MICROBIOLOGICAL EXAMINATION OF WILD RATS LIVING IN VARIOUS ENVIRONMENTS IN THE EPIZOOTIC ASPECT

Edyta Wincewicz

ABSTRACT

The goal of the research was to specify the share of rats, coming from different environments, in transmitting and remaining in the environment of bacterial, virus and mycotic infections.

Key words: rats, bacteria, viruses, fungi, environment, epizootiology.

INTRODUCTION

Rats belong to the most troublesome and detested plagues tormenting people from the beginning of mankind. An estimated number of 10 milliard rats are present in the world. In Poland it is difficult to evaluate their quantity, therefore we can only assume that among 40-million human population there may be 40-million rats population. These are synanthropic animals, i.e. animals that have to live near people providing them both with food and shelter. At present two species of rats dominate in Europe: wild brown and black rat. The oldest records of rat’s presence in Europe date back to the period of early and late Pliocene. The black rat currently prevails in our continent, particularly far into land. The brown rat is most frequently to be encountered in our region in ports, yet also far into land one may come across its larger clusters [36].

Among the two rat species encountered in Poland, the black rat (Rattus rattus) stays most eagerly in warm cellars, near central heating boilers, in dark gaps of building seats and in wall corners. The body length of this rodent amounts to 21 cm, and its tail is longer than its trunk. This species is good at climbing and jumping, but it does not like swimming. It is active mainly in the night. It lives in family colonies which can number as many as several hundreds of individuals. The female bears from 2 to 6 litters per year numbering on average 6 offspring each time. Unlike the brown rat, the other species – the brown rat (Rattus norvegicus) does not avoid water and swims eagerly. Its body length is 26 cm, and its tail is shorter than its trunk. The black rat settles down most frequently in water pipes and sewage pipes as well as in gas and telephone channels, and from these places it moves to store houses, inventory buildings and dwelling-houses. It is omnivorous and active 24 hours a day. It feeds everywhere: in cereal store houses, slaughterhouses, in waste dumps and shop store houses. It lives in family herds, in which all colony members originate from the same parent couple [1,14,16,32].

The economic damages inflicted on the human by rats are mainly caused by their feeding routines. One adult rat needs 40-100 g of food a day, thus it eats approx. 30 kg of bread a year. Generally, it feeds on people’s and animals’ food as well as on plants and small vertebrates, and it eats paper and fabrics. Since its constantly growing teeth must be abraded, it bites concrete, glass, metal plates, clothing, footwear and furniture. The high losses resulting from rats’ presence are also connected with mechanical damages and contamination of food products with faeces and urine [1,14,16,32,36].

However, rats constitute not only an economic and social problem. Serious hazard generated by them results from the undisputed role in the epizootiology of many infectious and invasive diseases. Research demonstrates a thoroughly widespread character of rats’ carrier state of numerous microorganisms and parasites causing dangerous diseases of humans and animals. The presence of rats contributes to the formation of permanent pathologic focuses in the environment, where the germ circulates among vertebrates and insects, and in favourable circumstances it also attacks the human [1,14,16,24,32,36]. A considerable increase of rat population after the flood of July 1997 was a reason for renewed interest in this rodent species and their current hazard for people and animals. There grows a need for improving deratization methods and ways of protecting groceries and animal fodder against rat access as well as determining the scale of risk of increasing the incidence of diseases carried by them.

Virus infections

Rhabdovirus. It has been assumed for a long time that small forest and field rodents, including rats [49,50], constitute a reservoir of rabies virus. According to Żmudziński and Smreczka’s data [58], 12 230 cases of rabies in animals were noted within a period of 5 years (1990-1994) in Poland, out of which 9 998 cases were diagnosed in animals living at large and 2 232 – in domestic animals. Among animals living at large, 82.7% of rabies cases concerned red fox, which is the main reservoir of this germ both in Poland and in other European countries. The fact of noting rabies in the rat should not disregard its role in the epizootiology of rabies only in 4 cases (0.04%), for the reason of the possibility of asymptomatic course of the disease and little chance of picking out rats infected with rabies virus [50,58].

Herpesviridae. The worldwide virus of Aujeszky - Pseudorabies (SHV-1) disease belongs (among others) to this family of viruses, and is characterised by diversified pathogenicity in relation to different animal species. In carnivorous animals (dogs, foxes, minks), SHV-1 causes an acute fatal disease in each age group [8,21,34]. In pigs the losses connected with the disease concern the period of reproduction and young animals, and the fact of asymptomatic carrier state is a significant problem hindering the disease eradication [34,36]. The occurrence of Aujeszky’s disease on swine farms frequently coincides with the period of rats’ number increase. Cases of rats falling ill are observed on such farms, proceeding with clinical symptoms, resembling Aujeszky’s disease; cerebral tissue pulp of ill rats serves for infecting rabbits, in which the disease develops with characteristic symptoms: pruritus and nervous signs [34]. An Alimentary type of getting infected with this disease was observed in pigs after they ate carrion or killed rats afflicted with Aujeszky’s disease [34].

The role of rats in carrying other herpesviruses cannot be excluded, the common characteristic of the herpesviruses being the ability to hamper replication and its repeated restoration, which is demonstrated by the occurrence of latent infections that are impossible to detect by means of routine diagnostic methods and cyclic spreading under the influence of different factors activating renewed replication [21,34]. In the face of these facts, the possibilities of detecting herpesviruses in rats and their role in the transmission of herpesvirus infections require broader research with the application of modern diagnostic methods.

Picornaviridae. Data from literature confirm rats sensitivity to aphthous fever virus infection, which indicates the role of rodents in developing and keeping of aphthous fever epizootia. Rats can be carriers of aphthous fever virus as well as mechanical vectors. If the virus has a high infectivity, a short rodent’s contact with the infected material, e.g. during rushing across excreta, saliva or food contaminated with the ill animal’s secretion, is enough to carry the virus to another place or farm [34].

