a bloodsucking arthropod and then enter the human
body via inhalation, swallowing or skin punctures (Ostfeld
and Holt, 2004). The most famous zoonotic disease associated
with rodent presence is probably the infection of rodent fleas
with bubonic plague caused by Yersinia pestis bacterium,
resulting in many millions of casualties worldwide.
6
Endoparasites of rodents play an important role in the
zoonotic cycles of many diseases, such as, schistosomiasis and
angiostrongyliosis. Parasites in rats, particularly helminthes,
belong to the four major groups; Nematoda, Cestoda,
Trematoda and Acanthocephala. Cestode and nematode
parasites in rat have been reported from all parts of the world.
Vampirolepis nana and Hymenolepis diminuta are commonly
found in rats and mice and they are potentially transmissible
(Zoonosis) to man. The occurrence of H. diminuta and V. nana
in certain rodents is of interest since the possibility exists that
rats and mice may serve as reservoir hosts and help in
dissemination of these worms to domestic animals and man
causing zoonosis (Jawdat and Mahmoud, 1980).
Also, rodents are suitable for hospitality of some groups
of arthropods that are known as ectoparasites. They are well -
adapted for living on the external surface of rodents bodies
(permanent or temporary). Rats are known to harbor four
groups of arthropod ectoparasites: fleas, ticks, mites and lice
(Ansari, 1953; Abu-Madi, et al., 2005).
Ectoparasitic arthropods as vectors of zoonotic
pathogens have an important role in causing diseases such as
anaplasmosis, ehrlichiosis, rickettsiosis, plague, lyme
borreliosis, viral encephalitis, tularemia, CCHF, zoonotic
leishmaniasis, murine typhus, etc. They can also transmit
disease to human by: feces, urine, saliva, milk and blood.
7
Among the ectoparasites infesting rats, the best known
and most dangerous to man is the rat flea, Xenopsylla cheopis
(Rothschild). This flea is the vector of Yersinia pestis, the
causative agent of plague, and Rickettsia typhi, the causative
agent of murine typhus. Rickettsial agents, such as
Anaplasma, Bartonella, Coxiella, Ehrlichia, and Rickettsia,
have been detected by molecular tools from Egyptian
ectoparasites, such as lice, fleas, and ticks (Reeves et al.,
2006).
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9
REVIEW OF LITERATURE
Indo and ectoparasites associated with Rattus
norvegicus
Rodents (rats and mice) follow man wherever he goes
carrying with them many serious zoonotic diseases (El Shazly
et al., 1991). Historically, R. norvegicus has played a major
role in diseases transmission. This fact is still important in
today's world as it acts as a reservoir and transmits many
serious diseases of man and animals like plague,
hymenolepiasis, leishmaniasis, trichinosis, babesiosis and
toxoplasmosis. (Louisiana, 2000).
1. A brief about Rattus norvegicus (Berkenhout, 1769)
Rattus norvegicus is a cosmopolitan rat species that may
has many common names like brown rat, Norway rat, sewer
rat or burrowing rat. Its usual habitat is away from houses, in
drains or in burrows. It is fleshier than R. rattus with broad
head, blunt muzzle, small eyes, short ears which, when drawn
forward, do not touch each other. Fur is rough, grey brown
above and whitish grey on the abdomen. The tail is shorter
than the length of the body and head combined. The faecal
pellets are sausage shaped and usually occur in groups. It is a
commensal rat and not a true domestic rat (Nowak, 1999).
Thought to have originated in northern China, R.
norvegicus has now spread to all continents and is the
01
dominant rat in Europe and much of North America. It is a
common pest wherever humans live particularly in urban areas
and degraded environments (Banks et al., 2003).
Classification of Rattus norvegicus (according to
Nowak, 1999)
Kingdom: Animalia
Phylum: Chordata
Sabphylum: Vertebrata
Class: Mammalia Linnaeus, 1758
Subclass: Eutheria Parker and Haswell, 1897
Infraclass: Eutheria Gill, 1872
Order: Rodentia Bowdich, 1821
Suborder: Myomorpha Brandt, 1855
Family: Muridae Illiger, 1815
Subfamily: Murinae Illiger, 1815
Genus : Rattus Fischer, 1803
Rattus norvegicus (Berkenhour, 1767)
2. Endoparasites of rats
The ecology, in particularly the component community
structure, of helminth parasites in small rodent population has
been well documented in temperate regions of Europe (Abu-
Madi et al., 1998). In contrast, and despite the wealth of
information on species lists and taxonomy, there is little
00
comparable data for rodents living in tropics (Behnke et al.,
2000).
Rats and mice in Egypt are well-known to be the
definitive hosts (reservoir hosts) of several helminthes (Arafa,
1968; Monib, 1980; Wissa, 1980). It has been known from the
previous work that rats act as reservoir hosts for many
parasitic helminthes as Trematodes, Cestodes and Nematodes.
a. Trematode
The Echinoparyphium recurvatunz is a trematod parasite
of the small intestine especially the duodenum of the domestic
duck, and pigeons. This parasite has also been recorded in
rats, dogs, cats and man in Egypt, Malaysia and Indonesia
(Soulsby, 1982). E. recurvatunz parasite causes emaciation,
anemia and sometime weakness of the legs; this is explained
by the marked enteritis which observed on autopsy (Bowman,
1999).
Prohentistoman vivax is a well-known parasite of fish
eating birds and mammals like Rattus norvegicus. It has been
recorded to be infectious to Man (Chandler and Clork, 1961).
Schistosoma mansoni is a blood fluke occurs in the
mesenteric veins of man in Africa, South America and the
Middle East where humans are the most important definitive
host. However, a variety of animals have been found to be
02
naturally infected with S. inansoni since it has been recorded
in gerbils and Nile grass rats in Egypt, rodents in Southern
Africa and Zaire, Various species of rodents and wild
mammals and cattle in Brazil and Baboons, and rodents and
dogs in East Africa (Soulsby, 1982).
Mansour (1973) in Egypt, reported that 3 out of 22
Arvicanthis niloticus caught from Giza were naturally infected
with S. mansoni and S. haematobium. He added that on
experimental work this animal can serve as a natural reservoir
host. Also, El-Nahal et al., (1982) and Morsy et al., (1982)
reported the presence of the bilharzial worms or its antibodies
in some species of rodents. Likewise, Fedorko (1999) reported
S. japoniam in different rat species in Philippines in
association with different other endoparasites.
b. Cestode
Hymenolepis nana is essentially a parasite of rats
(rodents) but it also infects humans especially children. It is
distributed all over the world and it is the most common
cestode infecting humans in the tropics and subtropics, but
human infection is most prevalent in areas where temperature
is high and sanitary conditions are poor (Miyazaki, 1991;
Smyth, 1996; Roberts and Janovy, 2001).
H. nana has an alternate mode of infection consists of
internal autoinfection, where the egg release their hexacanth
03
embryo, which penetrate the villi continuing the infective
cycle without passage through the external environment. The
life span of adult worms is 4 to 6 weeks but internal
autoinfection allows the infection to persist for years. One
reason for the facultative nature of the life cycle and
autoinfection is that H. nana cysticercoids can develop at
higher temperatures than can those of other hymenolepidids
(Smyth, 1996; Andreassen, 1998).
Infection of H. nana to the rat occurs by taking in an
intermediate hosts or eggs. Transmission of eggs from one
patient to another is considered the main route for human
infection, but insect hosts could also serve as sources of
infection (Bowman, 1999).
As long as the number of worms of H. nana in the
intestine is small, no symptoms are noted. As the
autoinfection progress, damage to the intestinal mucosa would
result from the invasion of cysticorcoids causing cellular
infiltration consisted of polymorphnuclear leucocytes and
lymphocytes (Andreassen, 1998). Also attachment of scoleces
of adults to mucosa could cause changes in the form of
disintegration of the villi, ulcers and haemorrhage in some
parts of the mucosa and cellular infiltration of submucosa
leading to hypertrophy and thickening of submucosa in other
parts (Crompton, 1999).
04
In light infection of H. nana, usually no symptoms
appears and it can pass unnoticed. But in heavy infection
patients may complain of loss of appetite, nausea, vomiting,
abdominal pain and diarrhea may arise. Nervous symptoms
such as insomnia, vertigo, headache, dizziness, irritability and
epileptiform convulsion (Lioyd, 1998).
H. diminuta is a cosmopolitan worm that is primarily
parasite of rats (Rattus spp.). Beetles of the genera Tribolium
and Tenebrio serve as an intermediate host for H. diminuta.
When provided with a choice of rodent faeces with or without
the tapeworm's eggs, the beetles preferentially consume the
faeces containing the eggs (Pappas et al., 1995).
Human infection with rat tapeworm, H. diminuta, is
considered rare and usually accidental (Schantz, 1996;
Andreassen, 1998) and almost always occur in children (Tena
et al., 1998).
Rat nests almost always contain larvae and pupae of
fleas that frequently harbor cysticercoids in their
haemocoeles. As the cysticercoid persists also in adult fleas
parasitizing rats, infection may result when the fleas are taken
in by the animal. In other words, the life cycle of this cestode
can be maintained within a rat nest. Infected rats disseminate
eggs with the faeces, which may be ingested by insects that
would in turn serve as infectious sources for humans. Human
05
infection could occur by eating food containing infected
insects. Since rat fleas can parasitize humans, crushing such
fleas with finders may result in infection via fingertips
contaminated with cysticercoid (Miyazaki, 1991).
H. diminuta parasites in the upper middle part of the
small intestine. Autoinfection does not occur; as a result, the
number of worms inhabiting a human host is accordingly
small. Symptoms are therefore slight, if there; only such light
ones as reduced appetite, abdominal pain and diarrhea may
occasionally be encountered (Lioyd, 1998).
Cysticercus fasciolaris is the heabatic larval stage of
tapeworm Taenia taeniaeformis. It infects rabbits, black rat,
cotton rat and other wild rodents. The adult tapeworm is
usually found in small intestine of cats (rat eater) and wild
carnivorous and may be found accidentally in dogs. The
hepatic larval stage and the adult stage occur worldwide
(Wanas et al., 1993). Strobilocercus is embedded in the liver
parenchyma in a pea-sized nodule (Esch and Self; 1995). The
main interest behind this species lies in its larval stage which
does not form a cysticercus but a strobilocercus that may
induce sarcoma in host liver.
Reaching the liver in the intermediate host, the
strobilocercus develops and rapidly becomes infective after
30 days (Smyth, 1996). The strobilocercus, in the final host,
has only the scolex which develops in cat small intestine into
06
an adult tapeworm of about 60 cm long (Lioyd, 1998)
c. Nematode
Syphacia species, the natural oxyurid nematodes of rats,
are considered zoonotic parasites. Human infection is resulted
from accidental contamination of human food or drink with
droppings of infected rodents (Wescott, 1992). This occurs in
localities with highly infected rodent population and poor
sanitation (El-Shazly et al., 1994).
Inhabiting the caeca of domistic rats and mice, the
oxyurid Syphacia spp. is a common parasitic nematode with a
direct life cycle (Tattersall et al., 1994).
Aspiculuris tetraptera is a pinworm of rats and mice,
occurs in the large intestine. The cuticle is transversely
striated with broad cervical alae terminating abruptly at the
level of oesophageal bulb. when a narrow lateral flanges run
to the posterior extremity. The mouth is with three lips.
Oesophagus is club-shaped followed by a well-developed oval
bulb. The life cycle of A. tetraptera is direct. Eggs pass in
faeces and the infective stage is reached in about six days.
Infection is by ingestion of eggs and the prepatent period is
about 23 days. Negligible pathogenicity is associated with the
infection; it is not a zoonotic infection (Arafa, 1968).
07
Protospirura marsupialis is a spiruroid nematode of
rodents, inhabiting the stomach. It is large and
semitransparent. The body is attenuated anteriorly, without
lateral flanges, the mouth has two large trilobed lateral l ips,
each lobe bearing a papilla externally at the base, and three
teeth on its inner surface. There are cervical papillae anterior
to the nerve ring. Buccal capsule is long, cylindrical with very
long oesophagus which is divided into two parts. Females
measure 67.5-79.0 mm in length and 1.45-1.60 mm in breadth
with very short conical tail. Males are shorter than females
measuring 40-50 mm in length. Its posterior extremity is
spiral, with caudal alae well developed. Male has two unequal
spicules. It is not recorded to be of zoonotic importance
(Yamagoti, 1962; Wanas et al., 1993).
3. Ectoparasites of rats
The intimate association of commensal rodents with
man, and the role of ectoparasites in transmission of
pathogens to man led several workers to pay attentions to
study their host parasite fauna (Allam et al., 2002).
In Egypt, many scientists gave an account of the
parasite species of Acari found on rodents. Hoogstraal and
Traub (1956) studied the fleas of Egypt and Johnson (1960)
studied the sucking lice. Also, Abdou (1981) made a study of
08
the commensal and wild rodents and their ectoparasites in
Assiut area
Rifaat et al., (1969) studied the relative incidence and
distribution of the medically important ectoparasites in the
various geographical zones of the country. The rodents and
fleas were studied at Ismailia Governorate (Morsy et al.,
1982), Suez Governorate (Morsy et al., 1986), Sharkia
Governorate (Zeese et al., 1990) and South Sinai Governorate
(Shoukry et al., 1993).
Rodents reserve and transmit many serious diseases of
man and animals as plague, hymenolepiasis, leishmaniasis,
trichinosis, babesiosis and toxoplasmosis. Man is infected
with these diseases by contagion as well as by the arthropod-
ectoparasites of rodents (Hilton. 1998). Ectoparasites could be
from-rat-to-rat or from-rat-to-man vectors. Man becomes an
incidental host of disease when bitten by ectoparasites or
when ectoparasite faeces contaminate the bite wound
(Shoukry et al.. 1991).
Ectoparasites obtain some of their requirements, like
oxygen, from the physical environment, and to some extent,
are influenced by factors that affect their non-parasitic
associates. They are also dependent on their hosts for
nutritional requirements and for developmental and maturation
stimuli (Soliman et al., 2001a).
09
a. Fleas
In general, fleas are not very host specific, although
they have preferred hosts. Most can transfer from one of their
hosts to another or to a host of a different species. Their
common names (for example, rat flea or human flea) refer
only to the preferred host and do not imply that they attack the
host exclusively. At least 19 different species have been
recorded as biting humans (Harwood and James, 1997).
Fleas could transmit many zoonotic diseases from rat to
man. Plague (black death) is essentially a disease of rodents
from which it is contracted by humans through the bites of
fleas, particularly Xenopsylla cheopis (Ryckman, 1971). It is
caused by a bacterium, Yersinia pestis. The bacterium releases
two potent toxins that have identical serious effects. Some
animals such as rats and mice, are more sensitive to the toxins
than others (rabbits and dogs) (Lewis, 1993).
