Immunology 1977 33 581

Trichinella spiralis infection in congenitally athymic (nude) mice PARASITOLOGICAL, SEROLOGICAL AND HAEMATOLOGICAL STUDIES WITH OBSERVATIONS ON INTESTINAL PATHOLOGY

E. J. RUITENBERG, ANNEKE ELGERSMA, W. KRUIZINGA & FERRY LEENSTRA Laboratory of Pathology, National Institute of Public Health, Bilthoven, The Netherlands

Received 24 January 1977; acceptedfor publication 3 March 1977

INTRODUCTION

Summary. In six experiments the course of a Trichinella spiralis infection in congenitally athymic (nu/nu) mice and their heterozygous thymus-bearing littermates (+/nu) was followed. In the +/nu mice worms were expelled at day 10 post infection. In nu/nu mice worms remained in the intestine until the end of the observation period (83 days post infection). In testing the yield of muscle larvae in + /nu and nu/nu mice 4-5 times more muscle larvae were isolated from nu/nu mice than from infected +/nu mice. The following phenomena were observed in + /nu mice only: anti-T. spiralis antibodies detected by immunofluorescence, intestinal plasma-cell production and intestinal eosinophilia. In nu/nu mice no blood eosinophilia was observed in contrast to the induction of eosinophilia both in infected + /nu and infected nu/nu mice reconstituted with thymuses from heterozygous littermates. Intra-epithelial lymphocytes, more numerous in + /nu than in nu/nu mice, were not attracted by Trichinella antigen.

The intestinal phase, or adult T. spiralis worms are expelled from the intestinal tract by the host's immune response (Ruitenberg, 1974; Love, Ogilvie &

McLaren, 1976). Since previous studies emphasized the role of T cell-mediated immunity in the host response (Gore, Burger & Sadun, 1970; Kozar, Karmatnska, Kotz & Seniuta, 1971; Ruitenberg, 1974; Ruitenberg & Steerenberg, 1974), experiments were carried out in congenitally athymic nu/nu mice, lacking functional T cells, and their heterozygous thymus-bearing littermates (+ /nu). Firstly, the expulsion of adult worms, the yield of muscle larvae and the production of specific anti-T. spiralis antibodies was studied. Then secondly, the number of pyroninophilic cells, intra-epithelial lymphocytes and eosinophils in the small intestine were counted. Thirdly, blood eosinophilia was examined in +/nu and nu/nu mice, and also after thymus reconstitution of nu/nu mice. An attempt was made to correlate the changes in intestinal mucosa with the expulsion of T. spiralis adult worms from the gut.

The data supported the hypothesis that worm expulsion is a T cell-dependent phenomenon. Plasma cell and antibody production as well as tissue and blood eosinophilia were shown to be thymusdependent in a T. spiralis infection.

MATERIALS AND METHODS

Correspondence: Dr E. J. Ruitenberg, Laboratory of Pathology, National Institute of Public Health, P.O. Box 1, Bilthoven, The Netherlands.

Mice

Male SPF BIOLP nude (nu/nu) mice were used. For 581

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Expts 1-5 nude mice, 6 weeks of age, were obtained from the Central Laboratory for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands, where the mice were maintained by conventional back crossing with strain BIOLP. Comparisons were made with age- and sexmatched mice heterozygous for the nude gene (+ /nu). After an acclimatization period of 2 weeks, the animals were used for the experiments. Animals were maintained under conventional conditions. For Expt 6 animals from the same source were obtained at an age of 4 weeks. Parasite and parasitological methods The T. spiralis strain used was originally isolated from an infected pig in Poland and maintained in our Institute in laboratory rats (Wistar) from 1960 onwards. After 1972 the strain was also maintained in Swiss mice. Passages were performed every 6 weeks. Larvae used for oral infection were obtained from infected Swiss mice after conventional digestion with HCI and pepsin (Kdhler & Ruitenberg, 1974). Digestion was not prolonged beyond 2 h in order to prevent possible interference with the infectivity of the larvae.