Parvoviridae. Parvoviruses demonstrate a special predilection to cells that are subject to fast cell division, therefore they particularly attack intestinal villis (Canine Parvovirus-2, Feline Parvovirus, Racoon-dog Parvovirus) or fast dividing cells of a developing embryo (Porcine Parvovirus, Blue Fox Parvovirus) [21,34].

Rat’s parvovirus, similarly as pigs’ or blue foxes’ parvovirus, occurs in adult rats in the form of persistent infection, while in pregnant females there comes to intrauterine infection and decay of developing embryoes or fetus [21]. Because of high variability of parvoviruses and their ability to change tropism, the active mode of carrying animals’ and people’s parvoviruses by rats cannot be excluded [21,34].

Hantavirus. In people the Hanta virus occurs under the condition of a direct contact with rats, and it causes hantavirus fever and hemorrhagic fever with the renal syndrome. The disease manifests itself in fever, headache, backache, extravasation quality and acute renal failure [6,12]. Hanta fever occurs in the Far East. The highest intensification of this disease was noted during Corean War. Over 3 thousand ONZ soldiers became ill, and mortality reached 5-10% [11].

Hantavirus occurs in wild rodents, which are at the same time its biggest reservoir. The mode of the virus transmission inside the rat population is not fully recognised, but it probably spreads along the aerogenic route with secretion from the nose or through blood during fights among individuals [6,11,12]

Examinations conducted in Great Britain on 173 rats in order to prove the hantavirus presence revealed that only 5 of them were seropositive. No predisposition concerning age or sex was proved; no antibodies occur in young individuals [11,13]. The Portugese, on the other hand, when examining the population of 131 rats - Rattus rattus i Rattus norvegicus - in order to prove the hantavirus, showed its presence in the lungs of 4 individuals, and the occurrence of specific antibodies was found in 12 individuals [6,27]. Rats and international navigation are the only reliable link through which the virus can spread into the whole world [11,12,39].

Bacterial infections

Leptospira spp. Leptospire infections common in mammals are manifested by a diversified clinical picture. Depending on the serovariant, these germs cause a disease of a septic character, or with changes of the urinary system, liver and also the nervous system. They are also a reason for disorders in females’ reproductive system as well as spontaneous abortions [35].

Rats suffer from leptospirosis and, like in other animals, long-lasting carrier state of leptospires occurs in their renal tubules, from where they are excreted with urine. People and domestic animals get infected through a contact with germ contaminated water or wet soil or through a direct contact with rats [35]. Scientific and popular science literature often mentions that the rat population is infected with leptospires, nearly always with L. Icterohaemorrhagiae serotype, widespread in 70-90% [30, 51]. The British, when carrying out investigations in 1994, proved that the infection of these microorganisms in the rat population amounts only to 14% in their country, and that two serotypes are widespread: L. icterohaemorrhagiae i L. Bratislava, concluding at the same time that the role of rats in carrying leptospirosis is smaller than stated before [50]. Russian researchers, on the other hand, revealed leptospire carrier state in rats in 10-40% [35].

The percentage of leptospire carriers in the rat population depends on the type of environment where they stay. Independently of that, the fact of leptospire spreading with urine creates a high risk of infecting people and animals through rats, owing to contaminating food and animal fodder with their urine. Leptospirosis enzootias on animal farms are most frequently connected with the presence of a large number of rats feeding on unprotected fodder and in feed store houses [35].

Listeria spp. Listerias are carried by many species of animals. They are detected in water and ensilage. Listeriosis occurs rather sporadically in Poland. It cannot be excluded, however, that the number of registered cases of this disease is much smaller than in reality [26]. The role of rats in spreading this zoonosis is not recognised in detail. Examinations conducted by British scientists on 44 rats revealed the occurrence of listerias in 11% of animals, among which 2 cultures were identified as L. monocytogenes, 2 as L. innocua and 1 as L. grayi or L. murrayi [53]. Similar results were obtained by Ogneva in Russia [32].

Yersinia spp. The role of rats in carrying plague, caused by Yersinia pestis, has been recognised for a long time. Another microorganism of this type - Yersinia pseudotuberculosis, causing pseudo-tuberculosis in animals and people, is also commonly associated with rodents, including rats, which is reflected even by accepting a customary name of the disease (reserved for rodents) as rodenciosis [9,18,47]. Currently Yersinia enterocolitica infection creates a serious hazard. The epidemiology of this zoonosis is not fully recognised in Polish conditions, even though the serological examinations results indicate frequent occurrence of this infection in people. Yersinia enterocolitica can also be isolated from intestines and organs of various animals [10,42,43,44]. A human is infected by rats and several other mammals and aves indirectly or directly through animal fodder contaminated with excreta. Yersinia pseudotuberculosis and Y. Enterocoliti ca were isolated from wild rats in Japan and the USA [20]. Therefore, rats are a serios reservoir of all kinds of bacteria of Yersinia type.

Francisella tularensis. This germ causes tularemia, one of the diseases in which the epidemiologic role of rats has been proved. The brown rats falling ill with tularemia was first successfully proved in 1925. The ill rodents disseminate tularemia germs in the external environment and are a dangerous infection source for animals. An important role in carrying the germ from ill rodents to other animals and people is played by ticks, which parasite on rodents in the phase of larva and nymph, and as adults – on other animals. It has been established that tularemia germ, passing from an adult female through an ovum in the phase of larva and then nymph, survives 247 days. It also survives in carcasse of carrion animals for several weeks [34,36].

Brucella spp. The participation of rats in falling ill and in carrier state of Brucella microorganisms has been proved. Poljakov detected brucellas in 11 individuals among 34 rats caught in rooms where there were animals suffering from brucellosis. In another case, the author managed to isolate brucellas from spleen, liver, lymph nodes and blood in 7 out of 18 rats caught on an infected farm [34].

Rats can be a reservoir of Brucella sp. rods on a farm not only through preserving infection among farm animals, but also through falling ill of the rats themselves becoming infected as a result of cannibalism, since healthy rats, when eating individuals suffering from brucellosis, also get infected and start to be ill. There were also cases when, after removing ill animals from the farm and after conducting disinfection, the healthy animals brought again fell ill. It was a result of leaving on the farm rats suffering with brucellosis, which contaminated fodder, feeders and rooms with excrement [34].