Yersinia pestis is widely distributed in rodents and
occurs across broad areas of every continent. The bacteria are
consumed by a flea along with its blood meal, and the
organisms multiply in the flea's gut, often to the extent that
passage of food through the proventricular teeth is blocked
(Hilton, 1998). When the flea next feeds, the new blood meal
cannot pass the obstruction, but is contaminated by the
bacteria and then regurgitated back into the bite wound. The
21
propensity of a particular flea species to have its gut blocked
by growth of Yersinia pestis is an important determinant of its
efficacy as a vector. Xenopsylla cheopis is a good vector
because it becomes blocked easily and feeds readily both on
infected rodents and humans (Roberts and Janovy, 2001).
The disease may exist in rodent populations in acute,
subacute, and chronic forms. Epidemics among humans
usually closely follow epizootics, with high mortality among
rats. When the rat dies, its fleas depart and seek greener
pastures (Allam et al., 2002).
The second important disease could transmitted from
rats to humans is murine typhus or flea-borne typhus. It is
caused by Rickettsia nzooseri or R. typhi and occurs in warmer
climate throughout the world. Murine typhus can infect a wide
range of small mammals but the most important reservoir is
Rattus norvegicus in which it causes slight disease symptoms.
Murine typhus can be transmitted from one rat to others by
Xenopsylla cheopis, Nosopsyllus fasciatus, Leptopsyllus
semis, Polyplax spinulosa (the rat louse); and the tropical rat
mite Ornithonyssus bacoti. In humans the disease is a rather
mild. But it may involves febrile illness of about 14 days, with
chills, severe headache, body pains, and rash. X. cheopis is
considered the primary vector transmitting the disease to
humans either through the bite or through contamination of
skin abrasions with flea faeces by scratching. Ingestion of
20
infected fleas and their faeces also can produce infection in
rats. The rickettsias proliferate in the midgut cells of the flea
but do not kill it. Rupture of the midgut cells releases the
organisms into the gut of the flea. (Farhang and Traub, 1985).
The incidence of murine typhus had been dropped
dramatically after the institution of a rat control program, use
of DDT, and increasing use of antibiotics (Roberts and
Janovy, 2001).
Lastly, Nosopsyllus fasciatus is a vector for
Trypanosoma Lewisi of rats. Ctenocephalides Canis, C. felis
and Pulex irritans serve as intermediate hosts of Dipylidium
caninum, a common tapeworm of cats and dogs. Nosopyllus
fasciatus and Xenopsylla cheopis can serve as vectors for the
rat tapeworm, Hymenolepis diminuta. The mouse tapeworm
Vampirolepis nana can develop in X. cheopis, C. felis, and P.
irritans; all of these fleas acquire the tapeworm as larvae
when they consume the eggs which pass in the faeces of the
vertebrate host, retaining the cysticercoid in their hemocoel
through metamorphosis to the adult. All these three species
can be transmitted to humans if the person inadvertently
ingests an infected flea (Robert and Janovy, 2001).
22
b. Lice
Lice are permanent ectoparasites on mammals including
rats and humans. Unlike fleas, lice are species-specific;
although rats may be infected with lice, those lice will not
cross over from one species of animal to another and if so; it
won't take long for it to realize this animal is not its food
source and will jumps onto a rat again (McArthur, 1999).
Hoplopleuro pacifica is the tropical rat louse
occurring on various species of rats throughout the world, it
is slender forms 1-2 mm in length with large paratergal plates
(Soulsby, 1982).
Polyplux spinulosa (the rat louse) is an anopluran
louse (Sucking louse) of rat causing restlessness, pruritus,
anaemia and debilitation in rats. Because lice are species-
specific, transmission to other animals or humans is not a
concern. P. spinulosa is a vector responsible for spread of
Haemobartonella muris (rickettsia, blood parasite) and
Rickettsia typhi between rats which may be passed to humans
via rat fleas (Hendrix, 1998; McArthur. 1999).
c. Mites
Mites are very important parasite on or in the skin,
the respiratory system or other organs of mammalian host.
Although some mites are not actually parasites of vertebrates,
they stimulate allergic reactions when they or their remains
23
come into contact with a susceptible individual (Bakr et al.,
1995).
Mites are temporally blood-sucking ectoparasites of
mammals (including rodents and human). Rat mites
frequently attack people living in rodent-infested-buildings.
Mites' bite may produce irritation, and sometimes painful
allergic dermatitis or mite respiratory allergy particulary in
children. This occurs especially in the absence of their natural
hosts. Rat mites are associated with groceries and warehouses
(Cook, 1997).
Animal in an environment infested with mites may be
anemic and exhibit a marked reproductive decline. The mite
can transmit rickettsial organisms in humans. Ornithonyssus
bacoti could transmit Yersinia pestis (the cause of plague),
Rickettsia typhi (the cause of murine typhus) and Coxrella
buinetii (the cause of Q fever). 0. bacoti is the intermediate
host of the filarial nematode of rodents Litomosoides
Allodermanyssus sanguineus may transmit Rickettsia akari
the cause of rickettsial pox of man (Hendrix, 1998).
Mites are transmitted to man by direct contact with an
infected animal, but also may arrive in contaminated bedding
or wood products (McArthur, 1999).
Rats may be infected with Radforia ensifera, the fur
mite of rats, which is not bloodsucker and is often endemic to
rats. Transmission between rats usually occurs by direct
contact. This species of mite is not known to infect humans
24
and it does not cause problems unless the infestation is heavy
or the rat is ill with another disease.
Burrowing mite of rats Notaedres inuitis is among the
ear mange mites. A skin scraping and a microscope are
needed to see these mites. They attack the ear pinnae, tail,
nose, and extremities. These mites are spread by direct
contact. Lesions caused by it are reddened crusty and itchy.
They may also infect other rodents, but are not known to
infect humans.
25
MATERIALS AND METHODS
1. Study Locations
Commensal Norway rats (Rattus norvegicus) were
collected from four governorates; Beni Suef (Wish-El-Bab
Village), Giza (El-Mansouria village), Qaliubiya (Tookh and
Beltan villages) and Behaira (El-Tayria village).
2. Collection and manipulation of rats
The study was carried out during the period from July
2012 to December 2013. Live Rats were captured using wire-
box traps of the usual spring-door type. Traps were distributed
in the evening at houses, poultry farms and drainage then
collected next morning. Bait materials were consisting of
tomato slices, fried fish or fried potato. Positive traps
provided with water using wet cotton and put in cloth bags
then transferred to laboratory for the study. The collected rats
were identified using the keys given by Arafa (1968) and
Osborn and Helmy (1980). Sex was determined by examining
the external genitalia of males and females and weight was
registered then a reference number was given to each
individual.
3. Examination of rats for endoparasites
a. Examination of intestinal parasites
The abdomen and chest of each rat were split opened
after killing. The lumen tract was then removed in one piece
26
and left in a separate petri-dish for some time in saline
solution to insure complete relaxation and easy removing of
the worm contents. Then it was slit opened in warm normal
saline. Freed helminthes if visible to the naked eyes were
picked out using a blunt forceps and transferred to petri-dishes
containing warm saline solution. The other contents were
evacuated into separate labeled jars full of water and were
taken thoroughly and left to sediment. The supernatant fluid
was decanted and the process of washing was repeated several
times with distilled water. Finally the sediment was placed in
a large petri-dish and examined for minute worms under a
stereomicroscope. Such worms were picked off using either a
wide mouthed pipette or a camel's hair-brush.
The mucous membrane of the stomach, on the other
hand, was examined under a dissecting stereomicroscope
utilizing a strong source of light of adherent worms and if
present could be picked out in warm normal saline.
Besides, careful searching for the smaller worms both in
the intestinal contents and scrapings of the mucosa was
carried out to extract the worms present inside.
Helminthes of large sized were easily spotted by the
naked eyes or by the aid of a hand lens. However, it should be
stated that some parasitic worms might have been missed due
to their minute size especially if they were scanty. In order to
27
overcome this difficulty, the mucosal surface of the
gastrointestinal tract was rubbed or lightly scraped to assure
complete transfer of worms to the container.
Worms were stirred vigorously for few minutes to allow
thorough relaxation, after which they were preserved in well
stoppered vials containing sufficient amount of glycerin-
alcohol (consists of 95 parts 70% alcohol and 5 parts glycerin)
and a label carrying the date, location and corresponding
serial number of each animal.
In the meantime the split opened abdomen and chest
were inspected for extra intestinal helminthes.
b. Examination of non-intestinal endoparasites
The liver, kidney, heart, lungs and reproductive organs
were inspected for cysts or worms which were then counted.
Particularly, liver was examined for cysts (e.g., Cysticercus
fascialaris) which dissected out and notched in warm normal
saline to free their worm contents.
c. Preparation of adult helminthes for examination
(according to Gardner et al., 1988)
1. Washing of adult helminthes
Before examining the worms, they were washed several
times in warm normal saline solution to separate them from
mucous and debris and to inspect their movement as
28
monitored while still living. Specimens preserved in glycerin-
alcohol were brought down to water (in descending grades of
alcohol 50% then 30% for 15 minutes each then to distilled
water several changes prior to staining).
2. Relaxation
By lifting the specimens in refrigerator for 2 h.
3. Fixation
Cestodes were roughly measured before being divided
into small pieces; head region, mature segments and gravid
segments and then gently compressed between two slides, and
fixed in 1% formalin for 24 h.
Nematodes were dropped in 70% hot alcohol (60°C)
then preserved in 70% alcohol containing 5% glycerine. For
studying the morphological feature of nematodes, they were
first cleared in lactophenol for 24h which was prepared from:
10 gm phenol, 10.6 ml glycerol, 8.2 ml lactic acid and 10 ml
distilled water. The worms were then mounted on glass slid
dipping in Canada balsam and left in an oven at 38°C to dry.
4. Staining
Cestodes were stained with acetic-acid alum carmine
formulated from: 20 gm. carmine, 25 ml acetic acid, 6 gm.
potassium alum and 100 ml distilled water.
29
The dye was boiled for an hour then cooled and the acid
was then added and left for ten days for maturation.
Thereafter, the solution was filtered. Working solution was 1
part of stock solution and 99 parts distilled water.
Half an hour was found sufficient to stain the
trematodes and small scolices of cestodes, while mature and
gravid segments were left for 2 hours. Helminthes were then
washed with water several times to remove the excess of the
stain.
5. Mounting
After staining, the specimens were dehydrated in
ascending grades of alcohol (30-50) % for half an hour each.
Destaining and differentiation of the over-stained specimens
were done in 1% acid alcohol (1 part of hydrochloric acid in
99 parts of 70% alcohol). The process was microscopically
checked until the specimens became well differentiated. The
specimens were then washed several times in 50% alcohol to
remove the residual hydrochloric acid. Specimens were then
dehydrated by passing through ascending grades of alcohol
70%, 95% and absolute alcohol half an hour each. Stained
specimens were then cleared in clove oil followed by two
washed of xylene. They were mounted in Canada balsam and
left in an oven at 38°C to dry for few weeks.
The detected helminth parasites were identified
according to Monib (1980) and El-Azzazy (1981).
31
4. Examination of rats for ectoparasites
a. Ectoparasites collecting
Rats skin with terminal parts of the four limps, tail and
head were put in modified tullgren funnel.
The ectoparasites received in petri dish filled with 70%
alcohol, were picked up with a moistened camel's hair brush
with the aid of a strong source of light. Then, the ectoparasites
were dropped in separate vials containing 70% alcohol and a
label comprising both the date, location and the corresponding
serial code number of each animal.
b. Ectoparasites' preparation, mounting and
identification
Arthropod ectoparasites preserved in 70% alcohol were
brought down to water in descending grades of alcohol 50-
30% 15 minutes each.
Fleas and lice were then removed to 10% potassium
hydroxide or lactophenol after puncturing the specimens on
the ventral side, and then left overnight until soft parts were
dissolve. The material was washed thoroughly in distilled
water slightly acidified with 10 drops of acetic acid to remove
the alkali and then treated with ascending grades of alcohol -
50%, 70%, 90% and 95% - 20 minutes each.
30
The individuals were then cleared in clove oil for 10
minutes. Mounting was performed in Canada balsam then left
to dry in oven at 38°C.
Mites, on the other hand, were mounted from 70%
alcohol after cleaning in water into Hoyer's medium.
Fleas species recorded were identified according to the
key given by Soulsby (1982), lice were identified according to
the key given by Johnson (1960) and mites were identified
according to Krantz (1978).
32
33
RESULTS AND DISCUSSIONS
1. Rattus norvegicus investigations
Rattus norvegicus was collected from four governorates:
Giza, Beheira, Beni Suef and Qaliubiya . the structure of its
population was studied, the whole number of Rattus
norvegicus live trapped was 83; 34 from Giza, 24 from
Beheira, 10 from Beni Suef and 15 from Qaliubiya .
Table 1. Rattus norvegicus population structure
Gov. No. Males' No. Females' No.
Mature Immature Total Mature Immature Total
Giza 34 12 7 19 10 5 15
Beheira 24 9 5 14 6 4 10
Bani-Suef 10 4 1 5 3 2 5
Qaliubiya 15 6 4 10 3 2 5
Total 83 31 17 48 22 13 35
Based on sex, the Norway rat population was consisted
of 48 male individuals and 35 female individuals. The male to
female ratio (sex ratio) was 1.37:1. The maturity status was
obtained, therefore, the population was divided into mature
individuals (53) and immature individuals (30), table (1).
This result showed that males' number is bigger than
females', and the reason behind may be that females stay in
borrows to lactate and to take care of offspring or to avoid the
harsh weather conditions during pregnancy and after giving
birth. While, on the other hand, males don‘t have all these
34
constrains; they usually explore and roam more than females.
This result is in accordance with that obtained by El-Bahrawy
and Al-Dakhil (1993) but it is in discrepancy with that of Soliman
et al. (2001b).
2. Parasites of R. norvegicus recorded
Rodents play an important role as hosts of parasites and
reservoirs of many zoonotic diseases. A total of twelve species
of parasites were found of which 11 were zoonotic including,
two Cestodes (Hymenolepis diminuta and Cysticercus
fasciolaris), three fleas (Xenopsylla cheopis, Echidnophaga
gallinacea and Ctenocephalides felis), two sucking lice (Hoplopleura
oenomydis and Polyplax spinulosa) and four mites (Ornithonussus
bacoti, Lealaps nuttalli, Liponyssoides sanguineus and Radfordia
ensifera).
a. Endoparasites
Indoarasites of rats, particularly helminthes, are
belonging to the four major groups; Nematoda, Cestoda,
Trematoda and Acanthocephala; Cestode and nematode
parasites in rat have been reported from all parts of the world.
