The effect of concurrent infection with Trichinella spiralis on Hymenolepis microstoma in mice

Summary The intestinal changes brought about by rejection of Trichinella spiralis from mice were studied in relation to their effects on a concurrent infection with Hymenolepis microstoma, a cestode not normally rejected from mice. The rejection phase of T. spiralis was associated with a marked stunting of growth of H. microstoma given just before, during, or just after rejection of the nematode. The survival of H. microstoma was affected only when rejection of T. spiralis coincided with the intestinal phase of the cestode: if T. spiralis rejection was timed to occur after the scolex of the cestode had entered the bile duct there was no loss of H. microstoma. It is suggested that the adverse effects on growth and establishment of H. microstoma were due to the non-specific inflammatory component of the host's response to infection with T. spiralis.


INTRODUCTION
The expulsion of Nippostrongylus brasiliensis from rats has been reported to require not only an antibody response and a specific cellular element but also a non-specific element derived from bone marrow (Dineen & Kelly, 1973). After reviewing the immune mechanisms of various nematode infections, Ogilvie (1974) suggested that 'whatever the exact sequence of immunological events which trigger expulsion (of nematodes) it is probably caused by the release of some nonspecific effector'. Bruce & Wakelin (1977) showed that the expulsion of Trichinella spiralis from mice resulted in the non-specific expulsion of a concurrent Trichuris muris infection and that this effect was reduced by the administration of the antiinflammatory drug indomethacin. A similar non-specific rejection of Hymenolepis diminuta from mice is associated with the rejection of a concurrent T. spiralis infection and it was suggested that this was attributable to the inflammation in the intestine initiated by the latter parasite (Behnke, Bland & Wakelin, 1977). These observations on interspecific interactions were extended in the present * Reprint requests to P. R. Christie. t Present address: Department of Zoology, University of Nottingham, University Park, Nottingham, NG7 2RD. report by determining the effect of a T. spiralis infection upon a concurrent H. •microstoma infection. In contrast to all the above parasites, H. microstoma is not normally rejected from mice (Moss, 1971;Howard, 1977), although a secondary infection grows more slowly than a primary infection (Tan & Jones, 1967, 1968Howard, 1976). The work presented in this paper was initiated to establish whether or not H. microstoma is susceptible to rejection processes, utilizing the severe host response to T. spiralis infection.

MATERIALS AND METHODS
Helminth-free male and female NIH (Anglia Laboratory Animals) mice were used in Experiments 1 and 2 respectively, male C3H (Bantin and Kingman Limited, Yorkshire) mice were used in Experiment 3, and outbred male CFLP (bred at the Wellcome Laboratories) mice were used in Experiment 4. All mice received food and water ad libitum.
The infection and autopsy procedures have been described previously for H. microstoma (Howard, 1976(Howard, , 1977 and T. spiralis (Wakelin & Lloyd, 1976a). Briefly, mice received cysticercoids of the cestode by stomach tube whilst under light ether anaesthesia. The cestodes were recovered by slitting open the bile duct and small intestine and incubating these tissues at 37 °C for 1-2 h. Infective larvae of T. spiralis were recovered from rats or mice by digestion of the animals in acid pepsin for 2 h at 37 °C These larvae were suspended in 0-2 % agar and the mice were infected with 500 (Exps 1 and 3) or 350 (Exps 2 and 4) larvae by oral inoculation into the stomach with a syringe and blunted cannula. The adult nematodes were recovered by a modified Baermann technique.
The dry weights and numbers of cestodes recorded from different groups of mice were compared using the Wilcoxon rank sum test (Remington & Schork, 1970).
NIH mice reject T. spiralis starting on day 8 of infection and rejection is complete by day 11-5 (Wakelin & Lloyd, 1976a); C3H and CFLP mice are slower in rejecting T. spiralis, but major worm loss generally occurs between days 10 and 12, rejection being complete by day 18 (Wakelin, unpublished observations).