Coxiella burnetti. It causes a disease called Q fever. At first it was regarded as zoonosis of relatively small significance. Two forms of the disease were found in people: acute and chronic. There occur influenza-like symptoms, hepatitis, encephalitis and meningencephalitis as well as endocarditis [28,46,52]. Infection in other mammals usually proceeds asymptomatically. Cattle, sheep, geese, cats and wild rats constitute a reservoir of Coxiella burnetti, which was particularly confirmed by examinations carried out in India and Africa [57].

The role of rodents in carrying other rickettsioses, including typhus fever (Rickettsia prowazekii), was also proved. It was established that rickettsias of the rat type cause infection in guinea pigs through injured skin, and injured mucosa of nose and oral cavity. The infected guinea pigs excrete rickkettsias of the rat type with urine [34].

Salmonella spp. Rats can be carriers and spreaders of Salmonella rods. It was proved that they are an important vector in spreading and maintaining salmonellosis in a pigsty, caused by Salmonella ser. choleraesuis [32].

Chlamydia spp. Among three genuses of Chlamydia type, two of them: Chlamydia pneumoniae i Chlamydia trachomatis cause infections mainly in people, while Ch. psittaci occurs commonly in animals, and its pathogenic strains are the reason for many diseases both in aves and in mammals [4,5,19]. However, the role of rats in epizootiology of chlamydiosis is not clear until today [45].

Borrelia burgdorferi. This treponema is widespread, particularly in the northern hemisphere, and the occurrence of borreliosis has been described in nearly all European and North American countries. It is carried to the human and animals by ticks of Ixodes type. The reservoir of B. burgdorferi is constituted by animals living at large, among others rats, which (even though they do not have a disease themselves) are the source of infection for ticks feeding in different development phases [23]. When feeding on the blood of the infected animal, the tick gets infected itself, and then carries the infection to successive hosts [48]. The influence of rats on the borreliosis transmission has not been proved so far, though the germ has been found in several other rodents [29]. The presence of Borrelia spp. was not noted in examinations carried out on rats by the British [53].

Mycotic infections

Dermatomycoses. These are mysoses concerning mainly the cornifying epidermis layer, though fungi causing these diseases can also be found on skin and hair of individuals not demonstrating any pathologic lesions. Trichophyton mentagrophytes and Trichophyton schloenleini oraz Microsporum spp. are the most frequently occurring fungi on rats’ skin. Rats have fungi relatively seldom, but they are passive carriers of pathogenic fungi spores [21]. There are known cases of carrying the disease to a farm with fodder and by domestic and field mice, which are often infected by fungi of Trichophyton type. Spiesiwcewa found considerable spreading of mycoses among rats [40]. In some geographic regions, the infection of these rodents varies between 10-15%. It is these animals that constitute a reservoir of fungi in the external environment and contribute to farm and domestic animals falling ill with mycosis [40].

Candidiasis. The disease is caused by yeast-like fungi of Candida type. These are saprophytes widespread in the external environment, and can live on various kinds of ground and in the soil. Animals and aves are carriers of these fungi [25,40]. Candidiasis can occur as a primary or secondary chronic illness involving various infectious and non-infectious diseases [17,40]. On chicken farms, apart from endogenic infections, exogenous infection is also possible. Fungus is carried to a flock of birds free from the disease by ill individuals or through water and fodder contaminated with rodents excreta [40].

Aspergillosis. Mildew fungi occur very commonly both in the air, soil and in water. Until recently, they were considered as impurities in case of being isolated from dermal pathologic lesions. At present, the pathogenicity of some mildew fungi seems unquestionable [33,38]. Fungi spores float in the air, and in case of mucous membrane function impairment as a result of, for example, virus infections, it may come to airways colonising with them and causing a disease [17]. Fungi of Aspergillus type most often causing pathologic lesions include successively: A. fumigatus, A. flavus, A. glaucus, A. nidulans and A. niger [54].

AIM OF RESEARCH

Due to the fact that rats play a considerable role in epizootiology and epidemiology of infectious and invasive diseases, research has been undertaken in order to establish the participation of rats in virus and bacterial infections transmission with particular regard paid to infectious factors not examined before in this group of rodents.

Within the main goal, detailed examinations were carried out:

1. Bacteriological and mycological examinations with particular regard paid to the presence of microorganisms in organs enabling their carrying, such as oral cavity, rectum and skin.

2. Serologic examinations to prove the presence of listeriosis, leptospirosis, borreliosis, yersiniosis and rodenciosis.

3. Virusological examinations to reveal infections of herpesviruses, parvoviruses, rotaviruses and rabies virus.

4. Examination to reveal chlamydiosis.

MATERIAL AND METHODS

The total number of wild rats included in the examinations was 85, classified into 5 groups depending on the environment of their living, and 6 laboratory rats Buffalo (test group), coming from the laboratory of the Medical Academy in Wrocław. The number composition of particular groups is presented below:

- group 1 (G 1) – rats coming from the Main Railway Station in Wrocław – 12 individuals,
- group 2 (G 2) – rats coming from central streets in Wrocław – 22 individuals,
- group 3 (G 3) – rats coming from swine farm – 25 individuals,
- group 4 (G 4) – rats coming from poultry farm – 15 individuals,
- group 5 (G 5) – rats coming from swine slaughterhouse – 11 individuals,
- group 0 (G 0) – test group – 6 individuals.

The bacteriological and mycological examinations of rats coming from the first three groups (G1, G2, G3) were conducted taking into consideration 2 seasons: winter-spring season and summer-autumn one.

The rats were caught into specially designed cages – traps, and then transported to a clinical laboratory and narcotised with the use of chloroform; then, inspection and postmortem examinations were carried out accompanied by drawing blood, swabbing, taking faces and organs for detailed examinations.

