PATHOLOGY OF TAENIA TAENIAEFORMIS INFECTION IN THE RAT BY Roger W. Cook A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1979 TO RUTH ii ACKNOWLEDGEMENTS I sincerely thank Dr. Jeffrey F. Williams for guiding and encouraging me during these studies and for broadening my perspectives on experimentation in animal disease. Dr. Allan L. Trapp, my academic advisor, and Dr. Janver D. Krehbiel have helped me throughout my training in the Department of Pathology at Michigan State University. I appreciate the assistance of my other committee members, Drs. Harold C. Miller and Robert W. Leader. My colleagues in the laboratory, Anthony J. Musoke, Bruce Hammerberg, John Picone, Joseph M. Ayuya, Carlos W. G. Lopes, Anne M. Zajac and Martha S. Calkins, have frequently helped, and I greatly value their friendship. Alma M. Shearer, Marla Signs, Sharon M. Garland and Elaine M. Dunlap have provided me with excellent technical assistance, for which I am grateful. I thank the New South Wales Department of Agriculture in Australia for granting me this period of study leave. My wife Ruth has been very patient and encouraging for so long and with my daughter Amanda made many sacrifices, especially towards the end. I thank them both. iii TABLE OF CONTENTS LITERATURE REVIEW. 0 O O O O O O O 0 O O O O O O O O O O O O O Morphologic ReSponse of the Host to Taeniid Metacestodes. . . . . . . . . . . . . . . . . . . . . General. . . . . . . . . . . . . . . . . . . . . Taenia taeniaeformis . . . . . . . . . . . . . . Immunity to Taeniid Metacestodes. . . . . . . . . . . . Humoral Immunity . . . . . . . . . . . . . . . . YE . . . . . . . . . . . . . . . . . . . . . . . Cell Mediated Immunity . . . . . . . . . . . . . Gastrointestinal Hormones and Parasitic Disease . . . . General. . . . . . . . . . . . . . . . . . . . . Gastrin. . . . . . . . . . . . . . . . . . . . . Trophic Action of Gastrointestinal Hormones. . . Gastrin and Disease. . . . . . . . . . . . . . . Gastrin and Parasitism . . . . . . . . . . . . . REFERENCES 0 O O O O O O O O O O O O O O O O 0 ARTICLE 1 - PATHOLOGY OF TAENIA TAENIAEFORMIS INFECTION IN THE RAT O O O O O O O O O O O O O O C I C O O O O 0 ARTICLE 2 - PATHOLOGY OF TRENIA TAENIAEFORMIS INFECTION IN THE RAT: GASTROINTESTINAL CHANGES . . . . . . . . ARTICLE 3 - HYPERPLASTIC GASTROPATHY IN THE RAT DUE TO TAENIA TAENIAEFORMIS INFECTION: PARABIOTIC TRANSFER AND HYPERGASTRINEMIA O O I 0 O O O O O O O O O O O 0 O - APPENDICES . O O O O O O O O 0 O O O O A SURGICAL REMOVAL OF THE PYLORIC ANTRUM IN WEANL I NG RATS I O O O O O O O O O O O O O O O O C B EFFECT OF INFECTION WITH TAENIA TAENIAEFORMIS ON BODY AND ORGAN WEIGHTS, FEED CONSUMPTION AND FECAL OUTPUT IN THE RAT. . . . . . . . . . . . . C CELLULAR TRANSFER OF IMMUNITY TO TAENIA TAENIAE- FOMIS IN TEE RAT O C O O O O O O O O O O O O O O VITA O O O C O O O O C O O O O O O O O O O O O O O O O O O O 0 iv Page momwww 10 ll 12 14 16 17 19 29 51 81 94 94 104 116 131 Table 18 1C LIST OF TABLES ARTICLE 2 Body and organ weights of rats 86 days after oral infection with 1000 eggs of Taenia taeniaefbrmis in Experiment 2 C C O C O I O O O O O O O O O C O O O O O 0 ARTICLE 3 Body and organ weights of Fischer 344 rats with gastric hyperplasia amongst parabiotic pairs with one partner infected with T. taeniaeformis. . . . . . . . . . . . . Serum gastrin levels in Spartan rats with gastroin- testinal hyperplasia 52 DAI with T. taeniaeformis . . . APPENDICES Body and organ weights of rats 86 days after oral infection with 1000 eggs of Taenia taeniaefbrmis. . . . Protective capacity of immune cells (IC) and immune serum (IRS) during the first 12 hours of primary infection with Taenia taeniaefbrmis . . . . . . . . . . Page 58 88 89 110 125 LIST OF FIGURES Figure Page ARTICLE 1 1 Thymic atrophy 75 DAI with T. taeniaeformis (above); thymuses from controls (below). . . . . . . . . . . . . . 