INTRODUCTION
A number of freshwater and marine fish can be definitive hosts for the thorny-headed worm, Pomphorhynchus laevis. Within the Polish Exclusive Economic Zone of the Baltic Sea this acanthocephalan has been recorded predominantly from flounder, Platichthys flesus (cf. Sulgostowska et al., Reference Sulgostowska, Banaczyk and Grabda-Kazubska1987; Rokicki & Morozińska, Reference Rokicki and Morozińska1994; Ziółkowska et al., Reference Ziółkowska, Chibani, Kijewska and Rokicki2000). This parasite settles in the lower part of the intestine, perforating its wall with its proboscis. In the course of such action the anterior part of the worm, consisting of the proboscis and bulbous anterior part of the body, protrudes into the peritoneal cavity, leaving the rest of the body within the intestinal lumen. Mass infection of flounder with this acanthocephalan causes not only occlusion of the intestine, but can also be the reason for acute inflammatory changes (Janiszewska, Reference Janiszewska1938). Physically damaged walls of the lower part of the alimentary tract are convenient sites for establishment and development of various micro-organisms. The bacteria living in flounder have been most frequently collected from skin lesions (Kroon, Reference Kroon1994; Wiklund, Reference Wiklund1994). Only sporadically such material has been isolated from the intestine. There has been no conclusive evidence, as to which bacteria, found within the intestine, are pathogenic. Ullrich (Reference Ullrich1994) believed that bacteria of the genera Vibrio and Aeromonas constitute natural components of the bacterial flora of this fish. It is commonly known that bacteria of the genus Vibrio cause vibriosis and one of the sites, from where they can be isolated, is the intestine. On the other hand, bacteria of the genus Aeromonas cause not only pathogenic changes on the external surface of fish, but also in their internal organs. Quite often, the organisms inhabiting the alimentary tract, and believed to be commensals, exhibit their harmful effects only under certain conditions. The above is evident not only for bacteria, but also for fungi, protozoans and worms. The presence of acanthocephalans in the intestine may disturb bacterial equilibrium.
The aim of the present paper was to study the possible effect of the Pomphorhynchus laevis infection and the composition of the host fish's intestinal bacterial flora.
MATERIALS AND METHODS
A total of 112 flounder, Platichthys flesus, was surveyed between May and December 2005. The fish, measuring 25–37 cm, were caught at the Łeba fishing grounds (ICES subdivision 25, statistical rectangles O, N-7). Each batch of the fish was refrigerated and promptly delivered to the laboratory. The fish were examined for possible presence of thorny-headed worms and for inflammatory changes. The worms were identified and counted.
The contents of the lower intestine of the fish were sampled and placed in brain–heart infusion (BHI) broth. Then, they were passed through dilution series. The suspended material, prepared in such way, was inoculated on nutrient agar and nutrient agar with blood. After 72 hours of incubation at room temperature, the bacterial colonies were counted and Gram-stained preparations were made. The isolated bacteria were inoculated on 5% of blood agar, MacConkey agar, and Hektoen agar. Individual species of bacteria were identified with the aid of API 20E biochemical diagnostic tests (BioMérieux), following the manufacturer's instructions.
RESULTS
The infection levels of flounder with Pomphorhynchus laevis were variable and a number of fish did not harbour that parasite (Table 1).
Table 1. The infection levels of flounder (N = 112) with Pomphorhynchus laevis.
The highest number of P. laevis, recorded in the lower portion of the intestine was 24.
The fish infected with the acanthocephalan often had a hyperaemic lower portion of the intestine and proctitis.
There was a relation between the total number of P. laevis and the overall bacterial count in the intestine (within the proposed infection-level groups) (Table 2).
Table 2. The overall bacteria count of the lower part of the flounder intestine, in relation to the number of Pomphorhynchus laevis acanthocephalans.
We determined that the number of bacteria, occurring in the intestine, increases with the number of parasites living there. Moreover, the genera and species of those bacteria, grown from the samples taken from intestines of infected- and non-infected fish, were similar and they differed in the prevalence of infection within three earlier defined infection-level groups (Table 3).
Table 3. Per cent of fish infected with the studied bacteria species, within three groups of flounder, infected with Pomphorhynchus laevis.
The most frequently isolated bacteria, represented the genera: Aeromonas, Pseudomonas and Shewanella. The least frequent were species of Chromobacterium, Escherichia, Providencia and Serratia.
The fish infected with parasites showed a two-fold increase in the intensity of Aeromonas hydrophila/sobria/caviae gr. 2, Burkholderia cepacia, Chryseobacterium indologenes, Ch. meningosepticum and Stenotrophomonas maltophilia, compared with the parasite-free fish. On the other hand, the fish without acanthocephalans harboured more frequently Pantoea spp. and Providencia stuartii.