Experimental design Experiment 1. Thirty-one nu/nu and thirty-one + /nu mice, 8 weeks old, were each infected orally at day 0 with 100 T. spiralis larvae. Four (or three at day 6) mice of each group were killed at day 6, 8, 10, 12, 16, 18, 20 and 22 post infection between 9 and 11 a.m. Experiment 2. Thirty-five nu/nu and eight +/nu mice, 8 weeks old, were each infected orally at day 0 with 300 T. spiralis larvae. Five mice of the nu/nu group were killed at day 20, 25, 30, 35, 40, 45 and 50 post infection. Three mice from the + /nu group were killed at day 40 and five mice at day 50. Autopsies were performed between 9 and 11 a.m. Experiment 3. Thirty-five nu/nu mice, 8 weeks old, were each infected orally at day 0 with 300 T. spiralis larvae. Five mice of each group were killed at day 45, 50, 55, 60, 65, 75 and 83 post infection, between 9 and 11 a.m.

Experiment 4. Thirty nu/nu and thirty + /nu mice, 8 weeks old, were each infected orally at day 0 with 100 T. spiralis larvae. Five mice of each group were

killed at day 3, 5, 7, 10, 13 and 16 post infection, between 9 and 11 a.m. Non-infected control mice were killed at day 0. Experiment 5. Twenty-eight nu/nu and 28 +/nu mice, 8 weeks old, were each infected orally at day 0 with 100 T. spiralis larvae. Four mice of each group were killed at day 1, 7, 10, 13, 16 and 18 post infection, between 9 and 11 a.m. Noninfected control mice were killed at day 0.

Experiment 6. Forty nu/nu and 20 +/nu mice, 4 weeks of age, were divided into the following groups, all consisting of ten animals: (1) + /nu infected; (2) +/nu control; (3) nu/nu infected; (4) nu/nu control; (5) nu/nu reconstituted and infected; (6) nu/nu reconstituted and control. Animals from groups 5 and 6 were reconstituted with two thymuses of 6 weeks old + /nu donor mice by s.c. implantation in the axillary region. Animals from groups 1, 3 and 5 were each infected orally with 300 T. spiralis larvae when 9 weeks of age.

Haematological techniques From the animals from Expt 6 blood samples were collected at 8.30 a.m., once every 4 days from the retro-orbital plexus by means of a glass capillary. Blood samples were examined for: (a) total number of leucocytes with a Coulter counter, and (b) number of eosinophils by cytological examination of a blood smear. From these combined data the absolute number of eosinophils was calculated and expressed as number of cells per ml. Autopsy Mice from all experiments were killed by cervical dislocation and the peritoneal and thoracic cavity was opened. Blood was drawn from the heart for serum collection (Expts 1, 2 and 3) and the small intestine was removed. In Expts 1, 2 and 3 the total number of adult T. spiralis worms present in the small intestine was determined. In Expt 2 the animals killed at days 40 and 50 were-after evisceration and skinning-processed by the conventional digestion technique (Kohler & Ruitenberg, 1974) for recovery of the total number of muscle larvae. In Expts 4 and 5 two 10-cm portions of the proximal jejunum were opened longitudinally and rolled up from the posterior end with the mucosa outwards on a wooden stick to form a so-called Swiss roll (Reilly & Kirsner, 1965) and

Trichinella spiralis infection in mice 20 _ FIG.

then transferred to the proper fixative (see histological techniques).