The effect of a T. spiralis infection on the growth and survival
of a pre-existing H. microstoma infection Investigation of the effect of T. spiralis infection on a pre-existing H. microstoma infection began with experiments (Exps 1 and 2) in which groups of mice were infected with either 5 cysticercoids of H. microstoma, or H. microstoma and T. spiralis 5 days later, or T. spiralis only. These groups were infected and killed as shown in Table 1, and the results are given in Table 2.
The establishment and rejection of the nematode were apparently not affected by concurrent infection with the cestode (Group B). Similarly, the survival of the cestode was not affected by the nematode (Groups B, E, H and K). However, * Hm, infected with 5 cysticercoids of H. microstoma; Tsp, infected with 500 (Exp. 1) or 350 (Exp. 2) larvae of T. apiralis.
•f K, day of autopsy. H. microstoma recovered from dual-infected mice were lighter than those from control mice, but the difference was significant only in mice killed immediately after rejection of the nematode had commenced (Groups E and H killed 11 and 12 days after infection with T. spiralis respectively). The weight of worms recovered from the dual-infected mice 30 days after T. spiralis infection (Group K) was lower than that from control mice but not significantly so, indicating that the stunting of the cestode was not permanent.
The establishment and growth of H. microstoma given before, during, and after the rejection phase of T. spiralis The results of the previous experiments showed that the growth, but not the survival, of H. microstoma was affected during the expulsion of T. spiralis. The cestode may not have been lost during the rejection phase of T. spiralis because (1) the scolex of H. microstoma is completely resistant to the changes in the gut associated with rejection of the nematode, or (2) the scolex was protected from contact with these intestinal changes by being inside the bile duct.
In an attempt to clarify this situation, an experiment (Exp. 3) was designed in which 4 groups of T. spiralis-infected mice were challenged with H. microstoma at various times so that (1) the scolex of the cestode was inside the bile duct (3-4 days after infection) before expulsion of the nematode had begun (Group B), (2) the cestode was migrating in the intestine during the early part of the expulsion phase of the nematode (Group D), (3) as (2), but during the latter part of the expulsion phase (Group F) and (4) the cestode was establishing after the nematode had been expelled (Group H).
The scope of this experiment was extended in Exp. 4 by infecting a group of mice with H. microstoma before infecting with T. spiralis, so that the cestode had only experienced the pre-rejection phase of T. spiralis at autopsy (Group J); and by infecting another group as for (4) above, but later, after expulsion of the nematode. All mice were autopsied 10 days after infection with H. microstoma and the design and results of Exps 3 and 4 are summarized in Table 3.
In mice infected with H. microstoma 3 days before giving T. spiralis (Groups I and J) there was a similar number of cestodes recovered from dual-infected and control mice and there was no loss of weight in the cestodes recovered from the dual-infected group: in fact, the average weight was slightly higher than that from the control group.
In mice infected with H. microstoma 1 day after infection with T. spiralis (Groups A and B) there was a similar number of cestodes recovered from both groups, but the weight of the cestodes from the dual-infected group was significantly lower than that from control mice.
In mice infected with H. microstoma 8 or 12 days after T. spiralis (Groups G-D, E-F and K-L) significantly fewer cestodes were recovered from the dual-infected mice than from control mice and these worms were significantly lighter than those from control mice (the differences between Groups F and L may be attributable to the use of different strains of mice and/or different numbers of T. spiralis administered). In mice infected with H. microstoma 21 or 29 days after giving T. spiralis (Groups G-H and M-N), the recoveries from the dual-infected groups were not significantly lower than from controls. Cestodes recovered from the group given T. spiralis 21 days before infection with H. microstoma were significantly lighter than controls, but no significant difference in weight was evident in the group given H. microstoma 29 days after infection with T. spiralis.

DISCUSSION
The results of the experiments presented in this paper suggest that the effect of concurrent infection with T. spiralis on survival and growth of U. microstoma depends greatly on the relative timing of the infections. In mice infected with H. microstoma before T. spiralis and autopsied before rejection of the nematode had commenced, there was no significant effect on the weight of the cestode, indicating that the pre-rejection phase of T. spiralis had no harmful effects on the cestode.
However, H. microstoma which had been exposed to the rejection phase of T. spiralis were always lighter than those from control mice, and this deleterious effect on the cestode appeared to persist until at least 21 days after infection with the nematode. There was no significant difference between the weights of H. microstoma recovered from dual-infected mice and those from control mice when infection with the cestode took place 29 days after infection with T. spiralis, indicating that this effect is unlikely to be due to an immunologically specific cross-reaction. Stunting of H. microstoma exposed to the rejection phase of T. spiralis was severe, but apparently not permanent: there was no significant difference between the weights of worms recovered, from mice infected with H. microstoma only and those infected with S. microstoma 30 days after infection with T. spiralis (Table 2, Exp. 2, Groups J and K).
Rejection ofT. spiralis from the mouse appears to involve both specific humoral and cell-mediated responses (Love, Ogilvie & McLaren, 1976;Wakelin & Lloyd, 19766), but the final effector mechanism may well be a non-specifically acting inflammation of the intestine (Larsh, 1967;Larsh & Race, 1975). As there appears to be no direct cross-immunity between H. microstoma and T. spiralis, it is apparently the non-specific inflammatory component that is responsible for the observed effects on survival and growth of H. microstoma. However, mice reduce their food consumption during rejection of T. spiralis (Larsh, Goulson & Van Zandt, 1962;Goulson & Larsh, 1964) and it is well known that reduced intake of carbohydrates may retard the growth of hymenolepids (Read & Rothman, 1957 a). The reduction in growth of H. microstoma may, therefore, have been at least partially due to the altered dietary intake of the mouse, although it is also conceivable that a reduced intake might be offset by the malabsorption of nutrients associated with T. spiralis infection (Castro, Olson & Baker, 1967). However, it is unlikely that altered intake would markedly affect the initial establishment of the worms (Read & Rothman, 19576): the significant reduction in numbers of H. microstoma recovered that did occur in some groups was probably, therefore, caused by the inflammation associated with the rejection of T. spiralis. The role of inflammation in retarding growth of the cestode is less clear.
In secondary infections of H. microstoma in mice, worm growth is retarded by the host response during the first 4 days of infection: after entering the bile duct, growth resumes at a similar rate to that in primary infections (Howard, 1977). This resembles the situation described in the present paper in which, following establishment of the scolex of H. microstoma in the bile duct, the worm was not sufficiently affected by inflammation for loss to occur: it may be that changes in the intestine during inflammation do not extend into the bile duct. It is also possible that the scolex itself changes after entry into the bile duct and becomes resistant to inflammatory products; the inflammation of the peribiliary tissues which is initiated by the cestode itself does not appear to affect its survival (Lumsden & Karin, 1970), but the components in this inflammation may differ considerably from T. spiralis-inAnced intestinal inflammatory products.