In this study, we have just recorded two cestodes:
Hymenolepis diminuta and Cysticercus fasciolaris, which are
commonly found in rats and mice and they are potentially
transmissible (Zoonosis) to man and one non-zoonosis
nematode, Spirura talpae.
35
Helmenthic parasites pose a major part of this study, as
65 individuals out of 83 rats were infected with one or more
helminthic parasites with an infection rate of 78.31 %. This
rate of infection among small mammals is slightly higher than
that obtained by Arafa (1968) and Monib (1980). A reasonable
explanations for that could be the contact increase between
man and rats in recent years or the environmental
contamination increase or even the climatic changes that
favour parasitic transmission. These findings are in harmony
with those showing that wild small rodents rarely remain
uninfected (Behnke et al., 2001). Also, the high prevalence of
infection with helminthic parasites in the Norway rats might
be attributed to its high reproductive activity, high population
density and its omnivorous way of nutrition (Hrgović et al.,
1991).
1. Types of Infection of endoparasites
The type of infection of helminthic parasites varies
among individuals. Some individuals were infected with only
one helminthic parasite, 27 individuals (32.5%) and some
were double infected, 32 individuals (38.5%) while triple
infection was recorded in just 6 individuals (7.2%), table (2).
In a similar study of endoparasites of Norway rat, Rezan et al.
(2012) stated that Single parasitic infection was the highest
(52%), followed by double infection, 16%, and two cases of
triple infection (8%). No more than four helminthic species
36
were found in one host (Kataranovski, 2011).
Table 2. Types of Infection of endoparasites
Gov. Single (%) Double (%) Triple (%)
Giza 11 13 2
Beheira 8 9 2
Bani-Suef 3 5 1
Qaliubiya 5 5 1
Total 27 (32.5%) 32 (38.5%) 6 (7.2%)
2. Endoparasite species recorded
Three different species of helminthic parasites were
recorded in Rattus norvegicus examined from different
locations comprising tow cestodes , Hyminolipis diminuta and
Cysticercus faciularis, and one nematode Spirura talpae. No
new species were recorded in the given areas of this study.
a. Cysticercus fasciolaris:
Cysticercus fasciolaris is a larval and cystic stage of
Taenia taeniaeformis and it is a feline tapeworm. The
intermediate hosts of T. taeniaeformis are mouse, rat, cat,
muskrat, squirrel, rabbit, other rodent, bat, and human that
may catch the infection through contaminated water or feed
materials with infected cat faeces (Al-Jashamy, 2010).
The C. fasciolaris was found in the liver of Rattus
norvegicus in the form of whitish prominent single to multiple
parasitic cysts. The sizes of the cysts varied from 4 to 12 mm
in diameter. Each cyst contained a single live characteristic
strobilocercus larva. Mature C. fasciolaris showed obvious
37
scolex, long neck and pseudo-segmentation, larva revealed
armed rostellum characterized by double rows of hooks and
four suckers which were clearly obvious, Fig. 1.
Fig. 1. Cysticercus fasciolaris; (A) Rat's Liver (3x) showing
pea sized cyst (B) Strobilocercus larvae of Taeniae
taeniaeformes (100x) with rostellum armed with double row of
hooks.
Hymenolepis diminuta:
H. diminuta (Fig. 2) is a cosmopolitan worm that is
primarily parasite of rats. It has been reported in different
parts of the world including Kuwait (Zakaria and Zaghloul,
1982), Great Britain (Webster and Macdonald, 1995), Croatia
(StojĈeviĆ et al., 2004), Qatar (Abu Madi et al., 2005),
Argentina (Gomez-Villafañe et al., 2008) and Kuala Lumpur,
Malesia, Southeastern Asia (Paramasvaran et al., 2009). H.
diminuta parasites mainly in the upper middle part of the
small intestine.
38
Fig. 2. Hymenolepis diminuta from R. rattus intestine (A)
Unarmed scolex (100x) (B) Maturing proglottids (100x) (C)
Maturing proglottids (400x) with a median ovary and three
testes. (C) Gravid segments (400x) (E) Eggs teased apart from
gravid segments.
b. Spirura talpae
S. talpae is the only nematode species found during this
study. It was picked from the stomach where it was parasiting
with capacity of 1-4 larvae. According To Gbif Backbone
Taxonomy S. talpae is classified as follows:
Kingdom: Animalia
39
Phylum: Nematoda
Class: Secernentea
Order: Spirurida Chitwood, 1933
Family: Spiruridae Örley, 1885
Genus: Spirura Blanchard, 1849
Fig. 3. Spirura talpae; (A) Anterior end of male (100x),
(B)Posterior end of male (100x), (C) Anterior end of female
(100x), (D) posterior end of female (100x).
41
3. Infection prevalence based on host location
Location of infestation may affect the infection prevalence.
However, in this study, the infection percentage does not considerably
differ among locations.
There was no tangible difference among the cestodes
infection percentages in three locations, as it was 70.59%,
73.33% and 75% at Giza, Qaliubiya and Beheira
governorates; respectively, but at Bani-Suef, it was higher
(90%). Likewise, the nematode infection percentages were
41.18%, 33.33% and 40% at Giza, Beheira and Qaliubiya;
respectively, and it was slightly greater at Beni Suef (50%).
The combined infection percentages of both cestodes and
nematodes exhibited the same pattern, table (3). These results
could be supported with that obtained by Allymehr et al.,
(2012) who stated that the rate of rodent infection with
nematodes and cestodes differs among locations.
Table 3. Endoparasites Infection prevalence at study locations
Governorate Cestodes Nematodes Total
Endoparasites
Total
infected
No.
Infection
%
Total
infected
No.
Infection
%
Total
infected
No.
Infection
%
Giza 24 70.59 14 41.18 26 76.47
Beheira 18 75 8 33.33 19 79.17
Bani-Suef 9 90 5 50 9 90
Qaliubiya 11 73.33 6 40 11 73.33
40
4. Infection prevalence based on host sex
Sex is an internal host factors that may have impact of
intestinal helminth fauna of Norway rats. Sex-related
differences were noted in the prevalence of infection with
some endoparasites, e.g., Capillaria sp. and Trichuris muris,
was higher in males than in females, (kataranovski et al.,
2011).
Both Rattus norvegicus sexes were examined for their
endoparasites. Regarding cestodes, males were more infected
than females as 39/(83) males were infected (46.99%) versus
23/(83) females (27.71%). The prevalence percentage on
males was 81.25% (the percentage of males infected out of the
total number of males) while, it was 65.71% on females. This
indicates that the rate of the infection prevalence on males is
greater than that on females. Similarly, nematodes infection
was greater on males, 20 (24.1%) than that on female, 13
(15.66%). But the prevalence of infection of male's population
was close to that of female's; 41.67% for male's and 37.14%
for female's; respectively, table (4).
Such conclude is in concurrence with that found by
Abu-Madi et al., (2005) who maintain that the abundance of
infection and worm burdens were affected with the sex of the
host. They stated that "the worm burdens in adult rats were
almost twice as heavy in males compared with females".
42
42
Table 4. Infection prevalence of endoparasites based on host sex
Governorate
Males Females
Infected
males'
No.
Infected
males'
%
Infection
prevalence
%
Infected
females'
No.
Infected
females'
%
Infection
prevalence
%
Cestodes
Giza 15 44.12 78.95 9 26.47 60.00
Beheira 11 45.83 78.57 7 29.17 80.00
Bani-Suef 5 50.00 100.00 4 40.00 80.00
Qaliubiya 8 53.33 80.00 3 20.00 60.00
Total 39 46.99 81.25 23 27.71 65.71
Nematodes
Giza 7 20.59 36.84 7 20.59 46.67
Beheira 5 20.83 35.71 3 12.50 30.00
Bani-Suef 3 30.00 60.00 2 20.00 40.00
Qaliubiya 5 33.33 50.00 1 6.67 20.00
Total 20 24.10 41.67 13 15.66 37.14
43
Also, These results are in solidarity with those gained
by (Udonsi,1998; Kataranovski et al., 2011). Taking into
consideration the fact that infected males have larger home
range than uninfected males and that the home range of males
tend to overlap which could increase their chance to
disseminate the infection and to increase the exposure by
uninfected rats (Brown et al., 1994) while reproductive
females show a stronger site-specific organization which
could explain low rates of transmission (kataranovski et al.,
2011), we can come up with an acceptable justification of the
high rate of prevalence of helminthic infection of males
compared with females. Brown et al., (1994),
correspondingly, proposed that the infected rodents move
more often and faster than uninfected rodents which proved an
over spread distribution.
Also, the adverse impact of the male hormone
(testosterone) on immune defense functions may represent a
greater tendency of males for helminthic infection (Folstad
and Karter, 1992). In the same way, Udonsi (1998) suggested
that increased estrogen level in females may increase
resistance to infection.
On the contrary, Nur-syazana et al., (2013) and Viljoen
et al., (2011) have different point of view, they claim that sex
and reproductive status contribute little to the parasite
prevalence and abundances or have no influence on the macro-
44
parasites community structure as both sexes share the same
burrow system.
5. Infection prevalence based on host age
In many reported studies, both abundance and
prevalence of infestation of endoparasites are host age
dependent.
In this study, 44 individuals out of 83 (53.01%) were
cestode infected mature and the infected immature individuals
were only 18 (21.69%). The prevalence of infestation among
mature individuals was greater than that among immature
individuals as 83.02% of mature individuals were infected
versus 60% of immature individuals.
As to nematode infection, 28 out of 83 (33.73%) were
infected mature individuals while 5 (6.02%) individuals were
infected immature. The prevalence of nematode infection
among mature individuals was 52.83% but it was only 16.67%
among immature individuals.
These outcomes are in harmony with those of Abu-Madi
et al., (2005) that The abundance of infection and prevalence
of H. diminuta was influenced by the host age. Adults of both
sexes harbored heavier infection than juveniles. Reasons for
this may lie behind the fact that older rats have a longer
exposure time to potential infection (Easterbrook et al., 2007).
45
45
Table 5. Infection prevalence of Endoparasites in mature and immature rats
Mature Immature
Governorate
Infected
mature
(No.)
Infected
mature
(%)
Infection
prevalence
(%)
Infected
immature
(No.)
Infected
immature
(%)
Infection
prevalence
(%)
Cestodes
Giza 16 47.06 72.73 8 23.53 66.67
Beheira 14 58.33 93.33 4 16.67 44.44
Bani-Suef 6 60.00 87.71 3 30.00 100.00
Qaliubiya 8 53.33 88.89 3 20.00 50.00
Total 44 53.01 83.02 18 21.69 60.00
Nematodes
Giza 12 35.29 54.55 2 5.88 16.67
Beheira 7 29.17 46.67 1 4.17 11.11
Bani-Suef 3 30.00 42.86 2 20.00 66.67
Qaliubiya 6 40.00 66.67 0 0.00 0.00
Total 28 33.73 52.83 5 6.02 16.67
46
In contrast, this result contradicts that revealed by
Udonsi, (1998) who justified his findings that juveniles or
immature individuals have a greater need for food materials
necessary for growth which containing infective parasite
stages while they are still immunologically naïve. This is in
line with Nur-syazana et al., (2013) who indicated that neither
intrinsic (host age, host sex) nor extrinsic (season) factors
influenced the macro-parasites community structure.
b. Ectoparasites
Rodents in particularly, Rattus norvegicus are usually
infected by certain groups of arthropods; fleas, lice and mites.
In this study 77.2% of Rattus norvegicus were infested with at
least one ectoparasite. This high rate of infestation could be
supported by the relatively small home range of the Norway
rat in addition to its neighborhood to domestic animals which
might pose an important source of infestation.
1. Ectoparasite species recorded in this study
Results of our study revealed that 938 ectoparasites,
comprising:140 (14.93%) fleas, 234 (24.95%) lice and 564 (60.1%)
mites, (Fig. 4), are belong to 4 orders, 7 families, 9 genera and 9
species, Fig. 5. Ectoparasite species collected from 83 individuals of
live trapped Rattus norvegicus include:
47
Fleas (Insecta: Siphonaptera)
Pulicidae: Xenopsylla cheopis,
Echidnophaga gallinacea
Ctenocephalides felis
Lice (Insecta: Anoplura)
Hoplopleuridae: Hoplopleura oenomydis
Polyplacidae: Polyplax spinulosa
Mites (Acari: Mesostigmata)
Macronyssidae: Ornithonyssus bacoti
Laelapidae: Laelaps nuttalli
Dermanyssidae: Liponyssoides sanguineus
(Acari: Prostigmata)
Myobiidae: Radfordia ensifera
Fig. 4. Relative frequency of ectoparasites groups
48
F
ig. 5
. Arth
rop
od
e ecto
para
sites record
ed o
n R
attu
s norveg
icus. (1
)Xen
opsylla
cheo
pis, (2
) Cten
ocep
halid
es
felis, (3) E
chid
noph
aga g
allin
acea
, (4) P
olyp
lax sp
inu
losa
, (5) H
oplo
pleu
ra o
enom
ydis, (6
) Lip
on
yssoid
es
san
gu
ineu
s, (7) L
aela
ps n
utta
lli, (8) O
rnith
on
yssus b
aco
ti an
d (9
) Radfo
rdia
ensifera
49
2. Infection prevalence and general indices of ectoparasite
according to location
Location of infestation could be a key factor of infection
prevalence. In this study, the numbers of infected individuals vary
among locations and the different ectoparasites general indices as well.
Regarding to the flea infection, Giza governorate had
the highest infection percentage (50%) and the highest flea
index as well (2.56). on the other side, Beni Suef had the
lowest flea infection percentage (20%) and the lowest flea
index (0.5).
Lice and lice index had a certain pattern which is
different from that in fleas. Although Beni Suef governorate
had the highest lice infection percentage (50%), Giza
governorate had the highest lice index (3.76). this means that
the lice burden is higher in Giza than that in the other three
locations. In the same context, Beheira governorate had the
lowest lice infection percentage (25%), but its lice index
(2.46) is bigger than that of Beni Suef (2.1) and Qaliubiya
(1.73) table (6).
With regard to mite infection, Beni Suef governorate
came first (70%) followed by Beheira governorate(66.67%)
while Qaliubiya had the lowest percentage of infection
(40%). Mite indices were relatively high; since it ranged from
4.27 in Qaliubiya governorate to 11.3 in Beheira governorate,
table (6).