33 2 A - Thymic atrOphy characterized by marked thymocyte depletion and narrowing of cortex due to cell lysis and phagocytosis. B - Thymus from control rat. . . . . . 33 3 Taenia taeniaeformis in the rat liver 4 DAI . . . . . . . 36 4 Taenia taeniaeformis 7 DAI. . . . . . . . . . . . . . . . 38 5 Taenia taeniaeformis 10 DAI . . . . . . . . . . . . . . . 38 6 Taenia taeniaefbrmis l6 DAI . . . . . . . . . . . . . . . 40 7 A high power view of the host capsule l6 DAI. . . . . . . 4O 8 Taenia taeniaefOrmis 22 DAI . . . . . . . . . . . . . . . 41 9 A higher power view of the cyst wall shown in Figure 8. . 41 10 Taenia taeniaefbrmis 42 DAI . . . . . . . . . . . . . . . 43 ll Taenia taeniaeformis 75 DAI . . . . . . . . . . . . . . . 43 12 Serum levels of enzymes alanine aminotransferase (above) and sorbitol dehydrogenase (below) following oral infection with 1000 eggs of T. taeniaeformis. . . . . . . 45 ARTICLE 2 1 Effect of T. taeniaefbrmis infection in Experiment 1 on mean stomach, small intestine, liver and net body weight 4.. — SE. C C O O C O C O O O O O O O O O O O O O O O O O I O 57 2 Macrosc0pic gastrointestinal changes in T. taeniaeformis infection of we rat. 0 O O O O O O O O O O O O O O O O O 61 vi Figure 3 Page A - Junction of forestomach and glandular stomach of control rat. B, C, D - Increasing degree of mucous cell hyperplasia of the glandular mucosa with replace- ment of parietal and zymogenic cells in infected rats . . 64 Papillary (A) and cystic (B) gastric mucous hyper— plasia with edema, increased cellularity and fibro- plasia within the lamina prOpria. . . . . . . . . . . . . 64 Hyperplasia of duodenal mucosa 75 DAI . . . . . . . . . . 66 Mast cell infiltration of lamina propria of jejunum 41 DAI in Group 1B O O O I O O O O O O O O O O O C O O I O 66 Mucosal mast cell counts in duodenum, jejunum and 118m t SE. 0 O O O O C I O O O O O O O O O O O O O O O O 67 Eosinophil counts in duodenal mucosa and peripheral blmd '1. SE C O O O O O O O O O O O O O O O O O O O O O O O 68 ARTICLE 3 Enlarged stomachs (S) in both infected and non- infected rats of a parabiotic pair. . . . . . . . . . . . 86 Marked mucosal hyperplasia of both stomachs from the parabiotic pair of rats in Figure l . . . . . . . . . . . 86 Gastric hyperplasia (left) despite antrectomy in 2 rats infected with T. taeniaeformis . . . . . . . . . . . 90 APPENDICES A - Diagram of rat stomach intact, depicting the loca- tion of forestomach (light stipple), fundus (plain) and antrum (dark stipple). The esophagus is oriented vertically, the duodenum horizontally and to the left. B - View of these areas from mucosal surface exposed by incision along the greater curvature. . . . . . . . . . . 98 A - Anterior surface of the stomach with the light- colored antrum (a) exposed. Duodenum, held by forceps, has been transected at the pyloric sphincter (single arrow). The second incision is being made from the greater curvature towards the esophagus (double arrows). B - Gastric transection has been completed. Antrum (a) has been isolated and cardiac fold of limiting ridge (arrow) is exposed. C - Antrum has been removed and remaining gastric section reduced in preparation for vii Figure 13 1C Page anastomosis with the duodenum. D - