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 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.
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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.
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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.
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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.
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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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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
9 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
01 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
00 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
02 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).
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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
04 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
05 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).
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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 lips, 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
07 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).
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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
09 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
21 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).
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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
22 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
23 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.
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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
25 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
26 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
27 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.
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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).
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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.
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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).
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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
33 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.
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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
35 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
36 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.
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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
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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).
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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 Infection Total Infection Total Infection infected % infected % infected % No. No. No. 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
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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".
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Table 4. Infection prevalence of endoparasites based on host sex Males Females Governorate Infected Infected Infection Infected Infected Infection males' males' prevalence females' females' prevalence No. % % No. % % 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 42 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
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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-
43 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).
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Table 5. Infection prevalence of Endoparasites in mature and immature rats Mature Immature Infected Infected Infection Infected Infected Infection Governorate mature mature prevalence immature immature prevalence (No.) (%) (%) (No.) (%) (%) 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
45 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
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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:
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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
47 sanguineus felis, Fig
. 5. Arthropode recorded ectoparasites on
(3)
Echidnophaga gallinacea Echidnophaga
, (7)
Laelaps nuttalli
, (8)
, (4)
Ornithony
Polyplax spinulosa, Polyplax spinulosa,
ssus bacoti ssus
Rattus norvegicus Rattus
and (9)and
(5)
Hoplopleura oenomydis
. (1)
Radfordia ensifera
Xenopsylla cheopis,
, (6)
(2)
Liponyssoides
Ctenocephalides
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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).
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Table 6. Infection prevalence and general indices of ectoparasite according to location Flea Lice Mite Gov. Total General Total General Total General infected flea index infected Lice infected Mite No, (%) No, (%) index No, (%) 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
51 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.
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Table 7. Infection prevalence of the ectoparasites on both male and female hosts Males Females GOV Infected Infected Infection Infected Infected Infection males' No. males' % prevalence% females' No. females' % 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
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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,
53 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.
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Table 9. Infection prevalence of ectoparasite on mature and immature individuals GOV Mature Immature
Infected Infected Infection Infected infected Infection Mature No. Mature % prevalence% Immature Immature prevalence % No. % 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
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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%).
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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
57 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).
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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.
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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.
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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
60 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.
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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.
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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
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(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).
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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
66 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
67 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
68 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
69 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
71 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
70 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
72 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.
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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
74 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
75 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,
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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
77 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%
78 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).
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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.
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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.
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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
83 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).
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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).
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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
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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.
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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).
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Fig. 8. The DNA sequence of VKORC1 gene amplified using specific primers, the product length about 550 pb from R. norvegicus
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Fig. 9. DNA sequencing result; the DNA sequence of VKORC1 gene amplified using specific primers, the product length about 550 pb from R. norvegicus
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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.
90
a GACATGGGCACCACC TGGAGGAGCCCTGGA CGTTTGCGGCTTGCA CTATGCCTCGCTGGC CTAGCCCTCTCACTG 1 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 1 TACGCACTGCACGTG AAGGCGGCGCGCGCC CGCAATGAGGATTAC CGCGCGCTCTGCGAC GTGGGCACGGCCATC 76 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 26 AGCTGTTCCCGCGTC TTCTCCTCTCGGTGG GGCCGGGGCTTTGGG CTGGTGGAGCATGTG TTAGGAGCTGACAGC 151 51 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 226 ATCCTCAACCAATCC AACAGCATATTTGGT TGCATGTTCTACACC ATACAGCTGTTGTTA GGTTGCTTGAGGGGA 76 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 301 CGTTGGGCCTCTATC CTACTGATCCTGAGT TCCCTGGTGTCTGTC GCTGGTTCTCTGTAC CTGGCCTGGATCCTG 101
376 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 126 TTCTTTGTCCTGTAT GATTTCTGCATTGTT TGCATCACCACCTAT GCCATCAATGCGGGC CTGATGTTGCTTAGC 451 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 151 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)
92
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.
93
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.
94
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. 95
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).
96
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
97 the prevailing mutation which if found considered as an indicator of resistance.
Table 12. VKORC1 mutations (SNPs) recorded in Rattus
norvegicus.
s
Mutant AA mut.
⇒ ⇒ Mutation Wild codon codon Mutation Position AA WT AA Function No. of specimen Position 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.
.
pos
. .
cid
Chr. position mRNA pos dbSNP rs# id cluster Function dbSNP allele Protein residue Codon pos Amino a
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
98
Contu. Table 13.
.
pos
. .
cid
Chr. position mRNA pos dbSNP rs# id cluster Function dbSNP allele Protein residue Codon pos Amino a
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
99
Contu. Table 13.
.
pos
. .
cid
Chr. position mRNA pos dbSNP rs# id cluster Function dbSNP allele Protein residue Codon pos Amino a
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
011
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.
010
012
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:
013 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
014
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.
015
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
016 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).
017 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
018 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
019 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
001 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:
000
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.
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