51
Table 6. Infection prevalence and general indices of
ectoparasite according to location
Flea Lice Mite
Gov. Total
infected
No, (%)
General
flea index
Total
infected
No, (%)
General
Lice
index
Total
infected
No, (%)
General
Mite
index
Giza 17, (50) 2.56 9, (26.47) 3.76 17, (50) 5.26
Beheira 6, (25) 1.16 6, (25) 2.46 16, (66.67) 11.3
Bani-Suef 2, (20) 0.5 5, (50) 2.1 7, (70) 5
Qalyobya 4, (26.67) 1.4 4, (26.67) 1.73 6, (40) 4.27
From the aforementioned data, it is obvious that the rate
and the indices of infestation of different ectoparasites vary
from one location to another. These findings are in accordance
with El Deeb et al., (1999) and Soliman et al., (2001b) that
the distribution of ectoparasites varied according to rodent
host and location. Also, Kia et al., (2009) stated "the
infestation rate to different ectoparasite depend on season,
size of rodents, host preference, sex of host, host age, location
of capture and co-evolution between rodent and
ectoparasites". Similarly, Telmadarraiy et al., (2007)
mentioned the Infection prevalence and general indices of
ectoparasite mainly depend on season, rodent species,
ectoparasite species, location, method of catch, and host
population dynamics. For instance, The indices of infestation
by the mites Laelaps nuttalli, the louse Polyplax spinulosa and
the flea Xenonpsylla cheopis, on Rattus norvegicus in Brazil
50
were related to seasonal period, sex of the host and area of
capture (Linardi et al., 1985).
In my point of view, location is the key factor affects
the Infection prevalence and general indices of ectoparasite
because location change involves many criteria like
geographical situation, ecological condition, rodent predators,
seasonal activities, human practices and sources of infection
that influence the ectoparasite prevalence and indices
3. Infection prevalence based on host sex
Rattus norvegicus population was divided into males and
females to find out if there is a variation of the infection prevalence of
different ectoparasites between both rat sexes. The male infection
prevalence percentage calculated as the percent of infected males'
number to the whole males' population.
Respecting fleas' infection, 19 infected male individuals
(22.89%) represented 39.58% of the whole males' population
(male infection prevalence percentage). Infected females were
10 individuals with a percentage of 12.05%. The prevalence of
infection among females was 28.57%. There no fleas were
recorded on females in Beni Suef (0%), while Giza was the
highest in both infected males and infected females
percentages, (29.41% and 22.59%); respectively. Also, the
flea infection prevalence was the uppermost in Giza since it
was 52.63% among males and 46.67% among females.
52
Table 7. Infection prevalence of the ectoparasites on both male and female hosts
Males Females
GOV Infected
males' No.
Infected
males' % Infection
prevalence%
Infected
females' No.
Infected
females' % Infection
prevalence%
Fleas
Giza 10 29.41 52.63 7 20.59 46.67
Beheira 4 16.67 28.57 2 8.33 20.00
Bani-Suef 2 20.00 40.00 0 0.00 0.00
Qaliubiya 3 20.00 30.00 1 6.67 20.00
Total 19 22.89 39.58 10 12.05 28.57
Lice
Giza 6 17.65 31.58 3 8.82 20.00
Beheira 2 8.33 14.29 4 16.67 40.00
Bani-Suef 1 10.00 20.00 4 40.00 80.00
Qaliubiya 3 20.00 30.00 1 6.67 20.00
Total 12 14.46 25.00 12 14.46 34.29
Mites
Giza 10 29.41 52.64 7 20.59 46.67
Beheira 9 37.50 64.29 7 29.17 70.00
Bani-Suef 4 40.00 80.00 3 30.00 60.00
Qaliubiya 5 33.33 50.00 1 6.67 20.00
Total 28 33.73 58.33 18 21.69 51.43
53
Regarding lice infection, a total of 12 male-individuals
(out of 83, the whole population) were infected with a
percentage of 14.46%. The infection prevalence among them
was 25% (12 out of 48 males). Infected females' number was
equal to that of males' (12, 14.46%) but the infection
prevalence among females (34.28%) was greater than that
among males.
Mite infection and prevalence was the greatest
comparing to other ectoparasites as 28 males (33.73%) and 18
females (21.69%) were infected. Also the prevalence of
infection among males (52.33%) and females (51.43%) was
the highest when compared with fleas and lice. There were no
differences of infection prevalence based on host sex.
General indices of ectoparasites based on host sex
Ectoparasites indices were calculated for both sexes for
determining if there is a relationship between the host sex and
the parasites' burden.
The flea index in males is bigger than that in females in
all governorates except for Giza but the total flea indices in
both males and females are equal (1.69). There was a big
difference between the male and female lice indices in Beheira
and Beni Suef governorates as they were 0.86 / 4.7 and 0.6 /
3.6; respectively, but the total lice index in males (2.85) was
almost bigger than that in females (2.77). With regard to mite,
54
the total mite index was approximately bigger in males than it
in females. But still there were some differences according to
locations, Table (8).
Table 8. General indices of ectoparasite based on host sex
Gov. Flea index Lice index Mite index
Male Female Male Female Male Female
Giza 2.26 2.93 4.42 2.93 6 4.33
Beheira 1.2 1 0.86 4.7 11.7 11.6
Bani-Suef 1 0 0.6 3.6 4.4 5.6
Qalyobya 1.6 1 2 1.2 6 0.8
Total 1.69 1.69 2.85 2.77 7.31 6.09
Overall outcome reflects that no host sex-associated
differences in the prevalence of infection were found for
ectoparasites. This result is in agreement with Nur-Syazana et
al., (2013) who did not find any strong independent effects of
host sex on the prevalence of ectoparasites although more
females were observed infested compared to males. But, at the
same time, this result contradicts the findings of Linardi et al.,
(1985), Botelho and Linardi (1994) and Kia et al., (2009) that
the ectoparasites preferentially infested male rodents, both in
wild and urban environments.
4. Infection prevalence based on host age:
We divided the host population into two groups, mature and
immature, to study the effect of the age on the infection prevalence of
ectoparasites.
55
Table 9. Infection prevalence of ectoparasite on mature and immature individuals
GOV Mature Immature
Infected
Mature No.
Infected
Mature %
Infection
prevalence%
Infected
Immature
No.
infected
Immature
%
Infection
prevalence %
Flea
Giza 12 35.29 54.55 5 14.71 41.67
Beheira 3 12.50 20.00 3 12.50 33.33
Bani-Suef 2 20.00 28.57 0 0.00 0.00
Qaliubiya 3 20.00 33.33 1 6.67 16.67
Total 20 24.10 37.74 9 10.84 30.00
Lice
Giza 9 26.47 40.91 0 0.00 0.00
Beheira 5 20.83 33.33 1 4.17 11.11
Bani-Suef 2 20.00 28.57 3 30.00 100.00
Qaliubiya 2 13.33 22.22 2 13.33 33.33
Total 18 21.69 33.96 6 7.23 20.00
Mite
Giza 14 41.18 63.64 3 8.82 25.00
Beheira 12 50.00 80.00 4 16.67 44.44
Bani-Suef 5 50.00 71.43 2 20.00 66.67
Qaliubiya 2 13.33 22.22 4 26.67 66.67
Total 33 39.76 62.26 13 15.66 43.33
56
A total of 20 (24.1%) mature individuals versus 9
(10.84%) immature individuals were infected with fleas. The
flea infection prevalence inside the mature population was
37.74% which was relatively higher than that inside the
immature population (30%). It means that mature individuals
are likely to be infected than immature individuals. Also, the
infection prevalence is likely to be the different between
mature and immature individuals with a slight tendency to be
higher in mature individuals.
Lice infection varied between mature and immature rats,
as a total of 18 mature individuals (21.69%) and 6 immature
individuals (7.23%) were infected. The infection prevalence of
lice inside the mature population (33.96%) was higher than
that inside immature population (20%). It is clear that
immature individuals are less likely to be infected.
Unlike fleas and lice, mite infection was higher and
more prevalent; as 33 mature individuals (39.76%) and 13
immature individuals (15.66%) were infected. When
comparing the infection prevalence between mature and
immature individuals, it was found that the infection
prevalence in mature individuals (62.26%) was greater than it
in immature individuals (43.33%).
57
General indices of ectoparasite based on host age:
General indices of the three main groups of arthropod
ectoparasites, fleas, lice and mites, were conducted for each age
stage as follows:
Generally, mature individuals tend to have bigger
ectoparasite index than immature individuals. As to flea index,
it was 1.96 in mature individuals versus 1.2 in immature
individuals, also lice index in mature individuals was three
times bigger (3.75) than it in immature individuals (1.17).
Likewise, the mite index was bigger in mature individuals
(7.15) than it in immature individuals (6.17). So it is
predictable for us to record high infection and high prevalence
of ectoparasite in mature individuals, while it tends to be low
in immature individuals, Table (10).
Table 10. General indices of ectoparasite based on host age
Gov. Flea index Lice index Mite index
Mature Immature Mature Immature Mature Immature
Giza 3.14 1.5 5.82 0 7.32 1.5
Beheira 1.27 0.89 3.27 1.1 14.53 5.89
Bani-Suef 0.43 0 1.7 3 2.85 9
Qalyobya 2.1 0.33 2 1.33 4.33 4.17
Total 1.96 1.20 3.75 1.17 7.15 6.17
Age is one of the key elements of a rodent host that may
affect the foraging choices of ectoparasites. The increased
prevalence and general infestation index of ectoparasites are
positively correlated to the increased densities of their hosts
(Anderson and Gordon, 1982). Randolph (1975); Thompson et
58
al., (1998) and Kia et al., (2009) stated that the catch rate and
infestation rate of different ectoparasite depend on host age.
Many important parameters in host–parasite dynamics, such as
infestation level of hosts and the consequent parasite
distribution among host individuals are often age-dependent
(Anderson and Gordon, 1982; Hudson and Dobson, 1997)
Juvenile rodents have larger surface to volume ratio and
thus, higher energy requirements for maintenance per unit
body mass (Kleiber, 1975). They also require additional
energy for somatic growth, maturation, and for mounting an
immune response. Thus, adult rodents under field conditions
usually represent a better nutritional resource than juveniles
(Buxton, 1984). Also, adult hosts show higher infestation
levels than juveniles because they have more time to
accumulate parasites (Hawlena et al., 2006).
59
Part II: Resistance of Rattus norvegicus to
warfarin, the first generation anticoagulant
INTRODUCTION
The best-known anticoagulant agent, warfarin, was
developed in the 1940s. Today, warfarin is used as a
rodenticide. It is added to grain meal in low concentrations
(usually between 0.005% and 0.1%) making the poisoned bait
product relatively safe for humans to handle. Warfarin causes
a slow death by gradual acting of internal bleeding. Within a
decade of the introduction of warfarin as a rodenticide, rats
and mice resistant to the poison were discovered. Among the
first resistant species described were Norway rats (Rattus
norvegicus), ship rats (R. rattus) and house mice (Mus
musculus). These initial discoveries were made in rural areas
of the United Kingdom and in other locations, not only in
Europe, but also in the United States, Asia, and Australia.
Decade ago, VKORC1 (vitamin K epoxide reductase
complex subunit 1), the target enzyme for coumarins, was
identified. VKORC1 is a key component of the vitamin K cycle
that reduces vitamin K epoxide and at the same time is
inhibited by warfarin. It was shown that mutations in VKORC1
confer resistance to anticoagulants of the Coumarin-type in
humans and rodents.
61
Because rodents carrying resistance mutations survive
poisoning, they are selected for survival in areas where
anticoagulant rodenticides are used. Genetic mutations
conferring resistance to anticoagulant rodenticides were
identified in both rats and mice. In rats and mice independent
mutations have arisen in different warfarin-resistance areas
throughout the world and affect different amino acid positions
of the VKORC1protein.
According to Rost et al., (2004), mutations in VKORC1
may cause a heritable resistance to warfarin, possibly by
preventing coumarin derivatives from interfering with the
activity of the reductase enzyme. So, resistance against
warfarin-like compounds poses a considerable problem for
efficacy of pest control.
60
REVIEW OF LITERATURE
1. Anticoagulant rodenticides
Control of Rattus norvegicus (Norway rat) depends
mainly on toxicants, either acute or chronic rodenticides to get
rid of its harmful in fields, houses and stores, and to
manipulate the diseases they carry. Acute poisons were used
for ages before the discovery of warfarin. It is well known that
R. norvegicus is very neophobic (being unfamiliar to new
items in their environment). Neophobic rats may eat a small
non-lethal dose of new bait. Survived rats learn to avoid the
bait that may consequently cause problems concerning the
rodenticides (Baert, 2012)
Anticoagulant rodenticides were first discovered in the
1940 s and have since become the most widely used toxicants
for commensal rodent control due to their convenience, safety,
and minimal impact on the environment. This new group of
rodenticides have been introduced as an alternative of acute
toxicants. Warfarin and related anticoagulant compounds
(coumarins) were massively used in the early 60s‘, and they
were a great choice to reduce or eradicate rat populations from
many area. Poisoned rodents die from internal bleeding as a
result of loss of the blood's clotting ability. Prior to death, the
animal exhibits increasing weakness due to blood loss.
anticoagulant baits are slow in action (several days following
62
the ingestion of a lethal dose), the target animal is unable to
associate its illness with the bait eaten. Therefore, bait
shyness does not occur. This delayed action also has a safety
advantage because it provides time to administer the antidote
(vitamin K1) to save pets, livestock, and people who may have
accidentally ingested the bait (Pelz et al., 2005)
There are two generations of anticoagulants; the first
generation anticoagulants: or multiple-feed rodenticides
(warfarin, pindone, diphacinone and clorophacinone). These
compounds are chronic in their action, requiring multiple
feedings over several days to a week or more to produce
death. First generation rodenticidal anticoagulants generally
have shorter elimination half-lives, require higher
concentrations (usually between 0.005% and 0.1%) and
consecutive intake over days in order to accumulate the lethal
dose, and less toxic than second generation agents. On the
other hand, second generation agents are far more toxic than
first generation. They are generally applied in lower
concentrations in baits — usually on the order of 0.001% to
0.005%. They are lethal after a single ingestion of bait and are
also effective against strains of rodents that became resistant
to first generation anticoagulants; thus, the second generation
anticoagulants are sometimes referred to as "superwarfarins.
63
a. Anticoagulant rodenticide
Where anticoagulants have been used over long periods
of time at a particular location, there is an increased potential
for a population to become somewhat resistant to the lethal
effects of the baits. Resistance to warfarin was first observed
in Scotland in 1958 (Boyle, 1960). Since then, resistant rats
have been reported all over the world, in Great Britain,
Denmark, Germany, Belgium, Finland and France, the USA,
Canada, Australia, and Japan (Mayumi et al., 2008). Warfarin-
resistance has led to failure of their control using warfarin as a
rodenticide. Rats and mice that are resistant to warfarin also
show some resistance to all first generation anticoagulants,
rendering control with these compounds less effective.