Examinations to reveal bacterial and mycotic infections

Bacteriological and mycological examinations

Swabs from oral cavity and pharynx, and liver and rectum were assigned for bacteriological examinations. Apart from the mentioned organs skin scrapings with hair were also taken for the purposes of mycological examinations. Additionally, organs inoculations were carried out in case of finding anatomicopathologic changes on the basis of postmortem examination.

The material was inoculated on standard agar, blood agar, Mc Conkey’s base and on Sabouraud’s base. Salmonella spp. isolation was conducted on fluid selenic base, from which another inoculation was carried out after 24 hours of incubation at the temperature of 37°C. The isolated bacteria from Enterobacteriaceae family were identified with the use of biochemical series. Identification tests Streptotest i Staphytest (LACHEMA) were used for staphylococci and streptococci. Inoculations to prove Listeria sp. infections were performed on fluid base (broth with nalidixic acid) and on solid base: standard agar, blood agar, tryptose agar with nalidixic acid and potassium telluryn and on Mc Conkey’s base. Incubation was carried out at the temp. of 26°C for 48 hours, and then the broth passage onto tryptose agar was performed and was subject to repeated incubation. Inoculation plates and the broth were placed at the temp. of 4°C, and the growth control was conducted e very day for the period of 10 days. Stained preparations were made with the Gram method out of colonies suspected as likely to belong to Listeria sp. The procedure was repeated 3-fold.

Serologic examinations

Examination to prove the occurrence of listeriosis

The serologic examination was conducted with the application of tube agglutination method. Undiluted antigen was added in the same volume to tubes including 0.5 ml of subsequent serum dilutions (from 1:20 to 1:1280) (Listeria monocytogenes, SO I, II, and 0, V, Bioveta). At the same time, the monitoring of positive and negative serum as well as of antigen was set. The results were read out after 18 hours of incubation at the temp. of 37°C.

Examination to prove the occurrence of leptospirosis

Serologic examination was conducted with a routine method of microscopic agglutination, elaborated in the Leptospirosical Centre of PZH branch and PIW Laboratory of Leptospirosis Research.

10-day old liquid culture of the following serovariants was used for the examination: Leptospira interrogans: icterohaemorrhagiae, grippotyphosa, sejroë, tarassovi, canicola and hebdomadis. Dilutions of serums from 1:50 in the geometric progression were carried out with tap water boiled for 20 min. and filtered through a filter paper. Drops of alive leptospires were added to particular drops of diluted serums put onto slides in the same volume (0.03 ml). Separate slides were used for each serotype. At the same time, checking was set with positive serum and negative checking with tap water. Samples were kept in a humid chamber at room temperature for 90 min. The read-out was conducted in a microscope with dark field of vision. In case of a negative test, the unchanged culture of alive leptospires was observed, while in positive tests – lysis and agglutination in a different degree of intensification.

Examination to prove the occurrence of borreliosis

Serologic examination made in order to prove the presence of antibodies anti–Borrelia burgdorferi was conducted with the use of ELISA test having the ISO 9001 certificate, called RIDASCREEN Borrelia burgdorferi, of R–biofarm company. The test was carried out according to the procedure provided by the producer. Two positive and two negative tests were always set with the examined samples to verify the correctness of testing.

The examined serums of room temperature were prepared to the test by adding, in equal quantity, 200 µl of the examined serum and diluent to the serums. The samples of prepared serums were put into the pits of polystyrene plates according to the procedure, and when subsequent incubations and rinsing were done and conjugate and substrate added, the results were read out in the spectrophotometer for microplates (Spectra) with recommended wavelength of 450 nm. The conjugate was prepared ex tempore, and diluted with rinsing fluid in the ratio of 1:11, until the quantity of reagent necessary for the examination was obtained. The results were interpreted after calculating the mean deviation values of positive and negative tests and after confirming the correctness of testing. According to the interpretation rules, the mean extinction of negative tests should be below 0.15, and the mean extinction of positive tests – above 0.8.

Examination to prove the presence of yersiniosis and rodenciosis

The serologic examination was made with the use of the tube agglutination method. Reference strains of Y. enterocolitica serotype I A and V as well as Y. pseudotuberculosis serotype I – V were used for the examinations. Antigens were prepared out of the strains mentioned, using a smooth form of microorganisms raised for 48 hours at the temperature of 20°C and for 24 hours at the temperature of 4°C on solid base (Agar – Triptose Difco). Formalinised antigenes were used for the serologic reaction (formalin concentration of 0.3%), which were kept at the temperature of 4°C. The agglutination reaction was conducted by diluting the examined serums with physiologic saline from 1:20, then a drop of antigene was added to each of them, and then they were incubated throughout the night at 37°C and 18–20 hours at room temperature, and then the read-out was made. The result of the serologic reaction was assessed with naked eye, and the titre was determined as the highest se rum dilution giving clear agglutination.

Examination to prove virus infections

Direct methods of virus infections detections

For the purpose of examination performed to prove the parvovirus and rotavirus infection, faces was taken directly from rectum. The parvovirus presence examination was conducted by means of ELISA test “Diagnostic Canine Parvovirus Test for dog, cat, mink” (On-Site-Biotech, Uppsala). A faces sample was placed in buffered solution (test component) and shaken for 2 min. After 5 min. the supernatant present above the sediment was stippled into the test window in the quantity of 5 drops. The results were read out after 5 min. on the basis of the presence of a stria in the result window.

The supernatant from the faces sample was also used for the rotavirus examinations, by means of the Slidex Rota set - kit 2 (Bio Merieux, Paris). A drop of monoclonal antibodies (R1) and a drop of negative check sample (R2) were placed on a suitable mat and black plate, to which a drop of supernatant from the examined sample was added. Both samples were mixed with a glass rod for 2 min., and then the result was read out. A positive result was shown by the occurrence of agglutination in R1 field, and in case of its lack – in R2 field. The test specificity was verified with positive check sample (R3).

