Norman F. Weatherly

Increased survival of Swiss mice given sublethal infections of Trichinella spiralis.

Male mice infected with 70, 160, 325, 420, and 535 larvae of T. spiralis lived longer than uninfected controls. Males infected with 35, 650, and 685 larvae had life spans similar to controls. Females infected with 35, 70, 160, 325, and 420 larvae lived longer than their controls, whereas females infected with 535 larvae had a life span similar to the controls. As expected, males and fe- males infected with more than 790 and 650 larvae, respectively, had shorter life spans than their con- trols. Reduced fighting among males, suppression of the development of breast cancers in females, and reduced food intake are proposed as possible explanations for the demonstrated longer life spans of the infected animals. Many studies have shown that mice infected with 30 to 75 larvae of T. spiralis per gram of weight will die within a relatively short period of time (Rappaport and Wells, 1951; Ewert and Olson, 1961; Kozar and Kozar, 1963; and others). To the authors knowledge, how- ever, no one has determined the effects of sublethal levels of infection on the life spans of mice or other animals. The present study shows that sublethal levels, in general, in- creased the life expectancy of Swiss mice.

Studies on Delayed (Cellular) Hypersensitivity in Mice infected with Trichinella spiralis. III. Serologie and Histopathologic Findings in Recipients given Peritoneal Exúdate Cells.

The present results confirm those of the previous paper in this series by showing that peritoneal exudate cells transferred from infected donors cause a significant loss of adult worms in recipients given a challenging infection. In addition, the mice of these various studies were shown to be an isologous strain, and peritoneal exudate cells from uninfected donors had no demonstrable effect on worm elimination. On the basis of serologic, and, especially, intestinal histopathologic findings, additional evidence is provided to support the hypothesis that worm elimination is associated with delayed hypersensitivity. In the previous paper of this series (Larsh et al., 1964), evidence was presented to support an hypothesis that delayed hypersensitivity is involved in the mechanism that brings about the expulsion of adult Trichinella spiralis from mice. The present study was designed in an attempt to reproduce these results, showing that recipients lost significantly more adult worms than controls after a challenging infection. In addition, it was decided to obtain other information needed to test this working hypothesis. In the first place, it was necessary to determine definitely whether the mice used in these various experiments are an isologous strain, since this is a prime requirement in such transfer studies. Second, it was desirable to determine whether peritoneal exudate cells from uninfected donors are incompetent in that they have had no contact with parasite antigens. Third, it seemed of value to test the serologic responses of various groups of mice at specified periods. While available evidence fails to support the view that humoral antibodies play an important role in adult worm elimination, it was felt that serologic findings might shed light on the possibility that transferred peritoneal exudate cells set up an adoptive immune response. Finally, and most important, it was necessary in testing the present hypothesis to determine whether or not intestinal inflammation precedes the loss of adult worms. A characteristic inflammation in the anterior small intestine of our mice has been shown in Received for publication 12 July 1965. 146 various experiments to be directly responsible for worm elimination (Larsh, 1963). MATERIALS AND METHODS

Effects on litter size and litter survival in Swiss mice infected with Trichinella spiralis during gestation.

Groups of mice during their first pregnancy were infected with 35, 70, 150, 275, or 400 larvae of Trichinella spiralis. The number of progeny born among infected mice was similar to that of uninfected controls. However, mice infected with 35, 75, or 150 larvae successfully reared litters averaging 4.6, 3.7, and 0.2 progeny at 30 days, respectively, whereas litters from uninfected controls averaged 7.1 progeny. Mice infected with 275 or 400 larvae gave birth to normal-sized litters, but no progeny survived 30 days. Females infected during their first pregnancy with 35, 70, or 150 larvae were reinfected during their second pregnancy with 150 larvae. Although the average sizes of the surviving litters that resulted from the second infection were somewhat less than those from the uninfected controls, they were larger than those from mice infected for the first time. Therefore, when compared with mice in the latter group, a prior infection with T. spiralis conferred a distinct advantage to mice during their second pregnancy and second infection. The effects of certain forms of physical stress applied during pregnancy of mice, such as hypoxia and elevated temperatures, have been reported (Ogle, 1934; Curley and Ingalls, 1957). Maternal and fetal responses to injection of formalin in rats were reported by Schniirer (1963). Because the adrenal glands are intimately involved in stress situations, adrenalectomy and adrenocorticotropin injections on pregnancy and litter size have been investigated (Velardo, 1957; Fernandez-Cano, 1958). The literature is replete with studies on the effects of viral, fungal, bacterial, and protozoal infections on pregnancy, gestation, and malformation of progeny. However, to my knowledge, no one has reported the effects of helminthic infections on litter size and litter survival. Related studies, such as those of Augustine (1934) using Trichinella spiralis and Webster (1956) using Toxocara canis, were primarily concerned with congenital infections. Oshima (1961) investigated the influence of pregnancy and lactation on the migratory habits of the larvae of T. canis, but did not report results concerning litter size and survival. This study shows the effects on gestation, litter size, and litter survival in mice infected with T. spiralis during pregnancy. MATERIALS AND METHODS The Swiss mice used in this study came from a colony randomly inbred in the Department of Received for publication 6 July 1970. Parasitology and Laboratory Practice, University of North Carolina at Chapel Hill, for over 27 years. The strain of T. spiralis was originally isolated from a pig in 1936. Although it was maintained in laboratory rats for 7 years after isolation, it had be n maintained for the past 27 years in the Swiss mice described above. Larvae for infection were obtained from mice infected not less than 45, nor more than 75, days. The technique used for collecting and standardizing the infecting inoculum were those described by Weatherly (1970). Experience has shown that the numbers of worms becoming established in mice are considerably less than the numbers of larvae in the infecting inocula. Consequently, 6 mice were used as viability controls for each group of mothers, and the levels of infection recorded in Tables I and II are the average counts from these controls. The recovery of adult worms for viability counts was done with the techniques of Larsh and Kent (1949). EXPERIMENTAL PROCEDURES AND RESULTS