Bailey and Eason (2000) stated that resistance to
anticoagulants can develop in a population after 5-10 years
sustained use of anticoagulant rodenticides. No enough data
are existed on the baseline susceptibility of rodent populations
in Egypt to anticoagulants or their changing patterns of
susceptibility in areas of sustained use. Monitoring systems
for wild target populations and changes to poisoning methods
will assist Egypt rodent control groups in avoiding the
resistance-induced control problems now seen outside Egypt.
Sustained control of rodents on the mainland is likely to be
substantially dependent on toxicants and anticoagulant
poisons in particular for the foreseeable future.
64
b. Mode and site of action of anticoagulants
Coumarins act as a vitamin K antagonist and block the
vitamin K cycle in the liver, preventing the reduction of
vitamin K epoxide to vitamin K by vitamin K epoxide
reductase (VKOR). Vitamin K is an essential co-factor in the
activation of several vitamin K-dependant coagulation factors
through which it plays an important role in blood coagulation.
When coumarins bind with VKOR, intoxication with
anticoagulants will lead to a deficiency of vitamin K and
coagulation factors, causing coagulation disorders such as
spontaneous bleeding and eventually death (OldenBurG et al.,
2008).
Anticoagulants act by interfering with the synthesis of
prothrombin, disturbing the normal clotting mechanisms and
causing an increased tendency to bleed.
The anticoagulant action of rodenticides arises from
inhibition of vitamin K metabolism in the liver. Vitamin K is
essential for the production of several blood-clotting proteins
and, when greatly reduced in concentration, results in fatal
hemorrhaging. Vitamin K in its reduced form (vitamin K
hydroquinone) is a co-factor for a carboxylase active in the
production of proteins such as clotting factors II,VII, IX, and
X. During this process, vitamin K is oxidised to vitamin KO
and is then unavailable until recycled to vitamin K
hydroquinone by the enzyme vitamin K epoxide reductase
65
(VKOR). It is this enzyme that is inactivated by the action of
anticoagulants, which have a similar structure to vitamin K
and bind strongly to the enzyme, leaving it unavailable for the
recycling of vitamin KO (Oldenburg et al., 2000).
All anticoagulants work by inhibiting the generation of
an active form of vitamin K1 via inhibition of vitamin K1
epoxide reductase. The presence of vitamin K as a cofactor is
required to the activation of clotting factors II, VII, IX, and X.
The VKORC1 gene produces the enzyme vitamin K1 epoxide
reductase, an essential enzyme in the vitamin K cycle and the
one blocked by all anticoagulant rodenticides (Buckle, 1994)
Anticoagulants can inhibit two different enzymes of the
vitamine K cycle: the epoxyde reductase and the vitamine K
reductase (although some scientists consider these two
enzymes are, in fact a single protein). The epoxide reductase
is the rate-limiting step and inhibition by anticoagulants will
result in the accumulation of Vitamine K epoxide, which is
not active. The second step is not as critical, since other
pathways may lead to the activation of vitamine K, such as the
diaphorases. Inhibition of this vitamin K cycle results in a
decreased production of active coagulation factors which, in
turn, will result in coagulation disorders and hemorrhages
(Berny, 2011).
66
2. Vitamin K and blood coagulation
Vitamin K1 is found mainly in green leafy vegetables
such as kale, spinach, and broccoli while vitaminK2 is found
in liver, milk, cheese, and fermented soy products such as
Natto. Menadione is a chemically synthesized derivative used
for animal feed.
a. The role of Vitamin K on blood coagulation
Synthesis of prothrombin, factors VII, IX and X are
dependent on vitamin K. besides, three other proteins are
vitamin K-dependent, in addition to other non-plasma
proteins. calcium ions are essential for activation of all these
proteins. The characteristic feature of the vitamin K-
dependent proteins is that they contain a modified glutamic
residue which has an extra carboxy-group attached to the γ-
carbon. This carboxy-group is added at a post-translational
vitamin K-dependent process (Mayumi et al., 2008).
Calcium ions are required as a co-factor for the action
of all the vitamin K-dependent proteins and the γ-
carboxyglutamic acid residues form the high affinity calcium
binding sites in these proteins (Jackson, 1972). As mentioned
above, γ-carboxyglutamic acid is formed by a vitamin K-
dependent process. The carboxylation of the specific glutamic
acid residues in the N-terminal regions of these proteins
occurs as a post translational event, and unlike other
67
biological carboxylation reactions, there is no dependence on
biotin or high energy phosphate. The only requirements are
reduced vitamin K, molecular oxgen and carbon dioxide and
an enzyme present in liver microsomes (Suttie, 1985).
The mechanism by which the carbon is activated and
transferred to the γ-carbon is not fully understood. However,
it is thought that the reduced vitamin reacts with molecular
oxygen to form a peroxy intermediate (a peroxy radical)
which then reacts with carbon dioxide to form a
peroxycarbonate adduct of the vitamin which decomposes to
carboxlate the glutamic acid residues in the presence of the
carboxlase1 and the vitamin is converted to the epoxide.
Under physiological conditions, the epoxide is converted back
to the reduced from through the vitamin cycle (Olson et al.,
1984; Suttie, 1985).
b. Vitamin K cycle, site of action and target molecule
of warfarin
Vitamin K functions as a cofactor for the γ-carboxylase,
an enzyme that resides in the endoplasmic reticulum (ER)
membrane and participates in posttranslational γ-
carboxylation of newly synthesized vitamin K-dependent
proteins. The γ- carboxylase converts a limited number of
glutamic acid residues in the amino-terminal part of the
targeted proteins to γ-carboxyglutamic acid (Gla), calcium-
binding residues. Members of the vitamin K-dependent protein
68
family include the coagulation factors prothrombin; factors
VII, IX, X, protein S, protein C, and protein Z, as well as
several other proteins synthesized outside the liver. These
proteins include osteocalcin, matrix Gla protein, Gas6, protein
S, and some recently discovered proline-rich transmembrane
proteins (Wallin et al., 2001).
Before serving as a cofactor for γ-carboxylase, vitamin
K must be reduced to the hydroquinone (vitamin K1H2).
When one Gla residue in the targeted protein is formed, one
hydroquinone molecule is converted to the metabolite vitamin
K1 2,3-epoxide. The epoxide is reduced back to the
hydroquinone form of the vitamin by an integral membrane
protein complex of the ER, the vitamin K epoxide reductase
(VKOR). This cyclic conversion establishes a redox cycle for
vitamin K known as the vitamin K cycle. VKOR is the target
for the anticoagulant drug warfarin, (Wallin et al., 2001)
Vitamin K-dependent proteins require carboxylation for
activity. The amount of vitamin K in the diet is often limiting
for the carboxylation reaction. It has been commonly assumed
that vitamin K may also be provided by enteric bacteria;
however, if coprophagy is prevented, rats fed a vitamin K-free
diet develop severe bleeding problems in weeks. Of more
interest is the recent observation that vitaminK1 appears to be
taken up primarily in the liver while vitamin K2 appears to
69
preferentially accumulate in arteries and extra hepatic
locations (Stafford, 2005).
The production and activation of coagulation factors
VII, IX, X and prothrombin are dependent on the vitamin K
cycle. Post translational modification of glutamate to gamma
carboxyl glutamate is required for the activity of vitamin K-
dependent proteins (Stafford, 2005). The carboxylated Glu
residue is converted to a Gla amino acid and a reduced
vitamin K molecule is converted to vitamin K epoxide. Before
vitamin K can be reused in the vitamin K cycle, vitamin K
epoxide must be converted back to reduced vitamin K by
vitamin K 2,3-epoxide reductase (VKOR). Recently, Wajih et
al. identified the novel endogenous molecules that transfer the
electron to VKOR and regenerate the vitamin K cycle (Wajih
et al., 2005; Wajih et al., 2007).
Warfarin blocks the vitamin K cycle and inhibits the γ-
carboxylation of the vitamin K-dependent blood-clotting
factors. An inadequate supply of vitamin K blocks the
production of prothrombin and leads to hemorrhaging
(Thijssen et al., 1989).
3. Resistance to anticoagulants
Resistance is defined according to the European and
Mediterranean Plant Protection Organization as follows;
"Rodenticide-resistant rodents should be able to survive doses
71
of rodenticide that would kill ‗normal‘ or ‗susceptible‘
conspecifics‖ (EPPO, 1995). Greaves, (1994) describes
anticoagulant resistance as "a major loss of efficacy in
practical conditions where the anticoagulant has been applied
correctly, the loss of efficacy being due to the presence of a
strain of rodent with a heritable and commensurately reduced
sensitivity to the anticoagulant". Monitoring for resistance is
important to reveal the secret behind its spread and to manage
resistant populations (Buckle, 2006).
a. Techniques used in resistance detection in rodents
There are few relevant techniques for detection of
resistance to anticoagulants. They are either in vivo assays,
like, feeding test and blood clotting response test (BCR) or in
vitro assays, like, VKOR activity, CYP450 metabolism and
VKORC1 testing. Each technique has its pros and cons as
follow.
1. Feeding tests
Basically, it is a no-choice feeding test over 6 days with
a 50 ppm warfarin, bromadiolone or difenacoum bait for
instance. Rodents surviving the 5-day test period (+14 days
observation) are classified as resistant to the anticoagulant
tested. This method may involve some modifications i some
cases. Some authors consider that this test has several
limitations, especially with regards to local variations in the
70
resistance of the strain, which would need adaptation of the
exposure period to cover a wider range of susceptibility.
Unfortunately, this approach requires a large number of
animals and is ethically critical (Gill and McNicoll, 1991).
2. BCR testing
It first developed by Martin et al., (1979). In its present
form, the BCR can be conducted in two ways: The first
approach is to determine the rat capacity to use the vitamin K
epoxide substrate (1 mg/kg) as a vitamin K source in the
presence of an anticoagulant (warfarin) (5 mg/kg).
Determination of the clotting response (Prothrombin time)
24hours later is a good indicator of the resistance status. A
new modified methos relies on the administration of a low
dose (1 mg/kg) warfarin, with no vitamine K epoxyde and
investigation of the clotting capacity 24hours later. The recent
developments of this approach are based on the works by Gill
and McNicoll (1991) and Prescott and Buckle (2000), who
tested several protocols with various anticoagulants. The
second approach investigates the rat vitamin K deficiency
status (Martin et al., 1979), this approach has been less
developed.
3. Measuring the activity of the hepatic vitamin K
epoxide reductase (VKOR)
Several protocols may be used on liver microsomes or
any other enzyme system (Lasseur et al., 2007). This approach
72
provides a very good estimate of the enzyme activity and the
resistance status of a population. Hepatic VKOR as-sessment
is carried out in vitro by monitoring the activity of VKOR in
the presence and absence of the toxicant. Susceptible samples
show minimal VKOR activity when anticoagulant is present,
while enzyme activity in resist-ant samples remains above
20% of original levels (MacNicoll, 1993).
4. CYP metabolism
It is an in vitro approach, like the VKOR activity assay,
and requires microsoms and analytical devices to look at
warfarin metabolites produced. It is not a standard tool for the
monitoring of resistance so far (Ishizuka et al., 2006).
5. VKORC1 sequencing
Sequencing of VKORC1 appears as one of the most
interesting and cost effective tools till now. It can be applied
rapidly on large scale samples, even across a country. It can
also provide a good indication of the resistance level
conveyed by a given mutation. The PCR dependent test aims
at detecting mutation in VKORC1 gene that may confer
resistance to anticoagulants. Sequencing of VKORC1 only
requires a piece of animal tissue (tail, ear, fur may be used)
and does not necessitate live-trapping of rodents. This
approach may be simpler even further with the use of qPCR
and specific primers, especially when only one mutation is
73
expected or known to occur in a certain area (Grandemange et
al., 2010).
b. The mechanisms of anticoagulant rodenticide
resistance.
The resistance mechanism mainly involves VKORC1,
the molecular target for coumarin drugs. Mutation of VKORC1
may constitute the genetic basis of anticoagulation resistance
in R. losea (Wang et al., 2008). Resistant to anticoagulants
involves VKOR modification through point mutations of the
DNA. While still remaining functional, VCOR displays a
reduced affinity for the toxicant or the toxicant is more easily
replaced by the vitamin KO. This modification is inheritable
(Bailey and Eason, 2000).
In 2004, two research groups identified and reported a
novel molecule that contributes to VKOR activity in the rat
and named it vitamin K epoxide reductase complex subunit 1
(VKORC1) (Rost et al., 2004; Li et al., 2004).
Pelz et al. (2005) identified which part of the genetic
code of rats and mice carried the DNA sequence, or gene,
which alters when rodents become resistant to anticoagulants.
The gene they discovered produces the enzyme vitamin K1
epoxide reductase, a crucial enzyme in the vitamin K cycle
and the one blocked by all anticoagulant. The gene was given
the name VKORC1 and the sequence of amino-acids used in
its construction was decoded.
74
In the first report on VKORC1, Rost et al. (2004)
suggested that warfarin resistance in rats was attributable to
the Tyr139Phe mutation in the VKORC1 gene. The VKORC1
139 mutation in warfarin-resistant rats causes a structural
conformation of the VKORC1 protein, and this conformation
prevents warfarin blocking (Rost et al., 2005).
c. The chemical structure of VKORC1
The enzyme is mainly found in liver cells. It is seen as a
chain of 163 amino-acids which passes several times through
the membrane of the endoplasmic reticulum (ER). The amino-
acids are numbered in the chain (Tie and Nicchitta, 2005).
VKORC1 is an 18 kDa hydrophobic protein resident in the
endoplasmic reticulum membrane. Hydrophobicity plots and
secondary structure predictions suggest a topology of
VKORC1 protein that includes 3 to 4 a-helical transmembrane
segments (Goodstadt and Ponting, 2004), Fig. 6.
All anticoagulants target the site of the vitamin K
epoxide reductase enzyme complex (VKOR) and the binding
of anticoagulants to the VKOR inhibits the essential
production of prothrombin, thus destroying blood clotting
ability. Today, it is possible to differentiate between rats that
are either susceptible or resistant to anticoagulants by means
of a blood clotting response (BCR) test, where changes in
blood coagulation during anticoagulant exposure are
75
visualized (Martin, 1973; Gill, et al. 1993). The genetic
background behind this inheritable trait has been investigated
over the last three decades (Pelz, et al. 2005).
Fig. 6. The chemical structure of VKORC1
d. Mutations in VKORC1 conferring resistance to
warfarin
Mutated genes are given names which describe the
position of the mutated amino acid in the DNA sequence of
the enzyme, e.g. In the case of the common French resistance
mutation this is at position 139. The name of the original
(wild-type) amino-acid is tyrosine and that of the mutant
amino-acid is phenylalanine. These are put before and after
the position number, hence tyrosine139phenylalanine. The
76
names of the amino-acids are commonly abbreviated, i.e.
tyr139phe.
