This experimental study is the first among other experimental animal models to provide detailed information about the pathogenicity, the fecundity and egg excretion patterns of H. pumilio infections.
An acute phase occurred within the first weeks of infection: From week 2 p.i. anorexia occurred in all infected foxes which resulted in weight loss and was associated with a depletion of lymphocytes, eosinophils, and moderate anaemia. Egg excretion also peaked in this initial phase. Appetite, body weight and most haematological parameters were back to normal at the end of the study and egg excretion had found a constant level from day 24 post infection. Important biological information, like finding the pre-patent period to be 9 days and estimating the reproductive capacity to be 58 ± 40 eggs daily per worm, was also gained. The knowledge we obtained on the clinical impact on H. pumilio infection in a host relevant to known reservoir hosts in endemic areas can be useful for both clinical management of human infections and in the planning of control programs in aquaculture.
A natural infection pattern with H. pumilio is likely to consist of multiple infections over time since farm dogs probably eat raw fish continuously rather than on a single occasion. However, an initial step to describe biological data and host reaction is to use single infections. The infection dose of 2000 metacercariae used for the foxes can be considered at the high end of what, for example, a dog would acquire by a single meal of raw fish based on findings in natural infections in fish. The density of metacercariae in the wild-caught fish from canals and fish from ponds in the Nam Dinh Province in Vietnam was 0.5 and 0.03 metacercariae/g fish tissue . However, metacercariae are not evenly distributed in the fish tissue [26, 27] and if a dog happens to eat parts of a fish containing predilection sites for metacercariae, e.g. basis of fins, or several fish or fish parts per day, a dose of 2000 metacercariae is probably not unlikely. Light infections of 500 H. pumilio metacercariae in dogs have been found to be asymptomatic  but clinical signs have been reported from a study in cats using 80,000 metacercariae of M. yokogawai in which infected cats lost their appetite, and became physically inactive during the first two weeks of infection.
This study described a shorter pre-patency period of H. pumilio, being only 9 days, than an experimental study in dogs . However, the finding in the foxes is in accordance with detection of gravid H. pumilio worms in experimentally infected mice day 9  and the prepatency period of the heterophyid, Heterophyes heterophyes reported in humans . Egg excretion in the present study increased rapidly after the infection became patent, peaked on two sampling days, then dropped again and became stable at a level below 500 egp. A similar pattern with an acute phase with high egg output is seen for other trematodes like echinostomes . The peak in egg excretion also coincided with anorexia and hence lower faecal output. A part of the increase could therefore probably be explained by an up-concentration of the eggs in the faeces. Since the total amount of faeces was not measured during this period it was impossible to determine to what extent the increase was attributed to up-concentration or increased egg output by the worms. The number of eggs excreted by the foxes can be considered low compared to the liver flukes (C. sinensis 2500–3000 eggs/worm/day)  but is comparable to what was estimated for H. taichui (82 eggs/worm/day) . Information describing worm burden and reproductive capacity is warranted for developing better diagnostic tools e.g. copro-DNA analysis for detection of minute intestinal flukes .
The life expectancy of Haplorchis spp. remains unknown but for other heterophyids the life span of the worms is speculated to be less than a year . In our study, all eight foxes harboured adult worms at the time of necropsy and were excreting eggs throughout the 8 week study period. After the initial peak in egg excretion no signs of declining fecundity of the worms were seen. In a previous study with low dose of 500 H. pumilio metacercariae in dogs the infection also prevailed until necropsy around 8 weeks p.i. . Hence, a single infection can result in at least two months of patency showing no obvious signs of immune related expulsion of worms; however future studies should aim at describing the host’s immune regulation towards repeated infections mimicking natural circumstances, which might differ from the pattern seen here.
The mean establishment of 47% in this study was considerably higher than in a similar study with dogs (mean 12%)  and in an experimental M. yokogawai infection in dogs (15-20%) . One might speculate that the metacercariae present in the fish tissue used for infection in this study had a higher infectivity and therefore gave a higher establishment proportion than in studies, where artificially digestion and storage of metacercariae has been performed prior to infection. The high establishment proportion and the persistent egg excretion underline that foxes can serve as a good experimental model for H. pumilio infections and perhaps for other zoonotic heterophyids as well [9, 35, 36].
The predilection site for the flukes being the lower part of the jejunum confirms earlier findings with H. pumilio. Whereas previous studies on M. yokogawai and H. heterophyes described predilection site of the worms in the small intestine to differ somewhat (M. yokogawai being found in the duodenum and more anterior than H. heterophyes which is located further back [11, 29]) both species are reported attached to the mucosa in the crypts of Lieberkühn . The finding of an adult worm in a histological section and the methods for recovering the worms (more than three times as many found by processing the intestinal wall than the content) in the present study also indicated that the majority of the worms were embedded between the villi, probably attached to the mucosa. Case reports with H. taichui have shown the worms to be attached to the epithelium with their oral sucker and causing damages like microhaemorrhages and villuos atrophy with eosinophil and lymphocyte infiltration . None of these pathological changes were seen in the present study, nor was any macroscopic changes in the mucosa found. A previous experimental study with M. yokogawai in dogs showed that mucosal damage was reversible and that the mucosa returned to a normal four weeks post infection . Future investigations should include necropsies in the early, acute phase of infection to evaluate possible histopathological changes.
Depletion of blood eosinophils was seen in week 2 p.i. but subsequently the level did not differ from the control foxes. The depletion in blood eosinophils in the acute phase of the infection could perhaps be due to an up-regulated, local immune response taking place shortly after infection. Although many helminth infections often are associated with eosinophilia, this is normally not the case for intestinal trematodes  and was not the case in this study, either. Hence blood eosinophilia is not a good diagnostic criterion in H. pumilio infections in foxes; however, confirmation is needed from other hosts.
The changes in biochemical markers were also mainly seen in week 2 p.i. The decrease in albumin has also been seen in other cases of gastrointestinal diseases and intestinal parasitism . Due to the small size of the protein it is likely to leak into the intestine if the integrity of the mucosa is disrupted. The transient anaemia could be a result of excessive blood cell destruction (haemolysis) or caused by haemorrhages e.g. micro-bleedings caused directly by the flukes. The anaemia was associated with a rise in total bilirubin, the breakdown product of the heme catabolism but this was only seen in week 2 p.i. After a few weeks the acute phase with symptoms declined and most markers analysed in the present study were back to normal. In the infected foxes we observed sporadic increased serum ALT, which is normally used as a marker for hepatic injury in dogs . We are not able to explain these sporadic increases by the fluke infection since no damage was seen in the livers, however, increased ALT activity is known to occur in a wide range of other disorders including hypoxia secondary to anaemia , which could perhaps be the case in this instance.