HELMINTH FAUNA OF RATTUS NORVEGICUS BERKENHOUT , 1769 FROM THE BELGRADE AREA , SERBIA

The aims of this study were to provide baseline knowledge about intestinal parasites in Norway rats (Rattus norvegicus) that inhabit the Belgrade area, and to analyze the associations among helminths. Of 302 trapped rats, 52% were females and 48% males, with 39% and approx. 37% of juvenile-subadult individuals, per sex, respectively. The following parasites were detected (with their respective prevalence): Cestoda – Hymenolepis diminuta (30.46%) and Rodentolepis fraterna (12.58%); Nematoda Heterakis spumosa (36.75%), Nippostrongylus brasiliensis (16.22%), Capillaria sp. (5.96%), Trichuris muris (5.96%), Syphacia muris (4.30%) and Strongylus sp. larvae (0.33%). Flukes (Trematoda) were not recorded. Of all examined rats, 68.54% were found to harbor at least one parasite species, with higher prevalence in male hosts and in adult individuals. There were no age-related differences in the prevalence of infection with individual helminth species. Multiple infections occurred with up to four species per rat showing different combinations of parasite infections. These are the first records of the gastrointestinal helminth fauna of Norway rats in Serbia.


INTRODUCTION
Extreme adaptation to life under the most distinct habitat conditions and types, euryvalency toward numerous environmental factors, tendency to synanthropy, high reproduction potential, expressed genetic polymorphism and the ability to acquire resistance to some rodenticide preparations represent the main characteristics of representatives from the genera Rattus and Mus.These characteristics enable their optimal existence in the immediate human environment, at the same time making the problem of their population abundance much more complex than it seems at the first sight (Kataranovski, 1988).Investigations in the field of biology and the population ecology of pest synantropous rodents are of multiple interest and significance because of economic and hygienic-epidemiological rea-sons; they pose a threat to public health, both through the consummation of different food and by serving as reservoir for pathogens that can be transmitted to humans and may cause outbreaks of diseases often with high morbidity and some mortality (Robinson and Olsen, 1960;Marchette, 1966;Cabrera, 1976;Jones and Twigg, 1976;Hoogstraal, 1979;Kaplan et al., 1980;Dick, 1983;Meehan, 1984;Kumar and Bubare, 1986;Gratz, 1988Gratz, , 2005;;Horgović et al., 1991;Kliks and Palumbo, 1992;Pozio et al., 1992Pozio et al., , 2009;;Gustafson, 1994;Young et al., 1998;Crompton, 1999;Markell et al., 1999;Serbezov et al., 1999;Salazar-Bravo et al., 2002;Garber et al., 2003;Petavy et al., 2003;Kumar et al., 2003;Calisher et al., 2005;Giraldo-Gomez et al., 2005;Satoh et al., 2005;Klimpel et al., 2006;Landais et al., 2007;Kijlstra et al., 2008;Samardzic et al., 2008;Cook et al., 2009;Salkeld and Lane, 2010).
The aims of this study were to determinate the parasitic fauna of R. norvegicus in the Belgrade area and to analyze the associations among helminths.

MATERIALS AND METHODS
Rat samplings were conducted during the period of four consecutive years, from May 2005 to July 2009.A total of 302 rats were collected in the Belgrade area (44 0 N, 20 0 E; approximate geometric center of Belgrade 44 0 49'14"N, 20 0 27`44"E) using snap live traps (31 x 16.5 x 14 cm).The traps were baited with pieces of smoked bacon and/or fresh-water fish.The captured rats were euthanized and necropsied in the laboratory of the Institute for Biological Research "Siniša Stanković", Belgrade.
For each rat examined, the data of trapping locality, body length (head and body), weight and sex were noted.The rats were separated into two age groups: juveniles-subadults (< 2.5 months old) and adults (>2.5 months old).The following criteria were used for separating adults from juvenilesubadult animals 1) the body weight (borderline value approx.200 g, according to Podloucky, 1977;Savić and Kataranovski, 1981;Kataranovski et al., 1991), and 2) the weight of the dry eye lens pairs (14.04-14.30mg, according to Savić and Kataranovski, 1981;Kataranovski, 1988;Kataranovski et al., 1994).The material was analyzed using the standard parasitological procedure according to Kataranovski et al. (2008).The identification of helminths was based on the Key for Helminths of Rodents of the Fauna of the USSR (1978,1979), and taxonomic keys to cestode and nematode parasites given by Anderson et al. (1974), Genov (1984) and Khalil et al. (1994).. The parasitological terminology and quantitative parameters are according to Buch et al. (1997).The quantitative descriptors of parasite infection were calculated, including prevalence (P), mean intensity of infection (MI) and mean abundance of infection (MA).Also, an index of infection (I) was calculated according to Kisielewska (1970).Statistical analysis was performed using the statistical software package STATISTICA 7.0 (StatSoft Inc., Tulsa, Oklahoma, USA).