Since the establishment of a correlation between
mutations within the VKORC1 and anticoagulant resistance in
rodents, various mutations in VKORC1 have been identified as
conferring anticoagulant resistance in rats and mice (Pelz et
al., 2005; Pelz, 2007). Recently, missense mutation in a
protein of the VKOR complex, named VKORC1, was
identified as being related to warfarin resistance (Rost et al.
2004). The VKORC1 mutations are currently believed to be
the genetic basis of anticoagulant resistance, conferring
resistance to, at the very least, the first-generation
anticoagulant warfarin, (Rost et al., 2004; Pelz et al., 2005).
One particular VKORC1 mutation, a change in an amino acid
from tyrosine to cysteine in exon 3 at codon position 139
(Y139C) coincides with anticoagulant resistance in Danish
and German rats (Pelz, et al,. 2005).
VKORC1 polymorphisms in rats from warfarin-
resistance areas in Europe, Asia, North-and South-America
have been reported; England Ile821Ile; Hungary Ile821Ile;
Korea Ile821Ile; Indonesia Ile821Ile, Ile90Leu, Ser103Ser,
Ile107Ile, Thr137Thr, Ala143Val; USA, Santa Cruz
Arg12Arg, Ile90Leu, Leu94Leu, Ile107Ile, Thr137Thr,
Ala143Ala; USA, Chicago Ile821Ile; Argentina Arg12Arg,
77
Ile90Leu, Leu94Leu, Ile107Ile, Thr137Thr, Ala143Ala (Rost
et al., 2009).
More than 30 missense mutations in the gene VKORC1
in humans, rats and mice have been found, 16 of which have
been confirmed to confer a certain degree of resistance or
insensibility to warfarin. A change of amino acids at positions
120 to 139 is connected to the strongest degree of resistance
observed. The tyrosin-cystein substitution at position 139 in
VKORC1 is probably the most widespread mutation. It is
common in Denmark and northwestern Germany, and was
found in parts of Hungary, France and England. Other
widespread mutations are the tyrosin-phenylallanin
substitution at position 139, which is common in France and
Belgium and was also found in Korea, the leucin-glutamic
acid substitution at position 128 (―Scottish-type resistance‖,
Scottland, northern England and parts of France) and the
leucin-glutamic acid substitution at position 120 (Hampshire-
and Berkshire-resistance, Southern England, parts of France
and locally in Belgium. The well-known Welsh-type
resistance can be attributed to a tyrosin-serin substitution at
position 139, however, the effect upon the degree of resistance
seems to be less pronounced than in the other two
substitutions at position 139 mentioned above. The occurrence
of resistance described for the Chicago (Illinois, USA) area
seems to be due to an arginin-prolin substitution at position 35
that was also detected in a wild rat from central France. Again
78
the degree of resistance mediated by this mutation seems to be
relatively low (Pelz, 2008)..
More than 250 rats and mice from anticoagulant-
exposed areas in Europe, East Asia, South Africa and both
North and South America were screened for mutations in the
VKORC1 gene. Pre-screening revealed a panel of mutations
and SNPs (single nucleotide polymorphisms) in the VKORC1
gene. Three already described mutations could be detected in
rats trapped in different English counties: the Tyr139Cys,
Tyr139Ser and the Leu128Gln substitutions. All three
mutations confer a moderately reduced VKOR activity and are
resistant to warfarin inhibition to a variable degree. Arg33Pro
substitution was observed in two confirmed warfarin-resistant
rats from Nottinghamshire. A Phe63Cys substitution was
detected in rats from Cambridge, including two rats with an
additional Ala26Thr or a Tyr39Asn amino acid exchange.
While Ala26Thr has – similar to Ala26Ser – only a moderate
effect on VKOR activity with a reduction to approximately
56% of wild-type activity, the Phe63Cys and the Tyr39Asn
substitutions reduce the VKOR activity to about 30% of
normal. Since both amino acids Phe63 and Tyr39 are highly
conserved in vertebrates and also in the mosquito, a
substitution of these amino acids is expected to have an
influence on protein function. VKOR activity measurements
of the Glu67Lys variant (observed in six rats from Japan)
showed a reduced vitamin K epoxide turnover of about 33%
79
compared to the wild-type protein. The most drastic effect on
VKOR activity was observed for the Trp59Arg substitution.
Only 16% residual VKOR activity could be measured after
recombinant expression of this VKORC1 mutant (Rost et al.,
2009).
Tyr139Cys, Tyr139Ser, Tyr139Phe, Leu128Gln and
Leu128Ser mutations dramatically reduce VKOR activity. It is
suggest that mutations in VKORC1 are the genetic basis of
anticoagulant resistance in wild populations of rodents,
although the mutations alone do not explain all aspects of
resistance that have been reported. These mutations may
induce compensatory mechanisms to maintain blood clotting.
These findings provide the basis for a DNA-based field
monitoring of anticoagulant resistance in rodents. However,
the ability to maintain a functional blood clotting mechanism
under anticoagulant exposure can be attributed to a
physiological response of the individual rat, which may be
enhanced by a genetic change in the VKORC1. (Pelz et al.,
2005; Pelz, 2007).
81
80
MATERIALS AND METHODS
Warfarin resistance study
1. Determination of the susceptibility level of rats to
warfarin Using a laboratory feeding test(no-choice)
The study population consisted of 42 R. norvegicus,12 from
Giza, 12 from Beheira, 9 from Beni Suef and 9 from Qaliubiya . All
animals were healthy adults and females were not pregnant. They were
confined in individual cages and received the same food and water ad
libitum. All animals received humane care to be in good conditions as
urine and droppings were being removed daily. Resistance to warfarin
was assayed by feeding studies, the no-choice-feeding test developed
by the World Health Organization (WHO) was used with some
modifications: an acclimatization period, followed by a pretest diet
assessment of 7 days, then by a 6-day no-choice feeding schedule of
0.005% (50 ppm) warfarin-containing yellow corn. Diet consumption
was monitored and recorded daily), and 22 days of post-treatment
observation are maintained. Survival during the test with the amount
of active ingredient consumed greater than 10 mg/kg body weight,
were considered as evidence of resistance
2. VKORC1 analysis using Polymerase Chain Reaction
(PCR)
Livers were excised, rapidly frozen in liquid nitrogen, and stored
at -20 °C. The total RNA was isolated and reverse transcribed to
cDNA. VKORC1 was amplified using primers based on corresponding
sequences for Rattus norvegicus in Gen-Bank (Accession No.
82
NM_203335). The primers amplify a 511 bp fragment spanning the
whole ORF of VKORC1 mRNA. Nucleotide sequences for the sense
primer and antisense primer were 5'-GTGTCTGCGCTGTACTGTCG-
3' and 5'-CCTCAGGGCTTTTTGACCTT -3'; respectively. PCR
products were electrophoresed and visualized under UV light and the
picture taken with a gel documentation system The following DNA
fragment was sequenced by the Sanger method (Sanger and Coulson
1975).
Procedure:
a. RNA extraction
Total RNA purification protocol
Liver tissue samples were grind in a mortar using liquid nitrogen
then a 0.1 gm was measured immediately in a 1.5 ml tube. 300 μl lysis
buffer and 6 μl of 14.3Mβ-mercaptoethanol were added immediately
and 10 min vortex. 600 μl of diluted Proteinase K (10 μl of the
included Proteinase K diluted in 590 μl of TE buffer) were added.
Vortex to mix thoroughly was done and then incubated at 15-25°C for
10 min. After that; Samples were Centrifuged for 5 min at 12000 xg
and supernatant was transferred into new RNase free tube. 450 μl
ethanol (96-100%) were add and mixed by pipetting. Up to 700 μl of
lysate were transferred to the GeneJET RNA Purification Column
inserted in a collection tube then centrifuged for 1 min at ≥12000 x g.
The flow-through was discarded and the purification column was
placed back into the collection tube.
83
Once all of the lysate has been transferred, 700 μl of Wash
Buffer 1 were added to the GeneJET RNA Purification Column and
centrifuged for 1 min at ≥12000 x g. 600 μl of Wash Buffer 2
(supplemented with ethanol) were added to the GeneJET RNA
Purification Column and centrifuged for 1 min at ≥12000 x g. 250 μl of
Wash Buffer 2 were added to the GeneJET RNA Purification Column
and centrifuged for 2 min at ≥12000 x g.
50 μl of nuclease-free water, were added to the center of the
GeneJET RNA Purification Column membrane, then centrifuged for 1
min at ≥12000 x g to elute RNA. the purification column was discarded
and the purified RNA became ready for Using in downstream
applications or to be stored at -20°C until use.
b. Synthesis of cDNA from RNA
11 μl RNA were added in a 0.2ml tube that placed in ice then 1
μl Oligo (dT) primer was added to get 12 μl total volume. The 12 μl
mix was Incubated at 65oC for 5 min then the tube placed back on ice
to add: 4 μl (5x) reaction buffer, 1 μl RiboLock RNase Inhibitor, 2 μl
10mM dNTP Mix (nucleotides) and 1 μl RevertAid M-MuLV Reverse
Transcriptase. The 20 μl total volume was mixed gently and put into
the PCR machine at 42oC for 60 min then at 70
oC for 5 min.
PCR reactions
Gentl vortex and brief centrifugation of DreamTaq Green PCR
Master Mix (2X) were done after thawing. In a thin-walled PCR tube
84
placed on ice, the following components were added for each 25 μl
reaction: 12.5 μl DreamTaq Green PCR Master Mix (2X), 1 μl Forward
primer, 1 μl Reverse primer, 2 μl Template DNA and nuclease-free
water to get 25 μl total volume. PCR was performd using the
recommended thermal cycling conditions consisted of 94C for 5 min,
followed by 35 cycles of 94°C for 45 s, annealing 60°C for 45 s, and
72°C for 60 s and a final extension at 72°C for 10 min.
c. DNA electrophoresis
PCR products were electrophoresed on a 1.0% agarose gel
stained with ethidium bromide and visualized under UV light and the
picture taken with a gel documentation system The following DNA
fragments were cut and purified using a TI-ANgel Midi purification kit.
d. DNA Sequence
The DNA fragment was sequenced by the Sanger method (and
Coulson, 1975).
e. DNA analysis
Sequence alignments were performed using gene-bank data base
(http://www.ncbi.nlm.nih.gov). Coding region sequence and predicted
amino acid sequence of VKORC1 were deduced from nucleotide
sequences according to the coding frame in R. norvegicus. Mutation
and polymorphism screens were then performed with ClustalW
(Thompson et al. 1994).
85
RESULTS AND DISCUSSIONS
Warfarin resistance
Resistance can hinder management strategies with bad
consequences for stored products, constructions protection,
hygiene and animal health. In many parts of the world,
anticoagulants of the first generation are not an option for the
control of resistant Norway rats. The spread of resistant rats
and conditions supporting and reducing resistance should be
investigated in order to improve resistance management
strategies and avoid the misuse of anticoagulants.
1- Monitoring resistance to warfarin using feeding test
42 Norway rats were collected from four governorates (12 rats
from Giza, 12 from Beheira, 9 from Qaliubiya and 9 from Bani-Suef)
for resistance study. No significant deference between males and
females average body weight (P< 0.05). Out of the 42 individuals, 5
rats were survived the 28-days no choice feeding test (6-day no-choice
feeding schedule of 0.005% warfarin and 22 days of post-treatment
observation). The resistance rate was 11.9%. There were two resistant
individuals found in Bani-Suef, while the other three governorates have
one individual each. Four survivals were males and the fifth one was
female. They consumed amount of active ingredient was greater than
10 mg/kg body weight. There was no significance difference between
the total consumption of active ingredient of resistant and susceptible
individuals (p < 0.05), table (11).
86
Table 11. Warfarin feeding test results for resistance monitoring.
Animals
Body weight (g)
Mortality Total consumption of active ingredient (mg/kg)
(no) % Survived Died
Site Sex, No. Mean ± SD Range Mean Range Mean ± SD Rang
Giza
Male,6 256.50±50.68 178-340 5/6 83.33% 12.45 9.47±0.68 7.02-11.92
Female,6 238.33±61.79 145-303 6/6 100% --- 10.89±0.16 7.70-18.3
Beheira
Male,7 242.43±84.09 125-368 6/7 85.70% 11.55 10.54±1.79 7.24-12.14
Female,5 272.6±71.36 212-354 5/5 100% 11.88±1.81 10.16-14.38
Bani-Suef
Male,5 268.5±115.44 136-401 4/5 100% 10.77 10.27±5.03 5.98-18.38
Female,4 315.75±125.15 148-450 3/4 100% 10.00 8.36±2.28 6.58-11.69
Qaliubiya
Male,6 271.75±90.16 120-350 5/6 83.33% 13.88 13.60±3.44 9.93-19.21
Female,3 242.17±106.66 173-365 3/3 100% - 14.83±4.45 10.64-19.50
87
2. VKORC1 analysis using Polymerase Chain Reaction
This approach has also been associated with recent genetic
advances and the identification of the first gene involved in the
synthesis of the VKOR enzyme. This first gene (VKORC1) is clearly
located on the chromosome 1 of the rat, associated with the D1Rat219
microsatellite. The embedded in the endoplasmic reticulum protein has
three trans-membrane domains. Mutated forms are associated with
severe changes in VKOR activity (Rost et al, 2004). This small protein
(18kDa) with 3 exons and encoding a small trans-membrane protein
(163 AA) was computed and a suggested structure that has been
published (Tie et al, 2005).
VKORC1 gene of 35 samples, 5 resistant (feeding-test survivals)
and 23 susceptible (died during the feeding test) was extracted,
amplified, sequenced and analyzed for mutation. Besides, 7 specimens
trapped from suspected resistance area in Giza and sent to the lab.
directly (did not undergo the feeding test).
The total RNA was isolated and reverse transcribed to cDNA.
VKORC1 gene was amplified using specific primers based on
corresponding sequences for Rattus norvegicus in Gen-Bank. The
cDNA was amplified and the PCR products were run on a 1.0%
agarose gel (Fig. 7). The corresponding cDNA fragments were cut and
purified and then sequenced according to Sanger and Coulson (1975), as
shown in Fig. 8, 9. Finally, sequence alignments were performed.
88
Fig. 7. PCR products were subjected to electrophoresis on a
1.5% agarose gel. "s" tested to be susceptible individuals, "R"
tested to be resistant individuals. "M" represents the DNA
marker, (R6 did not undergo the feeding test).