The herpevirus and adenovirus examinations were carried out by means of direct immunofluorocsence method. Impression preparations were made from liver, spleen, kidneys and lungs, and they were then fixed in ice acetone for 20 min. after drying for 24 hours at room temperature. Then, a proper conjugate was placed: anti-CPV/FITC and anti-CAV/FITC, and the preparations were kept in a humid chamber at the temp. of 37°C for 30 min. After 3-fold rinsing in PBS fluid, the preparations were embedded in buffered glycerin, and then observed in a fluorescent microscope (JENAMED 2) with the magnification of 10x12.5, 10x25 and 10x50.

Attempt at virus isolation in cell cultures

Material preparation. The material was constituted by organs (lung, liver), from which cubes were cut out aseptically with a side of approx. 1 cm, and then they were ground in mortars with aseptic sand. Then, 10% suspension was prepared in physiologic saline, in antibiotic cover (200 i.u. of penicillin, 200µg of streptomycin and 10µg of amphotericin B fell on 1 ml of suspension). Samples prepared in this way were filtered through 4-times folded aseptic gauze, and were subject to centrifugation (2000 rotations./min. for 10 min.). The obtained supernatant, after putting through bacterial filters, constituted inoculum for cell cultures.

Cell cultures. The following constant cell lines were used for the examinations: RK-13 (rabbit’s kidney) and Vero (monkey’s kidney). Cell cultures in a medium with the composition of: Minimal Essential Medium (MEM) - 90%, bovine fetus serum (FBS, Sigma) - 10%, with 1% addition of glutamine and in antibiotic cover (with the composition provided above) were poured into 12-pit polystyrene plates (Corning) in the quantity of 2 ml. The plates were kept in a thermostat with 5% inflow of CO₂, at the temp. of 37°C for 24 hours.

Cell culture infecting. Twenty-four hour cell cultures were infected with the examined material in the volume of 0.2 ml. Each of the 2 organs coming from a given rat was inoculated for both types of cell cultures. The plates were incubated in conditions described above, and they were observed every day in an inverted microscope to prove the occurrence of the cytopathic effect (CPE). The examined samples were passed every 4 days, transmitting 0.2 ml of fluid from the culture to the new cell cultures, prepared in the way described before. Each of the examined samples was passed 7-fold. Apart from subsequent plates, also the cultures on microscopic slides were infected with the fluid from over the culture during the seventh passing. After 3 days of incubation, despite the lack of CPE, direct immunofluorescence was carried out with anti-CHV/FITC and anti-CAV/FITC conjugate.

Rabies virus infection examination

The examination was conducted in the Laboratory of ZHW in Wrocław according to the routine procedure.

Chlamydia psittaci infection examination

The examination was carried out with the direct immunofluorescence method by means of IMAGEN^TM Chlamydia test (DAKO). The examination material was composed of th efollowing organs: spleen, liver and lungs, out of which impression preparations were made.

Conjugate (monoclonal antibodies anti-Chlamydia/FITC) in the quantity of 0.025 ml was put on preparations - dried and fixed in acetone, which were then incubated at the temp. of 37°C for 15 min., and then rinsed twice in PBS buffered fluid for 5 min. After removing the excess of rinsing fluid, the preparations were embedded in buffered glycerin, and then observed in a fluorescent microscope (JENAMED 2). At the same time, the positive and negative control was conducted. The assessment was carried out with the presence of at least 50 cells in the preparation. The result was regarded as positive with the presence of at least 10 characteristically gleaming Chlamydia bodies.

RESULTS

The results of this investigation comprising bacteriological and mycological examinations as well as virus and Chlamydia infections examinations are presented in detail in subsequent tables.

In clinical and postmortem examinations, preceding laboratory examinations, no significant aberrations were noted in most animals. The caught alive rats, kept in cages, behaved normally, took chow and water, and discharged normal excreta. Woundings or subcutaneous extravasations were observed in some individuals, mainly adult males, during the postmortem examination, probably caused by fighting among individuals. Edema, hyperemia and possibly bleeding from finger pulps as a result of forefeet partial freezing were observed in all rats coming from the swine farm (group 3). In rats coming from the slaughterhouse (group 5), cutaneous lesions were noted in the form of desquamated epidermis and few crusts. Anatomicropathologic lesions in particular internal systems and organs were observed only in single individuals. They are presented below together with the results of extra examinations to provide a more clear description. In one female (group 2), blood infiltrations in mucous membrane of uter us and its hypertrophy were noted, and b-hemol. Escherichia coli. was isolated bacteriologically. Lesions in lungs (white focuses, occurrence of exudate) were observed in one rat (group 3). Aspergillus flavus was isolated by means of mycological examination. In two old individuals, liver adipose degeneration was found (histopathologically). The results of bacteriological examinations were negative in both cases.

Bacteriological examinations results

The organs inoculations results (oral cavity and pharynx, final part of intestine) of examined rats are presented in detail in Tables 1–4.

The most frequently isolated microorganism was colon bacillus - Escherichia coli, which was found in 18.6% of examined rats in oral cavity and pharynx swabs, and in 80% - in rectum. E. coli was found in the rectum in high percentages, in all groups (from 60% in group 4 to 100% in group 3). The frequency of occurrence of E. coli in the oral cavity in particular groups was similar and ranged from 12% (group 3) to 27.7% (group 2). The isolation of such numerous colon bacillus from healthy rats may prove that, like in other animals, it constitutes an element of physiological microflora of the alimentary tract. A fact deserving attention is that b-hemolytic E. coli strains were isolated only from two rats. Among other bacteria from Enterobacteriaceae family, proving the occurrence of Salmonella spp. in 4.8% of examined rats (from group 2, 3 and 4) is significant.

Streptococci were represented in specially high quantities, particularly in isolations from the oral cavity and pharynx. Streptococcus spp. a-hem. was found in this part of the alimentary tract in 56.5% of examined rats, in all groups (from 40% in group 1, 3 and 4 to 77.2% in group 2 and 5). Streptococcus zooepidemicus was proved in smaller quantities (5.8%). Streptococci were isolated more rarely from the rectum, where apart from Streptococcus spp. a-hem. (18.8%), the occurrence of Streptococcus zooepidemicus and Enterococcus faecalis was also proved in single individuals.