RESULTS
Of the 302 rats sampled during the study, 48.01%were males and 51.99% were females.Two hundred and seven rats (68.54%) were infected with at least one parasite or more.A higher prevalence of infection was noted in the males compared to the female adult rats (Table 1).Similar numbers of male and female juvenile-subadult rats were infected.
Multiple infections were noted with up to four helminth species per host.Parasitism involving only one species was found in 51.21% of the infected rats.Multiple infections including two species of parasites were found in 32.37% of the infected rats.Three helminth species were found in 14% and four parasite species were found in 2.42% of the hosts (Table 3) Types of polyparasitism were assessed for all rats with multiple infections (Table 4).They were divided into classes with two, three and four species, respectively, representing the number of parasite species and specific combinations of parasites.The most frequent infection type was the H. spumosa/H.diminuta combination.Other infection types presented combined infection with different species.Fig. 1 shows types of polyparasitism among all the rats.S. muris were negatively associated with N. brasiliensis and T. muris, and T. muris is also negatively associated with Capillaria sp.

DISCUSSION
Urbanization and the growth of towns have resulted in an increase in the accumulation of garbage and refuse which create favorable conditions for the proliferation of rats, and they rarely remain uninfected or harbor just a single species' infections in nature (Behnke et al., 2001).As R. norvegicus is a synanthropic species, the potential for spillover of zoonotic agents poses a threat to public health.This study records the first data on  intestinal helminth parasites in the Norway rat from the Belgrade area along with recent data on Calodium hepaticum and Taenia taeniaeformis liver infections in this rodent (Kataranovski et al., 2010).This study shows that R. norvegicus from this area is host to two cestode species and six nematode species.The monoxenous nature of the life cycle of nematodes may be responsible for this parasitic group dominating the helminth community of small mammals worldwide, especially rodents.
Parasites with simple and direct life cycles may have more chance of following the dispersion of their hosts than parasites with indirect life cycles (Bellocq, 2003).The longevity of H. diminuta in its normal mammalian host can cause a high prevalence of infections with this parasite.Once established, it can live as long as the host (Read, 1967).
The moderate prevalence of H. spumosa is in accordance with the results of Seo et al. (1968) in South Korea and Stojčević et al. (2004) in Croatia.However, research results of Firlotte (1948) in Canada, Tscherner (1996) in Germany and in Buenos Aires, Argentina (Gomez Villafañe et al., 2008) show a high prevalence of H. spumosa in the Norway rat.
All parasite species showed a higher prevalence in male rats than in females.This could be explained by the fact that infected males have larger territories than uninfected males, and that the home range of males tends to overlap which could increase their exposure to infection while reproductive females show a stronger site-specific organi- zation (Davis et al., 1948;Pisano and Storer, 1948;Calhoun, 1962).Also, the male hormone testosterone has a negative effect on the immune function (Grossman, 1989;Folstad et al., 1992).Another hypothesis assumes that among mammals the larger bodies of males are easier targets for parasites (Arneberg, 2002).
The prevalence and abundance of helminths may also be influenced by various types of associations between species of parasites, which can be both synergistic (positive) and antagonistic (negative).Such associations can arise for ecological/behavioral reasons, although there remains the possibility that the host immune system may also be involved.In this study, the most frequent infection type was the H. spumosa/H.diminuta combination, which is probably a consequence of the fact that these are the two most prevalent parasites in this study, and does not imply a connection between these two species.It is possible that these parasites are in positive associations because they are located in different parts of the host; H. spumosa is localized in the lower intestine and/or caecum-rectum and H. diminuta is localized in the upper intestine, so the interaction is minimized and competition is reduced.The negative associations between Capillaria sp. and T. muris, N. brasiliensis and S. muris, S. muris and T. muris may be the result of competition between them or the different times of year when infection occurs.For the N. brasiliensis and S. muris association the first alternative is less probable because these species are located in different parts of the host's intestine.
The eight helminth species reported in this study is a new record for the Belgrade area.Norway rats represent some very important links in the food chains of the ecosystems they inhabit and, on the other hand, they often come into contact with people and may act as transmitters of various pathogens.The sparse knowledge of their parasites indicates the necessity of further parasitological studies.

Table 1 .
Prevalence of intestinal helminth infection in rats of different sex and age.Significantly different from females at * p<0.01; from juvenile-subadults at # p<0.05.

Table 2b .
Quantitative indices of individual intestinal helminth infection of R. norvegicus.n-number of animals infected, N-total number of parasites, P-prevalence, I-index of infection, MI-mean intensity, MA-mean abundance.Significantly different from females at * p<0.05.

Table 3 .
Prevalence of intestinal helminth infection in rats of different sex and age.Significantly different from females at * p<0.01; from juvenile-subadults at # p<0.05.