89
Fig. 8. The DNA sequence of VKORC1 gene amplified using specific primers, the product length
about 550 pb from R. norvegicus
91
Fig. 9. DNA sequencing result; the DNA sequence of VKORC1 gene amplified using specific primers, the
product length about 550 pb from R. norvegicus
90
a. Analyses of VKORC1 for SNPs
The sequence obtained was aligned with Rattus norvegicus
vitamin K epoxide reductase complex subunit 1 (VKORC1) mRNA,
complete cds Sequence ID: gb|AY423047.1| (Fig. 10) and
polymorphism screening were carried out by sequence alignment.
b. Identification of the point mutation of VKORC1 gene
Nucleotide sequences were analyzed for SNPs (single nucleotide
polymorphism) or point mutations. Nucleotide substitution name takes
the form: (position) (original nucleotide) > (substituted nucleotide),
e.g., 87C > T means the original C nucleotide at the position 87 is
changed into T.
mRNA sequence is converted into correspondent amino-acids
(Fig. 11a), then aligned with vitamin K epoxide reductase complex
subunit 1 precursor (Rattus norvegicus) amino-acid Sequence
ID: ref|NP_976080.1| (Fig. 11b) and screened for mutations.
Amino-acid substitution or mutation name takes a certain form:
original amino acid (position) mutated amino acid, e.g., when the
original amino-acid Histidine (H) is substituted at the position 28 with
Tyrosine (Y), the mutation name will be H28Y. Mutations detected in
both resistant and susceptible individuals that involved nucleotide
alteration do not have the same effect; however, there were different
types of mutations detected as follows.
92
a 1
1
76
26
151
51
226
76
301
101
376
126
451
151
GACATGGGCACCACC TGGAGGAGCCCTGGA CGTTTGCGGCTTGCA CTATGCCTCGCTGGC CTAGCCCTCTCACTG
D M G T T W R S P G R L R L A L C L A G L A L S L TACGCACTGCACGTG AAGGCGGCGCGCGCC CGCAATGAGGATTAC CGCGCGCTCTGCGAC GTGGGCACGGCCATC
Y A L H V K A A R A R N E D Y R A L C D V G T A I AGCTGTTCCCGCGTC TTCTCCTCTCGGTGG GGCCGGGGCTTTGGG CTGGTGGAGCATGTG TTAGGAGCTGACAGC
S C S R V F S S R W G R G F G L V E H V L G A D S ATCCTCAACCAATCC AACAGCATATTTGGT TGCATGTTCTACACC ATACAGCTGTTGTTA GGTTGCTTGAGGGGA
I L N Q S N S I F G C M F Y T I Q L L L G C L R G CGTTGGGCCTCTATC CTACTGATCCTGAGT TCCCTGGTGTCTGTC GCTGGTTCTCTGTAC CTGGCCTGGATCCTG
R W A S I L L I L S S L V S V A G S L Y L A W I L TTCTTTGTCCTGTAT GATTTCTGCATTGTT TGCATCACCACCTAT GCCATCAATGCGGGC CTGATGTTGCTTAGC
F F V L Y D F C I V C I T T Y A I N A G L M L L S TTCCAGAAGGTGCCA GAACACAAGGTCAAA AAGCCCTGAGGT
F Q K V P E H K V K K P * G
b Query 16 CCTGGACGTTTGCGGCTTGCACTATGCCTCGCTGGCCTAGCCCTCTCACTGTACGCACTG 75
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
VKORC1 27 CCTGGACGTTTGCGGCTTGCACTATGCCTCGCTGGCCTAGCCCTCTCACTGTACGCACTG 86
Query 76 CACGTGAAGGCGGCGCGCGCCCGCAATGAGGATTACCGCGCGCTCTGCGACGTGGGCACG 135
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
VKORC1 87 CACGTGAAGGCGGCGCGCGCCCGCAATGAGGATTACCGCGCGCTCTGCGACGTGGGCACG 146
Query 136 GCCATCAGCTGTTCCCGCGTCTTCTCCTCTCGGTGGGGCCGGGGCTTTGGGCTGGTGGAG 195
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
VKORC1 147 GCCATCAGCTGTTCCCGCGTCTTCTCCTCTCGGTGGGGCCGGGGCTTTGGGCTGGTGGAG 206
Query 196 CATGTGTTAGGAGCTGACAGCATCCTCAACCAATCCAACAGCATTTTTGGTTGCATGTTC 255
|||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||
VKORC1 207 CATGTGTTAGGAGCTGACAGCATCCTCAACCAATCCAACAGCATATTTGGTTGCATGTTC 266
Query 256 TACACCATACAGCTGTTGTTAGGTTGCTTGAGGGGACGTTGGGCCTCTATCCTACTGATC 315
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
VKORC1 267 TACACCATACAGCTGTTGTTAGGTTGCTTGAGGGGACGTTGGGCCTCTATCCTACTGATC 326
Query 316 CTGAGTTCCCTGGTGTCTGTCGCTGGTTCTCTGTACCTGGCCTGGATCCTGTTCTTTGTC 375
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
VKORC1 327 CTGAGTTCCCTGGTGTCTGTCGCTGGTTCTCTGTACCTGGCCTGGATCCTGTTCTTTGTC 386
Query 376 CTGTATGATTTCTGCATTGTTTGCATCACCACCTATGCCATCAATGCGGGCCTGATGTTG 435
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
VKORC1 387 CTGTATGATTTCTGCATTGTTTGCATCACCACCTATGCCATCAATGCGGGCCTGATGTTG 446
Query 436 CTTAGCTTCCAGAAGGTGCCAGAACACAAGGTCAAAAAGCCCTGAGG 482
|||||||||||||||||||||||||||||||||||||||||||||||
VKORC1 447 CTTAGCTTCCAGAAGGTGCCAGAACACAAGGTCAAAAAGCCCTGAGG 493
Fig. 10. a) VKORC1 gene nucleotide sequence and amino-acid residues
sequence, Accession number NM_203335.2 b) Pairwise alignment between
VKORC1 and the gene sequence of a susceptible sample (the query)
93
a GRLRLALCLAGLALSLYALYVKAARARNEDYRALCDVGTAISCSR
VFSSRWGRGFGLVEHVLGADSILNQSNSIFGCMFYTLQLLLGCLR
GRWASILLILSSLVSVAGSLYLAWILFFVLYDFCIVCI
b Sample 7 GRLRLALCLAGLALSLYALYVKAARARNEDYRALCDVGTAISCSRVFSSRWGRGFGLVEH 66
GRLRLALCLAGLALSLYAL++KAARARNEDYRALCDVGTAISCSRVFSSRWGRGFGLVEH
VKORC1 9 GRLRLALCLAGLALSLYALHLKAARARNEDYRALCDVGTAISCSRVFSSRWGRGFGLVEH 68
Sample 67 VLGADSILNQSNSIFGCMFYTLQLLLGCLRGRWASILLILSSLVSVAGSLYLAWILFF 124
VLGADSILNQSNSIFGCMFYTLQLLLGCLRGRWASILLILSSLVSVAGSLYLAWILFF
VKORC1 69 VLGADSILNQSNSIFGCMFYTLQLLLGCLRGRWASILLILSSLVSVAGSLYLAWILFF 126
Sample 125 VLYDFCIVCI 134
VLYDFCIVCI
VKORC1 127 VLYDFCIVCI 136
Fig. 11. a) Amino-acids correspondent to VKORC1 nucleotide sequence.
b) Pairwise alignment between amino-acids of VKORC1 and the
sequence of a resistant sample
1. Silent mutations (synonymous)
Some variants do not alter the amino acid sequence of the
protein as the new triple codon gives the same amino-acid. Thus, they
are likely to represent no-effect polymorphisms. This kind of mutations
is called silent mutation (synonymous mutations). Since the genetic
code is degenerate, several codons produce the same amino acid.
Especially, third base changes often have no effect on the amino acid
sequence of the protein. These mutations affect the DNA but not the
protein.
94
Fig. 12. shows a silent mutation that resulting no change of
amino-acid sequence (I82I). There is a substitution at the position 246
where "A" nucleotide changed into "T". The triplet codon "ATA"
became "ATT" but still gives the same amino-acid Isoleucine. More
silent mutations detected are shown in table (12) and table (13).
Fig. 12. Synonymous mutation – part of susceptible sample sequence.
2. Neutral mutations
Mutation that alters the amino acid sequence of the protein but
does not change its function as replaced amino acid is chemically
similar or has little influence on protein function. e.g., I133L mutation
(Fig. 13 ) are neutral because Leucine and Isoleucine amino-acids are
close to each other and there is no much difference in their influence on
protein function. The aforementioned two neutral mutations detected in
a resistant individual from Giza.
95
Fig. 13. Neutral mutation – part of susceptible sample sequence.
3. Missense mutations:
Missense mutations substitute one amino acid for another
different one. There are some variants or substitutions of nucleotides
predicted to alter the protein structure and could lead to functional
impairment or change of VKORC1 activity. Mutation screening
revealed some missense mutations, e.g., V29G, in which Valine
changes into Glutamine, Fig. 14. More missense mutations detected are
shown in tables 12 and 13.
Fig. 14. Missense mutation – part of resistant sample sequence.
96
c. VKORC1 mutations and resistance to warfarin
Since it has been discovered in 2004 by Rost et al., VKORC1
has been the main subject of many studies to explain susceptibility and
resistance of rodents and human to anticoagulants. It was published that
there is a correlation between mutations within the VKORC1 and the
anticoagulant resistance in Rattus norvegicus. Many mutations have
been reccorded in VKORC1 as conferring anticoagulant resistance in
rats and mice (Lasseur et al., 2005 and Pelz, 2007).
VKORC1 of susceptible individuals was sequenced to make
comparison between susceptible and resistant rats. Two silent and one
neutral mutations were detected, I82I, P154P and I133L. The I82I
polymorphism was identified in both susceptible and resistant rats, i.e.
two resistant and 7 susceptible rats. Since it is silent mutation, it has no
effect on the amino acid level and this was considered irrelevant to
resistance. Similarly, the I133L mutation has no tangible effect as the
Isoleucine converted to Luecine which is close to it, table (12).
The I82I mutation was previously detected in many countries
and it is among the VKORC1 mutations recorded in genebank with
dbSNP rs# cluster id: rs66459411, Table (13). This variant occurred at
high frequency in rats from all continents. Thus it may be an ancestral
variant or may have arisen several times independently (Rost et al.,
2009).
97
P154P mutation was found in two resistant individuals co-
existed with V29G. The NCBI database for short genetic variations
(dbSNP) currently includes SNPs for VKORC1. The 26 recorded
mutations (Table 13) include an SNP at position 154 where Proline
changes into Leucine (P154L) under the dbSNP entry rs8143495,
Which is a missense mutation that is different from our detected
synonymous mutation (P154P).
Of the variants which do cause amino acid substitutions, are
H28Y, V29M and E155K, table (12). All these missense mutations
were recorded in resistant individuals. One of them was previously
recorded that involves Glutamic Acid substituted with Lysine at
position 155 by Grandemange et al., (2010) in France.
In this study we found V29 is likely to be mutated; as is was
mutated in 5 resistant individuals. Rost et al., (2004) found that the
mutation V29L resulted in warfarin resistance. Also, the mutation
H28Y was found in accompany with V29. However, we might not
attribute Rattus norvegicus warfarin resistance to it unless
conformation studies are carried out to assess its effect on VKOR
activity.
Yet, to establish monitoring technique of resistance to
anticoagulants based on VKORC1 mutations we suggest that future
studies need to consider larger numbers of rats randomly collected from
local populations. Besides, more screening should be done to determine
98
the prevailing mutation which if found considered as an indicator of
resistance.
Table 12. VKORC1 mutations (SNPs) recorded in Rattus
norvegicus.
Mu
tati
on
Wil
d
cod
on
⇒M
uta
nt
cod
on
Mu
tati
on
Po
siti
on
AA
WT
⇒A
A m
ut.
AA
P
osi
tion
Fu
nct
ion
No
. o
f
spec
imen
s
Susceptible I82I ATA⇒ATT 246 I⇒I 82 Silent 7
I133L ATT⇒CTT 397 I⇒L 133 Neutral 1
Resistant H28Y CAC⇒TAC 82 H⇒Y 28 Missense 1
H28Y CAC⇒TAT 82,84 H⇒Y 28 Missense 4
V29M GTG⇒ATG 85 V⇒M 29 Missense 1
V29Q GTG⇒GAG 86 V⇒Q 29 Missense 1
V29L GTG⇒TTA 85 V⇒L 29 Missense 1
V29G GTG⇒GGG 86 V⇒G 29 Missense 2
I82I ATA⇒ATT 246 I⇒I 82 Silent 2
P154P CCA⇒CCT 462 P⇒P 154 Silent 2
E155K GAA⇒AAA 463 E ⇒K 155 Missense 1
Table 13. VKORC1 mutations (SNPs) recorded in genebank data base.
Ch
r.
posi
tion
mR
NA
pos.
db
SN
P
rs#
clu
ster
id
Fu
nct
ion
db
SN
P
all
ele
Pro
tein
resi
du
e
Cod
on
pos.
Am
ino
aci
d p
os.
206361639 483 rs8143495 missense T Leu [L] 2 154
contig ref. C Pro [P] 2 154
206361671 451 rs66459407 synonymous C Ala [A] 3 143
synonymous T Ala [A] 3 143
contig ref. G Ala [A] 3 143
206361672 450 rs66459409 missense A Glu [E] 2 143
missense G Gly [G] 2 143
contig ref. C Ala [A] 2 143
206361679 443 rs66459405 missense C Leu [L] 1 141
missense T Phe [F] 1 141
contig ref. A Ile [I] 1 141
99
Ch
r.
posi
tion
mR
NA
pos.
db
SN
P
rs#
clu
ster
id
Fu
nct
ion
db
SN
P
all
ele
Pro
tein
resi
du
e
Cod
on
pos.
Am
ino
aci
d p
os.
206361684 438 rs66459399 missense T Phe [F] 2 139
contig ref. A Tyr [Y] 2 139
206361689 433 rs66459397 synonymous A Thr [T] 3 137
synonymous G Thr [T] 3 137
contig ref. C Thr [T] 3 137
206361717 405 rs66459395 missense A Gln [Q] 2 128
contig ref. T Leu [L] 2 128
206361741 381 rs66459393 missense A Gln [Q] 2 120
contig ref. T Leu [L] 2 120
206361766 356 rs66459391 missense A Met [M] 1 112
missense C Leu [L] 1 112
contig ref. G Val [V] 1 112
206361779 343 rs66459389 missense G Met [M] 3 107
synonymous T Ile [I] 3 107
contig ref. C Ile [I] 3 107
206361791 331 rs66459387 synonymous A Ser [S] 3 103
synonymous G Ser [S] 3 103
contig ref. T Ser [S] 3 103
206362664 302 rs66459385 Missense A Ile [I] 1 94
Missense G Val [V] 1 94
contig ref. T Leu [L] 1 94
206362676 290 rs66459383 Missense T Leu [L] 1 90
contig ref. A Ile [I] 1 90
206362698 268 rs66459411 Synonymous T Ile [I] 3 82
contig ref. A Ile [I] 3 82
206362745 221 rs66459381 Nonsense T [Ter[*]] 1 67
Missense C Gln [Q] 1 67
contig ref. G Glu [E] 1 67
206362756 210 rs66459379 Missense A Tyr [Y] 2 63
Contu. Table 13.