The oral cavity infection with staphylococcus - Staphylococcus aureus was found in 7 individuals (46.6%) in group 4, in 6 individuals (27%) in group 2, in 3 individuals (27%) in group 5 and in one individual in group 1. The occurrence of Staphylococcus aureus in the rectum was found only in one group coming from a swine farm (group 3) in 2 individuals (8%).

Among other microorganisms, Table 1, the occurrence of bacteria such as: Corynebacterium spp. and Enterobacter spp. was proved only sporadically, in single individuals. Klebsiella spp. (approx. 13% both from oral cavity and rectum) and Proteus spp. (13% from oral cavity and 21% from rectum) occurred slightly less numerously. Neisseria catarrhalis was found in single individuals in group 2, and numerously in group 3 (24%).

Tables 5 and 6 present the results of bacteriological examinations of rats from three examined groups [1,2,3] depending on the season. Significant differences in the frequency of occurrence of microorganisms in the winter-spring and summer-autumn seasons were found only in case of Proteus spp. i Salmonella spp. In the examined animals, salmonella was isolated only in the summer-autumn season. The occurrence frequency of microorganisms of Proteus type in the summer-autumn season was higher (59.3%), in comparison with the winter-spring season (14.8%).

Mycological examinations results

Types of fungi isolated in the mycological examination are presented in Table 7.

For the purpose of interpretation it is necessary to distinguish between fungi causing organ mycosis and dermatomycoses. No increase of fungi was observed in liver inoculations of examined rats, while Aspergillus flavus was isolated from this organ of one rat, in which pneumonia was found during the postmortem examination.

In swab inoculations from oral cavity and pharynx, anascogenic yeasts – Candida spp. were the most numerously represented fungi, which were observed in all groups, from 6.6% (group 5) to 36% (group 3). Aspergillus flavus and Aspergillus fumigatus were also isolated from single individuals. The presence of Candida spp. was also proved in the rectum (from 6.6% in group 4 to 18% in group 2). Fungi of Aspergillus and Penicillium type were isolated individually. Numerously represented dermatophytes were observed in rats coming from all environments under investigation. In all examined groups of rats, Trichophyton spp. was isolated from inoculations of dermal and hair scrapings, while fungi of this type were found even in 36.6 % of rats in group 3 and in 58% - in group 1. In three cases (group 3 and 5) Microsporum spp. was also isolated. Aspergillus flavus, Aspergillus fumigatus and Aspergillus niger were found among oth er fungi as well as fungi of Penicillium type.

Table 8 presents the results of mycological examinations of rats in three examined groups depending on the examination season. The significance of the frequency of fungi occurrence in the alimentary tract was not specified, similarly as dermatophytes occurrence in the winter-spring and summer-autumn seasons.

Serologic examinations results

Listeriosis

The results of listeriosis examinations are presented in Table 9.

In some rats from groups 1, 3 and 5, the antibodies against serotype SO V were observed in low titres from 1: 40 – 1: 160. The occurrence of antibodies against serotype SO I – II (titre of 1:20) was found only in one individual from group 5.

Leptospirosis

The results of leptospirosis examinations are presented in Table 10.

The occurrence of antibodies against four serovariants Leptospira interrogans was observed in the examined rats. In group 1 coming from the railway station, the antibodies against serovariant icterohaemorrhagiae were observed in titres from 1: 100 to 1:1600 (in 3 individuals out of 4 examined ones). Some animals from group 1, 2 and 5 reacted positively with serovariant hebdomadis (titres 1: 100 to 1: 200). The occurrence of antibodies against serovariant canicola (titres 1: 100 – 1: 200) was proved in 50% of examined rats from group 2 and 5. Rats from group 2 also reacted with serovariant sejroë (2 out of 4 examined ones). Summing up, rats from group 3 and 4 did not react with any serovariant, while in the remaining groups there occurred antibodies against at least two serovariants Leptospira interrogans.

Borreliosis

The results of borreliosis examinations are presented in Table 11. The occurrence of antibodies anti-Borrelia burgdorferi was observed in 3 rats, in 2 from group 4 and in one from group 5. The rats from the remaining groups turned out free from borrelia infections.

Yersiniosis and rodenciosis

The results of yersiniosis and rodenciosis serological examinations are presented in Table 12. The presence of antibodies anti – Yersinia enterocolitica serotype I A in titres 1: 80 and 1: 1280 was observed in rats from group 4 and 5. The occurrence of antibodies against Yersinia pseudotuberculosis serotype V in titre 1: 160 was found only in group 3.

Results of virus and Chlamydia infections examinations

The results of virus and Chlamydia infections examinations are presented in Table 13.

With the application of direct methods of virus occurrence detection in feces (ELISA), the occurrence of parvoviruses and rotaviruses was not found in any rats.

On the basis of an official method of rabies examination it was proved that all examined rats were free from rabies virus infection.

Viruses in cell cultures were not isolated from any rats. During 7 passages, the cytopathic effect was not observed. The infected cultures did not differ morphologically from test cultures. All negative results were obtained also in the investigation with IF –direct method (with conjugates anti-CHV i anti-CAV) of infected cell cultures VERO and RK-13.

The occurrence of herpesvirus (CHV - canine herpesvirus) was observed in kidney in two rats (group 3) in the investigation with IF-direct method of impression preparations of liver, spleen, kidneys and lungs. The occurrence of adenovirus (CAV - canine adenovirus) was proved in three individuals (group 2,3 and 5) in the liver.

Chlamydia sp. infection was proved in 12 rats out of 64 examined ones – 18.75% (group 1, 2, 3 and 4) by means of IF-direct method. The presence of Chlamydia bodies was observed in impression preparations from lungs (1 in group 1 and 4 and 3 in group 2) and spleen (7 in group 3).

