The common frog (Rana temporaria) as a potential paratenic and intermediate host forAngiostrongylus vasorum

Parasitol Res (1993) 79:428-430

Parasitnlogy Research 9 Springer-Verlag 1993

Short communications The common frog (Rana temporaria) as a potential paratenic and intermediate host for Angiostrongylus rasorum G. Bolt 1, j. Monrad 1, F. Frandsen 2, p. Henriksen 3, H.H. Dietz 3 Department of Veterinary Microbiology/Parasitology Section, Royal Veterinary and Agricultural University Bfilowsvej 13, DK-1870 Frederiksberg C, Denmark 2 Department of Ecology and Molecular Biology, Royal Veterinary and Agricultural University Bfilowsvej 13, DK-1870 Frederiksberg C, Denmark 3 National Veterinary Laboratory Hangovej 2, DK-8200 ,~rhus N, Denmark Received: 12 January 1993 /Accepted: 23 February 1993

Abstract. Common frogs (Rana temporaria) were exposed either to third-stage larvae (L3) or to first-stage larvae (L1) of Angiostrongylus vasorum. Following exposure to L3, viable larvae could be detected in the frogs for at least 2 weeks. Following exposure to L1, the frogs developed viable L3 in their tissues within 30 days. In a test of the infectivity of these larvae, one fox was fed frogs previously infected with L3 and another fox was fed frogs previously infected with L1. On autopsy it was found that adult A. vasorum populations had been established in both foxes. Thus, it could be concluded that frogs can act not only as paratenic hosts but also as intermediate hosts for A. vasorum.

Angiostrongylus vasorum is a metastrongyloid parasite of the domestic dog (Canis familiaris), the fox (Vulpes vulpes) and other canid carnivores. Canine angiostrongylosis cause respiratory distress and may be fatal for the dog (Guelfi 1976). Most metastrongyloid parasites, including A. vasorum, have indirect life cycles including molluscs as obligatory intermediate hosts. In vitro, a variety of terrestrial and aquatic gastropod molluscs may act as intermediate hosts for A. vasorum (Eckert and L~immler 1972; Guilhon and Cens 1973). It is known that a number of metastrongyloid parasites are capable of incorporating paratenic hosts into their life cycle. This is particularly beneficial for parasite species whose final hosts feed infrequently on snails. Amphibians, reptiles, birds and rodents are typical paratenic hosts of metastrongyle parasites using carnivores as final hosts (Anderson and Strelive 1966; Ash 1968). Thus, A. cantonensis, a lung worm of rats, may utilize invertebrates and poikilothermic vertebrates, including frogs, as paratenic hosts (Ash 1968). In addition, experimental infections have shown that tadpoles of the clawed frog (Xenopus laevis) may act as intermediate hosts for Correspondence to: J. Monrad

A. cantonensis (Oku et al. 1980). With this knowledge in mind, we decided to investigate whether frogs could also be utilized as paratenic hosts and, possibly, as intermediate hosts by A. vasorum. The tropical freshwater snail Biomphalaria glabrata was used as the experimental intermediate host. The infection was established by the method described by Guilhon and de Gaalon (1969). The frog Rana temporaria was selected, since this is the most common frog in Denmark and Europe. A total of ten young frogs collected from one site were employed. The first-stage larvae (L1) of A. vasorum were extracted from the faeces of naturally and experimentally infected foxes and dogs by the Baermann method (Jorgensen and Madsen 1982). Five frogs (group A) were forcibly fed the soft tissues of snails (B. glabrata) containing infective third-stage larvae (L3) of A. vasorum. Another three frogs (group B) were orally infected with a suspension containing L1 A. vasorum by means of a soft plastic pipette. No estimation of the number of larvae received by each frog was done. Two non-infected frogs (group C) served as unexposed control animals. Two frogs from group A were euthanised by decapitation at 1 and 2 weeks after L3 exposure, respectively. The lungs, heart, liver, gastrointestinal tract with mesentery, head and hind legs were separated from the rest of the carcass and each section was comminuted and digested individually in a pepsin/HC1 solution at 30~ C (Wallace and Rosen 1969). The larvae liberated by digestion were inspected under a microscope. In all, 50 and 85 viable L3 A. vasorum larvae were recovered at 1 and 2 weeks after L3 exposure, respectively. Identification of the larvae were done according to the morphological descriptions given by Ash (1970) and by Guilhon and Cens (1973). Larvae were liberated from several tissues, but the majority were found after digestion of the gastrointestinal tract with mesentery. One frog from group B was euthanised at 30 days after L1 infection and examined for larvae by the abovedescribed technique. A total of 889 viable and at least