011
Ch
r.
posi
tion
mR
NA
pos.
db
SN
P
rs#
clu
ster
id
Fu
nct
ion
db
SN
P
all
ele
Pro
tein
resi
du
e
Cod
on
pos.
Am
ino
aci
d p
os.
Missense C Ser [S] 2 63
contig ref. T Phe [F] 2 63
206362769 197 rs66459377 Missense A Arg [R] 1 59
contig ref. T Trp [W] 1 59
206363714 188 rs66459375 Missense A Thr [T] 1 56
Missense G Ala [A] 1 56
contig ref. T Ser [S] 1 56
206363765 137 rs66459373 Missense A Asn [N] 1 39
contig ref. T Tyr [Y] 1 39
206363776 126 rs66459371 Missense C Pro [P] 2 35
contig ref. G Arg [R] 2 35
206363782 120 rs66459369 Missense C Pro [P] 2 33
contig ref. G Arg [R] 2 33
206363804 98 rs66459367 Missense C Pro [P] 1 26
Missense T Ser [S] 1 26
contig ref. G Ala [A] 1 26
206363819 83 rs66459365 missense C Pro [P] 1 21
missense T Ser [S] 1 21
contig ref. G Ala [A] 1 21
206363844 58 rs66459363 synonymous C Arg [R] 3 12
synonymous T Arg [R] 3 12
contig ref. G Arg [R] 3 12
Contu. Table 13.
010
GENERAL CONCLUSION
The presence of zoonotic ectoparasites that have medical and
veterinary importance confirms Rattus norvegicus as a reservoir for
different types of pathologies, which, therefore, constitutes a risk to the
public health. The information presented in this study enables us to
understand the major parasitic infections that Norway rat harbors and
transmits to people and domestic animals in Egypt. Periodical
surveillance and monitoring in local problem areas combined with
raising awareness help local authorities in the emergency situations
prevent rodent-borne diseases.
Polymorphisms in the vitamin K epoxide reductase complex
subunit 1 (VKORC1) gene and substitutions of amino acids in the
VKOR protein are the major cause for rodenticide resistance.
Monitoring resistance to anticoagulants should be periodically done to
avoid the use of ineffective rodenticides.
This work gives information about how it is important to
allocate mutations carried by some resistant rats. It is now possible to
monitor the resistance to warfarin by detecting only the mutation
repeatedly arose in resistant population. However this work need to be
supported with some complementary studies to measure the effect of
the new mutations on VKOR activity.
012
013
SAMMARY
Rodents are a group of the largest and most successful
groups of mammals; they have a high reproductive efficiency
and great ability to adapt over a wide environmental range.
Although rodents damages are mainly associated with
agricultural crops in stores and fields or farm animals'
attacking and the destruction of facilities, their health
problems are underestimated. Rodents can be reservoirs or
carriers for a number of dangerous pathogens of humans and
farm animals. Anticoagulant rodenticides are mainly used to
eliminate the rodents and undermine the chances of the spread
of diseases and associated parasites. The emergence of
resistance problems against anticoagulant rodenticides by
some members of the rodent threatens its usage in the
foreseeable future.
Norway rat was chosen as one of the important species
of rodents in Egypt to conduct the study . The first section
dealt with the study of its endo- and ectoparasites, while the
second section tackled the study of resistance to anticoagulant
rodenticides (warfarin). Four governorates were selected to
conduct the study, namely: Giza, Beheira, Qaliubiya and Beni
Suef. The present work covers the following points:
014
a. The study of the Norway rat endo- and ectoparasites
1- Studying the Norway rat species population structure at
four different governorates.
2- Identifying the Norway rat helminthic parasites and
determining their incidence and distribution at four
different governorates.
3- Identifying the Norway rat ectoparasites, and
determining their prevalence and general indices that is
useful to understand the role of arthropod vectors as
well as mammalian reservoirs in the maintenance of
various diseases in the study areas.
b. The study of the Norway rat resistance to warfarin
1- Monitoring the Norway rat resistance to warfarin (First
generation anticoagulant rodenticide) at four different
governorates by using the conventional method, non-
choice feeding test.
2- Monitoring the Norway rat resistance to anticoagulants
rodenticides (warfarin) at four different governorates
through VKORC1 analysis using Polymerase Chain
Reaction (PCR) technique.
The results obtained were as follows:
1. The study of the Norway rat endo- and ectoparasites
a. Rattus norvegicus investigations
Eighty three Rattus norvegicus were live trapped from four
governorates: 34 from Giza, 24 from Beheira, 10 from Beni Suef and
015
15 from Qaliubiya. Their population structure was studied to study the
effect of sex and age on parasits' infection. the sex ratio was 1.37
males:1 female. Based on age, the maturity status was 53 mature and
30 immature individuals.
b. Endoparasites
In this study, we have just recorded two cestodes:
Hymenolepis diminuta and Cysticercus fasciolaris, which are
commonly found in rats and mice and they are potentially
transmissible (Zoonosis) to man and one non-zoonosis
nematode, Spirura talpae. No new species were recorded
during the study.
Sixty five individuals out of 83 were infected with one
or more helminthic parasites with an infection rate of 78.31 %.
The type of infection of helminthic parasites varies
among individuals. Some individuals were infected with only
one helminthic parasite, 27 individuals (32.5%) and some
were double infected, 32 individuals (38.5%) while triple
infection was recorded in just 6 individuals (7.2%).
1. Infection prevalence of Endoparasites based on host
location.
Location of infestation could have a tangible effect on infection
prevalence. However, in this study, the rate of rodent infection
with nematodes and cestodes does not considerably differ
among locations.
016
There was no concrete difference among the cestodes
infection percentages in three locations, as it was 70.59%,
73.33% and 75% in Giza, Qaliubiya and Beheira; respectively,
but in Bani-Suef, it was higher (90%). Likewise, the nematode
infection percentages were 41.18%, 33.33% and 40% in Giza,
Beheira and Qaliubiya; respectively and it was slightly greater
in Bani Suef (50%). The combined infection percentages of
both cestodes and nematodes exhibited the same pattern.
2. Infection prevalence of endoparasites based on host
sex
Both Rattus norvegicus sexes were examined for their
endoparasites. Regarding cestodes, males were more infected
than females as 39/(83) males were infected (46.99%) versus
23/(83) females (27.71%). The prevalence percentage on
males was 81.25% (the percentage of males infected out of the
total number of males) while, it was 65.71% on females. This
indicates that the rate of the infection prevalence on males is
greater than that on females. Similarly, nematodes infection
was greater on males, 20 (24.1%) than that on female, 13
(15.66%). But the prevalence of infection of male's population
was close to that of female's; 41.67% for male's and 37.14%
for female's; respectively.
3. Infection prevalence of Endoparasites based on host age
In this study, 44 individuals out of 83 (53.01%) were
cestode infected mature and the infected immature individuals
017
were only 18 (21.69%). The prevalence of infestation among
mature individuals was greater than that among immature
individuals as 83.02% of mature individuals were infected
versus 60% of immature individuals.
As to nematode infection, 28 (33.73%) were infected
mature individuals while 5 (6.02%) individuals were infected
immature. The prevalence of nematode infection among
mature individuals was 52.83% but it was only 16.67% among
immature individuals.
c. Ectoparasites
Rodents in particularly, Rattus norvegicus are usually infected
with certain groups of arthropods; fleas, lice and mites. In this study
77.2% of Rattus norvegicus were infested with at least one ectoparasite.
Results of this study revealed that 938 ectoparasites, comprising: 140
(14.93%) fleas, 234 (24.95%) lice and 564 (60.1%) mites, are
belonging to 4 orders, 7 families, 9 genera and 9 species.
1. Infection prevalence and general indices of ectoparasite
according to location:
As to fleas' infection, Giza governorate had the highest infection
percentage (50%) and the highest flea index as well (2.56). On the
other side, Beni Suef had the lowest flea infection percentage (20%)
and the lowest flea index (0.5).
Although Beni Suef governorate had the highest lice infection
percentage (50%), Giza governorate had the highest lice index (3.76).
018
this means that the lice burden is higher in Giza than that in the other
three locations. In the same context, Behaira governorate had the
lowest lice infection percentage (25%), but its lice index (2.46) is
bigger than that of Beni Suef (2.1) and Qaliubiya (1.73), table (2).
With regard to mite infection, Beni Suef governorate came first
(70%) followed by Beheira Governorate (66.67%) while Qaliubiya had
the lowest percentage of infection (40%). Mite indices were relatively
high; since it ranged from 4.27 in Qaliubiya governorate to 11.3 in
Beheira governorate.
2. Infection prevalence of ectoparasites based on host sex
Nineteen infected male individuals (22.89%) represented
39.58% of the whole males' population. Infected females were 10
individuals with a percentage of 12.05%. The prevalence of infection
among females was 28.57%.
Regarding lice infection, a total of 12 male-individuals (out of
83, the whole population) were infected (14.46%). The infection
prevalence among them was 25% (12 out of 48 males). Infected
females' number was equal to that of males' (12, 14.46%) but the
infection prevalence among females (34.28%) was greater than that
among males.
Mite infection and prevalence was the greatest comparing to
other ectoparasites as 28 males (33.73%) and 18 females (21.69%)
were infected. Also the prevalence of infection among males (52.33%)
and females (51.43%) was the highest when compared with fleas and
019
lice. There were no differences of infection prevalence based on host
sex.
General indices of ectoparasites based on host sex:
The flea index in males is bigger than that in females in all
governorates except for Giza but the total flea indices in both males and
females are equal (1.69). There was a big difference between the
male/female lice indices in Beheira and Beni Suef as they were
0.86/4.7 and 0.6/3.6; respectively, but the total lice index in males
(2.85) was slightly higher than that in females (2.77). With regard to
mite, the total mite index was approximately bigger in males than it in
females. But still there were some differences according to locations,
table.
3. Infection prevalence of ectoparasites based on host age:
A total of 20 mature individuals versus 9 immature individuals
were infected with fleas. The flea infection prevalence inside the
mature population (37.74%) was relatively higher than that inside the
immature population (30%).
Lice infection varied between mature and immature rats, as a
total of 18 mature individuals (21.69%) and 6 immature individuals
(7.23%) were infected. The infection prevalence of lice inside the
mature population (33.96%) was higher than that inside immature
population (20%).
Unlike fleas and lice, mites' infection was higher and more
prevalent; as 33 mature individuals (39.76%) and 13 immature
001
individuals (15.66%) were infected. When comparing the infection
prevalence between mature and immature individuals, it found that the
infection prevalence in mature individuals (62.26%) was greater than it
in immature individuals (43.33%).
General indices of ectoparasites based on host age:
Generally, mature individuals tend to have bigger ectoparasite
index than immature individuals. Flea index was 1.96 in mature
individuals versus 1.2 in immature's, also lice index in mature
individuals (3.75) was three times bigger than it in immature's (1.17).
Likewise, the mite index was bigger in mature individuals (7.15) than it
in immature's (6.17).
Part II: Warfarin resistance study
The study of the Norway rat resistance to warfarin was done
through two methods. The first method involved the use of traditional
test known as no-choice feeding test while the second method, the
latest currently used, involves VKORC1 gene analysis to search for
mutations associated with resistance in some rodent individuals.
1-Feeding test (no-choice)
Forty two Norway rats were collected from four governorates
(12 from Giza, 12 from Beheira, 9 from Qaliubiya and 9 from Bani-
Suef). No significant deference between males and females average
body weight (P < 0.05). Out of the 42 individuals, 5 rats were survived
the 28-days no choice feeding test. The resistance rate was 11.9%.
There were two resistant individuals found in Bani-Suef, while the
000
other three governorates have one individual each. The survived rats
consumed amount of active ingredient greater than 10 mg/kg body
weight. There was no significance difference between the total
consumption of active ingredient of resistant and susceptible
individuals (p < 0.05).
2- VKORC1 sequencing using Polymerase Chain Reaction (PCR)
technique
VKORC1 gene of 35 samples, 5 resistant (feeding test survivals)
and 32 susceptible (died during the feeding test) was extracted,
amplified, sequenced and analyzed for mutation.
Analyses of VKORC1 for single nucleotide polymorphism
(SNPs)
The gene sequence obtained was aligned with Rattus norvegicus
vitamin K epoxide reductase complex subunit 1 (VKORC1) mRNA,
complete cds Sequence ID: gb|AY423047.1| and polymorphism
screening were carried out by sequence alignment.
mRNA sequence is converted into correspondent amino-acids,
then aligned with vitamin K epoxide reductase complex subunit 1
precursor (Rattus norvegicus) amino-acid Sequence
ID: ref|NP_976080.1| and screened for mutations. Three types of
mutation have been recorded as follows:
002
VKORC1 mutations and resistance to warfarin
Two silent and one neutral mutations have been detected, I82I,
P154P and I133L. The I82I polymorphism was identified in both
susceptible and resistant rats, 2 resistant and 7 susceptible rats. Since it
is silent mutation, it has no effect on the amino acid level, and this was
considered irrelevant to resistance. Similarly, I133L mutation has no
tangible effect as the Isoleucine converted to Luecine.
The I82I was previously detected in many countries and it is
among the VKORC1 mutations recorded in genebank with dbSNP rs#
cluster id: rs66459411.
P154P mutation was found in two resistant individuals co-
existed with V29G. The NCBI Database for Short Genetic Variations
(dbSNP) currently includes SNPs for VKORC1 of which an SNP at
position 154 where Proline changes into Leucine (P154L) under the
dbSNP entry rs8143495.
E155K, V29G, V29L, V29M, V29Q and H28Y are of the
mutations that involved amino acid substitutions. All these missense
mutations were recorded in resistant individuals. One of them was
previously recorded that involves Glutamic Acid substituted with
Lysine at position 155 by Grandemange et al., (2010) in France. Rost et
al., (2004) found that the mutation V29L resulted in warfarin
resistance. In this study we found that V29 is likely to be mutated; as is
was mutated in 5 resistant individuals. Also, the mutation H28Y has
been found in an accompany with V29.
003
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