DISCUSSION

Aversion towards the rat is equally strongly rooted in the consciousness of man as aversion towards snakes, and fear against rats as carriers of many different diseases has been present in our culture for centuries [21,36]. As early as in the olden days – despite the then primitive medical knowledge – epizootia present among rats were associated with epidemics, especially plague, and some connection was suspected between rats falling ill and cases of disease incidences among people [36].

The rat, as a synathropic species living in the direct neighbourhood of man, occupies a very important position in the infectious chain of many bacterial and viral diseases of humans and animals. The issue of transmissive diseases, broadly discussed in literature, indicates the significant role of rodents in the remaining and surviving of many germs, which can constitute a source of epizootias and epidemics. The possibility of spreading or passive transmitting of microorganisms by rats creates particular threat for example in meat-processing plants, slaughterhouses, food-processing plants, breeding-animal farms as well as in large city agglomerations.

Independently of active manifest infections or latent ones, the occurrence of microorganisms in the oral cavity, pharynx, in the final section of intestine and on body integuments is a special indicator of the possibility of spreading germs in the environment through secretions, excrements, hair and desquamated epidermis, and is also an indicator of the sanitary condition of the environment which the rodent comes from.

Numerous bacteria in the oral cavity, pharynx and rectum of rats were found while carrying out own research (Table 1). Most of the isolated strains can be treated as a standard element of alimentary tract bacterial flora, which mainly concerns the numerously observed bacteria from Enterobacteriaceae (Table 2). However, the microorganisms regarded as pathogenic for man and animals were also isolated, such as: b-hemol. E. coli, Salmonella spp., Klebsiella pneumoniae, Staphylococcus aureus, a-hemol. Streptococci (Table 3). A fact deserving attention is that the pathogenic bacterial strains mentioned above were isolated almost exclusively from rats coming from slaughterhouses and swine farms. It indicates the necessity for increasing the efficiency of rat extermination in meat-processing plants and on farms owing to the possibility of contact between rats , carcass and post-slaughter material, and the consequent contamination of these products. The fewest bacteria were observed in rats caught at the Main Railway Station in Wrocław, which may prove good sanitary conditions within the area of the railway station.

The occurrence of pathogenic bacteria Staphylococcus aureus were proved in own research in the oral cavity swabs and pharyngeal swabs in over 25%. It should be pointed out that these bacteria occurred most numerously on the poultry farm, on swine farm and in Wrocław streets. Similar results were obtained in Japan, where Staphylococcus aureus was isolated from 76.6 % among 59 rats from a fish market and from 34.6 % from a slaughterhouse [22].

Infections caused by Salmonella rods cause many chronic illnesses both in people and animals. Rats and mice are natural hosts of these bacteria, therefore carrier state among these rodents is very widespread. Germ excretion with feces and urine can last 5 months and longer [32,36]. As results from available data, Salmonella spp. carrier state in rats is very widespread and ranges from 1 to 50% depending on the place that they come from [36]. As a general rule, this percentage is very high in slaughterhouses and food-processing plants. In own research, Salmonella spp. carrier state was found in 6.6% in the slaughterhouse and in 8% on the swine farm. Small percentage – 4.5% was observed in the city area. In a way it confirms the fact of salmonella carrier state being dependent on the place of origin. What also results from own research is that bacteria spreading occurred in the summer-autumn season. It indicates quite a significant role of rats in the etiology of alimenta ry intoxications occurring among animals and people. On the other hand, an interesting fact is that in research conducted on the poultry farm in Great Britain the occurrence of salmonella was observed only in three out of 527 examined rats [53]. In own research carried out on the poultry farm Salmonella spp. occurred in one of 11 examined individuals.

Pasteurella multocida infection constituted a high percentage, as high as 41%, on poultry farms in Great Britain [5]. In own research, conducted in various environments, among others on a poultry farm, the occurrence of these microorganisms was not observed. Rats may have their share in transmitting many other bacterial infections, even if it is proved only by few data. One cannot exclude the role of these rodents in spreading, even mechanical, of salmonella and other infections specified as the “diseases of dirty hands”. Rat bites may also cause infection transmission, which was proved (for example) for Pasteurella multocida infections [5].

Rats can also be carriers of such microorganisms as Actinobacillus pleuropneumoniae and Streptococcus suis type 2 [32].

The role of rats in listeriosis spreading is not fully clarified. Examinations conducted by British scientists on a lot of 44 rats proved the occurrence of listerias only in 11% [31,50]. Own research showed the occurrence of Listeria spp. in 4.7% of individuals from direct inoculations, and in nearly 50% of examined rats. Titres above 1:320 are regarded as positive ones. Low titres (up to 1:160) obtained in own research can be of unspecific character, as a result of response to fractional antigens common for listerias and other microorganisms, such as enterococci, staphylococci, and others; they may, however, also indicate contact with listerias. Taking into consideration the fact of listeria isolation from some of the examined rats, one may assume that these rodents occupy a significant position as listeria reservoir in the environment. Routes of the disease and the infection spreading are not fully recognised. Rats are believed to be frequent carriers of this germ, similarly as for example hares and carnivorous animals [15].

Rats play an important role in carrying leptospirosis. The rat, mouse and other small rodents are of the main significance as the source of leptospires [34,35,36,50]. According to numerous authors, the carrier state of Leptospira interrogans is thoroughly widespread, yet more in older individuals. The highest percentage was observed in Third World countries, even up to 50%. The carrier state of Leptospira interrogans serovariant icterohaemorrhagiae amounted to approx. 27% in Wrocław in the 50s. Cyclic treponema excretion can last up to 2.5 years [36]. The British, in their examinations carried out in 1994, proved leptospire carrier state in 14% of examined rats, equally for serovariant icterohaemorrhagiae and bratislava [51,53]. Brem et al. [2], in summer 1992, observed cases of leptospirosis infection in people in the region of Tybinga. The occurrence of Leptospira interrogans serovariant copenhageni was proved in 11 rats caught in this regi on. In own research carried out at the turn of 1997/1998, the occurrence of antibodies against serovariant icterohaemorrhagiae was proved in 75% of examined individuals, against serovariant canicola in 50% and against serovariant sejroë also in 50%. Positive results may indicate the importance of wild rats as the main reservoir of Leptospira interrogans. Taking into account the fact of leptospire spreading with urine, one should assume that rats contribute to a large extent to the occurrence of leptospirosis in people and animals, which is indicated by data from world literature of the subject [2,51,53].