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116 dead L3 A. vasorum were recovered. Except for the lungs, larvae were liberated from all of the examined tissues, but the majority were found after digestion of the gastrointestinal tract with mesentery, in which a large number of dead nematode larvae were found. Only larvae that could be identified as A . vasorum with absolute certainty were counted. The individual length of the A. vasorum L3 recovered from this frog displayed an unusually wide dispersion (440-550 gin). Many larvae were shorter than both the minimal length of 508 gm stated by Ash (1970) and the minimal length of L3 larvae of the same strains of A. vasorum recovered from snails. The two control frogs were kept and fed the same way as the infected frogs for 40 days, after which they were euthanised and examined for larvae by the same technique used for the infected frogs. No A. vasorum larva was found in any of these frogs. In a test of the infectivity of the L3 larvae found in the frogs, the latter were fed to two foxes originating from a fox farm. Prior to infection, faecal samples from the foxes were subjected to the Baermann method so as to exclude the possibility of a pre-existing infection. The three frogs remaining in group A were euthanised, mixed with minced meat and fed to one of the foxes (fox 1) at 14 days after infection of the frogs with L3 A . vasorum. A suspension containing larvae recovered from the digested group-B frog together with the two remaining frogs of group B were fed to the other fox (fox 2) as described above at 38 days after infection of the frogs with L1 A. vasorum. Both foxes were euthanised 78 days after they had eaten the infected frogs. During this 78-day period they did not exhibit clinical signs of angiostrongylosis. Gross pathological and histopathological examinations of the heart and the lungs were performed. The adult worms were isolated by opening the right side of the heart and the pulmonary arteries. The peripheral parts of the lungs were chopped into pieces measuring approximately 1 cm in width, and the lung tissue was compressed and irrigated. Gross pathological and histopathological lesions typical of canine and vulpine angiostrongylosis were observed (Neff 1971 ; Prestwood et al. 1981; Poli et al. 1985). A total of 30 adult A. vasorum (26 females and 4 males) were found in fox 1, whereas 126 adult A. vasorum (96 females and 30 males) were found in fox 2. The histopathological examinations demonstrated L1 larvae in the lungs of both foxes, for which reason both of the infections must be considered patent. Apparently, this experimental study is the first demonstration of a poikilothermic vertebrate being a potential paratenic host as well as a potential intermediate host for A. vasorum. The finding of dead A. vasorum in the digested frogs may indicate that the larvae survive for only a limited period in the frog. Similar findings were obtained by Ash (1968) in a study of frogs naturally infected with A. cantonensis. In addition, the finding of many extraordinarily short L3 larvae in a frog infected with L1 larvae may indicate that frogs are not ideal intermediate hosts, although both an increase in larval mortality and a decrease in larval size may result from the crowding of many larvae in one individual frog.

The epidemiological significance of these results remains unknown. Examinations of the stomachs of wild frogs have established that snail tissue may constitute a considerable part of the diet of R. temporaria. Among the snails found in the stomach of these frogs were species known to be capable of acting as intermediate hosts for A. vasorum (Houston 1973; Blackith and Speight 1974; Pilorge 1982). The routes by which frogs may become infected with L1 A. vasorum are highly speculative. Apparently, European foxes feed on frogs infrequently (Brosset 1975; Reynolds 1979; Hewson and Leitch 1983; Robertson and Whelan 1987), and although jumping frogs are obviously attractive to young dogs, the importance of canine frog consumption remains to be elucidated. Acknowledgements. This study was supported by a grant from the Special Fund for Basic Research at the Royal Veterinary and Agricultural University. We are most grateful to M. Sorensen and K. Madsen for their technical laboratory assistance. The Danish Bilharziasis Laboratory, Charlottenlund, is thanked for the rearing of Biomphalaria snails.

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