Serological techniques Sera were tested for the presence of specific antibodies to T. spiralis with indirect immunofluorescence (IF) using cryostat sections of isolated T. spiralis larvae as antigen (Ruitenberg, Ljungstrom, Steerenberg & Buys, 1975a). The highest serum dilution giving definite fluorescence was taken as the endpoint titre. In the table results are expressed as positive (titre > 1: 2) or negative (titre < 1: 2). Histological techniques For general morphology and detection of intraepithelial lymphocytes tissues were fixed in buffered formalin, diluted 1 : 10, embedded in paraplast and sections were stained with haematoxylin and eosin (H and E). For staining of pyroninophilic (i.e. immunocompetent) cells, including plasma cells, tissues were fixed in 90 parts Zenker's fluid, 5 parts commercial formalin and 5 parts trichloro acetic acid (ZFT). Paraplast sections were stained with methylgreen pyronine (MP). For staining of eosinophils, formalin-fixed, paraplast-embedded sections were stained with Giemsa. Evaluation of the histological response The number of intra-epithelial lymphocytes in the jejunum was counted in twenty villi per animal. No difference was made between large and small intraepithelial lymphocytes (Ropke & Everett, 1976). The numbers of pyroninophilic cells and eosinophils in the intestinal mucosa were counted in twenty villus/crypt units per animal. Although the majority of the pyroninophilic cells showed the morphological characteristics of plasma cells and contained immunoglobulins (based on immunofluorescence studies), no reliable distinction can be made between the various pyroninophilic cells using the MP staining method. A villus/crypt unit represents a portion of gut mucosa, lying between two gland crypts and the lamina propria of the villus above (Jarrett, Jarrett, Miller & Urquhart, 1968). RESULTS

Parasitological data Recovery of adult worms The percentage recovery of adult worms in Expts 1, K

583

10 -e

6

8

10

12

14

K

18

20 22

0

FIG. 2 'D30

)45455050556065

75

83

Days post -infection Figure 1. Expulsion of adult T. spiralis worms from small intestine of groups of +/nu (open columns) and nu/nu mice (hatched columns) infected with 100 larvae each (Expt 1). Means of three to four per point ± s.d.

Figure 2. Expulsion of adult T. spiralis worms from small intestine of nu/nu mice infected with 300 larvae each Expt 2 (hatched columns) and Expt 3 (open columns). Means of five per point ± s.d. Table 1. Yield of muscle larvae from +/nu and nu/nu mice infected with 300 T. spiralis larvae each at various days postinfection + /nu

nu/nu

Day

No. of mice

Mean no. of larvae

No. of mice

Mean no. of larvae

40 50

3 5

35,700 29,900

5 5

170,400 176,700

and 2 and 3 are shown in Figs 1 and 2 respectively. In the +/nu mice a significant number of adult worms was present until day 8, whereas in the nu/nu mice T. spiralis worms were present until the end of the observation period of Expt 3 (day 83). In the nude mice the percentage recovery from the 100 larvae infection was lower than that from the 300 larvae infection of Expt 2 and comparable with that from the 300 larvae infection of Expt 3. Recovery of muscle larvae The numbers of larvae isolated at 40 and 50 days post infection (Table 1) from the infected nu/nu mice were 4-5 times higher than those of the infected + /nu mice.

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Table 2. Antibody response to T. spiralis in +/nu and nu/nu mice infected orally with 300 T. spiralis larvae each (groups of five animals per day) at various days post-infection

Table 5. Number of eosinophils in the jejunum of groups of four male +/nu and nu/nu mice infected with 100 T. spiralis larvae each at various days post-infection

IF titre Day

0; 8; 10 12; 14; 16; 18; 20; 22 20; 25; 30; 35; 40; 45; 50

No. of eosinophils*

+ /nu

nu/nu

Day

+ /nu

-* +t n.d.

-

0 1 7 10 13 16 18

1-31 ±0 94 0 93 0-82 1-61 ± 1-37 2-20±1-69 1-76±1-25

n.d. = Not done. * Titre < 1 : 2. t Titre >_1 : 2. Table 3. Number of pyroninophilic cells in the jejunum of groups of five male +/nu and nu/nu mice infected with 100 T. spiralis larvae each at various days post-infection

0-89±090 0-65 ±0-73

Pt

nu/nu

P

Trichinella spiralis infection in congenitally athymic (nude) mice. Parasitological, serological and haematological studies with observations on intestinal pathology.

Immunology 1977 33 581 Trichinella spiralis infection in congenitally athymic (nude) mice PARASITOLOGICAL, SEROLOGICAL AND HAEMATOLOGICAL STUDIES WIT...
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