In case of borreliosis, rats can constitute a biological germ reservoir for the tick parasiting on the rodent [48]. In own research three positive results were obtained, which can confirm this hypothesis.

Rodents (mice, rats) and animals of hare family (Leporidae) constitute the main reservoir of Yersinia pseudotuberculosis in nature [9]. Yersiniosis is often of saprozoonosis character. As a result of the external environment contamination with Yersinia rods by excreta of ill animals or carriers there comes to infecting of other animals or the human [10,43]. The level of antibodies in the serological examination 1:40 is regarded as doubtful, while 1:80 as positive [9]. In own research the antibodies titre in examined rats amounted to 1:160, which shows Yersinia pseudotuberculosis infection. Clinical observations and laboratory examination results demonstrate the increase of cases of yersiniosis, both in people and in different animal species. Wide spreading in nature and carrying Yersinia enterocolitica bacteria by animals may imply the animal-human direction of infection. The source of infection is mainly contaminated food [30]. The main reservoir of Ye rsinia enterocolitica infection for the human described in literature are pigs, which, however, may get infected from rats contaminating fodder with their excreta [30,31].

Rats play a significant role in viral infection maintaining. According to some authors, thay have an important role as rabies virus carriers, even though the percentage of examined individuals, in which the occurrence of the virus was proved, amounted only 0.04% [58]. In the years: 1995–1998, the virus was isolated only from three wild rats in the territory of Poland [41,58]. In own research, no case of rabies virus infection was observed in 30 examined rats; yet it does not write off the important role of these rodents in the epizootiology and epidemiology of this disease due to a limited number of examinations.

The role of rats in the transmission of other viral infections is underestimated. Some of them have not been examined in rats so far, e.g. there are no data in professional literature on herpesvirus and adenovirus infections in rats. Own research demonstrated the occurrence of CHV in two rats as well as CAV virus infection in three individuals, which may suggest that rats can constitute a source of infection of these viruses for dogs, and may also indicate the possibility of transmitting many other adenovirus and herpesvirus infections by rats, as it happens in case of Aujeszky’s disease on swine farms [34]. Obtaining all negative results in rotavirus and parvovirus infection examinations does not exclude the possibility of getting infected with these viruses in rats due to a limited number of groups included in the examinations.

There are no data in world literature concerning infections with Chlamydia microorganisms in rats. Finding Chlamydia spp infection in three rats coming from a city environment, in seven rats from a swine farm and in one from a poultry farm and one from the Main Railway Station may indicate the role of rats as Chlamydiosis vector in humans and animals.

Chlamydiosis is of considerable importance for the course of pregnancy and delivery. For example, the influence of Ch. ovis infection in the course of enzootic sheep miscarriage has been proved [19]. Chlamydiosis in domestic animals, e.g. in cats, is manifested by conjunctivitis, upper airways inflammation, and also miscarriages [37,45,55,56]. Similarly, pneumonia caused by Ch. psittaci was observed in dogs [7,9]. Simultaneous increase of the rat population shows that these rodents may have a big share in the epidemiology of this illness.

The isolation of numerous fungi types from examined rats is significant from the point of view of the common occurrence of mycotic chronic illnesses in people and animals. Few data in literature on the importance of rats as mycotic infections vectors should not exclude the role of these rodents as disseminators, which is confirmed by Spieciwcewa’s research [40]. Observing fungi of Candida and Aspergillus type both in the oral cavity and in intestine as well as very numerously on skin proves considerable importance of rats in the transmission and survival of spores of these fungi. This fact consitutes a particular implication in the light of the latest reports concerning the bigger and bigger share of Aspergillus sp. as a cause of chronic illnesses of the respiratory system in people and animals. In dogs the share of aspergillosis in the chronic illnesses of the respiratory system and lungs is estimated even at the value of 40% [8, 38]. Own research also indicates a sig nificant share of rats in dermatomycosis spreading, which is shown by the isolation of the most common fungus of the types causing dermatomycoses, i.e. Trichophyton in high percentage in all groups of examined rats.

Summing up, the results of own research confirm big share of the wild rat in the maintaining and transmitting of numerous viral and bacterial infections. Low percentage of positive results in case of some infections may result from big participation of young individuals included in the examinations, in which the probability of contact with microorganisms if much lower, and from resistance to microorganism infections particularly characteristic of these animals. These rodents can be treated as an indicator of the epizootic and epidemic situation of the given environment and the sanitary condition of a given object. Significant differences in the frequency of occurrence of particular microorganisms depending on the place of origin of examined rats were proved in the conducted examinations. The highest percentages of bacterial infections were observed in rats coming from a swine slaughterhouse. On the other hand, the examined rats coming from the railway station were in 100% infected with lept ospirosis. The highest percentage of animals infected with dermatophytes was observed also in this group.

High percentages of positive results in rats coming from a given environment indicate considerable threat from these rodents as a reservoir and infection source of particular microorganisms. Observing single infections should be a sign that in a specified area there is a potential possibility of occurrence of a given disease, whose spreading wild rats can cause.

CONCLUSIONS

1. The wild rat constitutes an important reservoir of germs causing viral, bacterial and mycotic infections in the environment.

2. Rats living at large are one of more important links in infectious disease spreading.

3. The highest percentages of bacterial infections were observed in rats coming from a swine slaughterhouse.

4. More infections were found in wild rats in the summer-autumn season.

5. Rats are of significant importance as the main reservoir of Leptospira interrogans, and to a large extent contribute to the occurrence of leptospirosis in people and animals.

6. Rats are an important indicator of the epizootic and epidemic situation of the environment, in which they live.

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