Studies on delayed (cellular) hypersensitivity in mice infected with Trichinella spiralis. IV. Artificial sensitization of donors.

In 3 experiments, it was demonstrated that 2 injections of supernatant material, collected after disruption and centrifugation of larvae, combined with Freunds complete adjuvant sensitized donor mice sufficiently so that after injection of their spleen cells recipients showed immunity as indicated by a significant reduction in adult worms compared with controls 12 days after infection. The adjuvant alone did not contribute to this effect, since recipients injected with cells collected from donors given 2 injections of adjuvant alone harbored about the same number of worms as the regular controls. The value of using spleen cells in such studies in mice, and of isolating a single provocative antigen for use in future studies, is discussed. In the last two papers of this series (Larsh et al., 1964, 1966), peritoneal exudate cells collected from infected donors conferred immunity upon recipients as proved by a significantly lower adult worm burden than in controls after a challenging infection. Although it has been shown in guinea pigs that a single small dose of a crude saline-extract antigen from T. spiralis larvae injected intradermally produced delayed hypersensitivity, as demonstrated by skin tests, that was transferred to recipients by spleen cells (Kim, 1966) or lymph node cells (Kim et al., 1967), this has not been reported from studies in mice. Therefore, the present study was designed to determine whether a crude extract antigen would sensitize our donors sufficiently to confer immunity upon recipients after the transfer of cells. In this case, the success of sensitization would be manifested by a significant reduction in adult worms after a challenging infection. In 10 unsuccessful experiments with peritoneal exudate cells, separate variables, such as methods of antigen preparation, size of sensitizing doses, and routes of injections and schedules for production of sensitivity, were manipulated in attempts to demonstrate immunity. At this point, it was decided to use spleen cells for transfer. In the report that follows, positive results were obtained in three experiments where spleen cells were taken from donors after two injections of an antigenadjuvant mixture. The antigen was the superReceived for publication 11 October 1968. natant extract obtained by disruption of T. spiralis larvae in a Ten Broeck Tissue Grinder followed by centrifugation at 1,000 g for 20

Studies on delayed (cellular) hypersensitivity in mice infected with Trichinella spiralis. V. Tests in recipients injected with donor spleen cells 1, 3, 7, 14, or 21 days before infection.

Three experiments are described. In the first, mice were injected twice into the footpads with an antigen prepared from larvae and mixed in equal volume with Freunds complete adjuvant. After an infection with T. spiralis, these mice harbored significantly fewer adult worms than noninjected controls thereby proving the immunizing effectiveness of the two antigen injections. In the last 2 experiments, donor mice, treated as the experimentals in the first experiment, were killed to collect spleen cells for transfer into recipients, which were challenged with T. spiralis 1, 3, 7, 14, or 21 days after transfer of the cells. The counts of adult worms in these recipients and their respective controls were not significantly different in the case of the 1-day and 3-day groups, but the 7, 14, and 21-day recipients harbored significantly fewer worms than their controls. On the basis of these results, it is concluded that the 2 injections of this antigen preparation produced effects that caused a significant expulsion of worms after challenge, and that the spleen cells from mice treated in this way were responsible for a similar effect noted in recipients challenged as early as 7 days after cell transfer. It was shown recently that recipient mice injected with spleen cells from artificially sensitized donors 21 days before infection with T. spiralis eliminated significant numbers of adult worms as shown by counts made 12 days after infection (Larsh et al., 1969). The 3-week period between transfer of cells and challenge was used for the reason that it was effective in earlier studies with peritoneal exudate cells (Larsh et al., 1964b, 1966) and lymph node cells (Larsh et al., 1964a). However, it requires a relatively brief period after transfer of sensitized lymphoid cells to demonstrate sensitivity in recipients. In fact, as shown by studies in guinea pigs sensitized with a crude saline extract of T. spiralis larvae, the optimal latent period for demonstrating delayed skin reactions in recipients was 3 days after transfer of lymph node cells (Kim et al., 1967). In view of this, and the obvious advantages of a shorter latent period in our studies with mice, it was decided to test the immunity in recipients infected 1, 3, 7, or 14 days after transfer of spleen cells from artificially sensitized donors. The 21-day period also was included as a check on previous results. Received for publication 26 March 1970. MATERIALS AND METHODS The Swiss white mice used in this study were a strain that had been randomly bred in our laboratory throughout the year for more than 27 years. The strain of T. spiralis and the methods for isolating larvae for infections and for recovering adult worms after challenge were those used by Larsh and Kent (1949). The antigen(s) for the sensitization of donors was made from larvae of