DISCUSSION
The present study demonstrated that the number of the acanthocephalans, occurring in the lower part of the intestine of flounder, had an effect on the quantities of the bacteria present there. An increase of the acanthocephalan number resulted in an increase of the bacteria numbers within the intestine. The reason behind that fact may be the damage to the intestinal wall, inflicted by the proboscis of the acanthocephalan. When, the proboscis of the acanthocephalan penetrates the intestinal wall further, it results in formation of inflammatory changes, visible as hyperaemia of the tissues (Wanstall et al., Reference Wanstall, Thomas and Robotham1988). The above-mentioned authors observed that the inflamed tissues contained granulocytes and phagocytes of the hosts and at the same time the integument of P. laevis was intact. The inflammation inflicted by acanthocephalans, and the related decrease of the fish defence mechanisms, stimulate additional bacteria build-up. High numbers of the acanthocephalans, anchored in the intestinal wall, cause an occlusion of its lumen and compromise its functions. This way the acanthocephalan introduces bacteria to the blood vessels and the peritoneal cavity.
Aeromonas hydrophila was present not only in the infected fish, but also in those without the presence of P. laevis. The percentage of fish, however, where these bacteria are present, increased with the rising number of the acanthocephalans. A similar tendency could also be observed in relation to bacteria representing the genera: Pseudomonas, Alcaligenes, Chryseobacterium, as well as Shewanella putrefaciens and Stenotrophomonas maltophilia.
Aeromonas hydrophila is a Gram-negative rod-like bacterium, widely distributed in the aquatic environment. It is an opportunistic micro-organism capable of causing pathologic changes in both freshwater (Shao et al., Reference Shao, Liu and Xiang2004) and marine fish (Lilley et al., Reference Lilley, Hart, Richards, Roberts, Cerenius and Soderhall1997). It has been found in pathological changes of the skin, ulcers, infected liver and kidneys (Johnson, Reference Johnson and Brown1993). The pathological changes are more likely to occur, when the fish shows decreased immunity levels, e.g. during spawning or under influence of external stress factors.
Aeromonas hydrophila has been considered a prime infection factor (Shome & Shome, Reference Shome and Shome1999) or a bacterium responsible for continuing pathological processes (Thampuran et al., Reference Thampuran, Surendran, Mukundan and Gopakumar1995).
Shewanella putrefaciens was observed in the majority of the flounder surveyed. It occurred in 80% of non-infected fish and in 100% of the flounder infected with 6 or more specimens of P. laevis. Species of the genus Shewanella are common in the marine environment. They are often isolated from sick people, who had contact with seawater (Heller et al., Reference Heller, Tortora and Burger1990; Leong et al., Reference Leong, Mirkazemi and Kimble2000). It is possible that those bacteria constitute a component of physiological bacterial flora of the intestine and are opportunistic micro-organisms that reproduce when the immunity of the organism decreases.
Stenotrophomonas maltophilia is a pathogen capable of infecting people (Robin & Janda, Reference Robin and Janda1996). Stenotrophomonas maltophilia has been isolated in cases of bacteraemia, endocarditis, inflammation of central nervous system, dermatitis, ocular inflammation and inflammation of urinary tract (Denton & Kerr, Reference Denton and Kerr1998). It naturally occurs in water, soil, on plants, animals and in hospitals (Denton & Kerr, Reference Denton and Kerr1998).
Bacteria of the genera Pseudomonas and Chryseobacterium are very common in nature (water, soil and air). They may also cause infections. Also the percentage of fish with those bacteria, increased with the number of the acanthocephalans.
In both fish groups, those non-infected as well as infected with the acanthocephalans, the bacteria detected were, in most cases, the same (Table 3). Some micro-organism species, however, were more frequent in the fish infected with P. laevis. It is possible that the attachment process of those acanthocephalans in the flounder intestine induces some stress. Opportunistic bacteria, which were not pathogenic as yet, increase in their numbers and cause inflammatory changes. At the same time, some species of bacteria, becoming predominant, take over available ecological niches and displace those micro-organisms, which are not so well adapted to the defined conditions.
The specific composition of the intestinal microflora of flounder can also be affected by the external environment, which is related to the pollution level of the Baltic Sea. Some evidence confirming this assumption is the fact that the bacteria species listed in Table 3, except Chryseobacterium spp., Flavimonas oryzihabitans, Ochrobactrum anthropi and Stenotrophomonas maltophilia have also been found in municipal sewage or in the air above a sewage treatment plant (Cyprowski et al., Reference Cyprowski, Szarafińska-Kraszewska, Dudkiewicz, Krajewski and Szadkowska-Stańczyk2005, Reference Cyprowski, Buczyńska and Szadkowska-Stańczyk2006).