Studies on delayed (cellular) hypersensitivity in mice infected with Trichinella spiralis. 8. Serologic and histopathologic responses of recipients injected with spleen cells from donors suppressed with ATS.

Spleen cells were collected from sensitized donors that had been suppressed with antithymocyte serum (ATS) for 16 consecutive days after a second sensitization with an antigen-adjuvant mixture. These cells were injected ip into mice of Recipient Group A. Ten days later, they were challenged with 100 T. spiralis larvae, and after 2 and 10 days some of the mice were killed for collection of tissue from the small intestine for histopathologic studies. Mice of Recipient Group B were injected with spleen cells from sensitized, nonsuppressed donors and those of Recipient Group C were injected with cells from nonsensitized, nonsuppressed donors. Other than the source of the spleen cells, the recipients of groups B and C were treated in the same manner as those of Group A. Blood for later serologic testing was taken from all recipients killed 10 days after infection. At 2 days after challenge, the tissues of the recipients (B) of cells from the sensitized, nonsuppressed donors showed evidence of acute inflammation, whereas such evidence was not detected in the mucosa and submucosa of recipient groups A and C. At 10 days, tissues from mice of all 3 recipient groups showed acute inflammation, but the response was much more severe in groups A and B than that observed in group C, and it was more severe in Group B than in Group A. Therefore, the transfer of cells from the sensitized, nonsuppressed donors resulted in an earlier initiation and a more acute degree of inflammation after challenge, and the cells transferred from the sensitized, suppressed donors resulted in effects in recipients that were intermediate between the former and those injected with cells from nonsensitized, nonsuppressed donors. As noted in various earlier histopathologic studies of the small intestine of our mice, the severity of the inflammation was associated directly with the numbers of worms recovered. The serologic results with a battery of tests were inconclusive. In the final experiment of the previous paper of this series (Larsh et al., 1972), it was demonstrated that donor mice sensitized twice by footpad injections of an antigenadjuvant mixture and suppressed with antithymocyte serum (ATS) for 27 consecutive days after the second sensitization harbored about the same numbers of worms after challenge as the regular controls that had not been sensitized or suppressed. Inasmuch as other donors that were sensitized in the same way as the former group but not suppressed harbored significantly fewer worms after challenge, it was clear that the immunosuppressive effect of ATS had abolished completely the expression of the immunity produced by the Received for publication 9 July 1973. * Supported in part by Grant AI-10671 from

Studies on delayed (cellular) hypersensitivity in mice infected with Trichinella spiralis. IX. Delayed dermal sensitivity in artificially sensitized donors.

A series of preliminary experiments was completed to check skin-testing techniques with both our crude saline extract antigen and Melchers antigen. Some of the sensitized mice that had been injected into the abdominal skin with test doses of 20 and 100 uLg of trichinella protein developed what appeared to be positive DH reactions, but reactions in some controls, due presumably to nonspecific irritation produced by particulate matter, made differentiation uncertain. In the 2 final experiments, Melchers antigen and the footpad swelling test were used and the results with peak reactions at 24 hr were unequivocal. It required 7 days after completion of sensitization to obtain consistently strong reactions, and 2 sensitizations produced much more swelling than one. Proof for a characteristic DH response was obtained in tissue sections that showed a predominant infiltration of mononuclear cells. We have shown repeatedly that mice sensitized twice by footpad injections of an antigen-adjuvant mixture develop striking immunity to a challenging infection with Trichinella spiralis (Larsh et al., 1969, 1970a,b, 1972, 1973). Moreover, spleen cells transferred from such artificially sensitized donors consistently conferred a significant degree of immunity upon recipients as measured by the numbers of adult worms in the small intestine 11 days after a challenging infection. Because macrophages were inhibited in migration when mixed with spleen cells from such sensitized mice in the presence of the antigen (Cypess and Larsh, 1970), we assumed tentatively that delayed hypersensitivity (DH) had been transferred adoptively to the recipients. However, despite the fact that macrophage migration inhibition, an in vitro correlate of DH, and delayed dermal sensitivity, an in vivo correlate, usually coexist, it seemed worthwhile to make a separate, direct study of the relation between dermal sensitivity and immunity produced by artificial sensitization(s). The present paper is limited to studies of this relation in sensitized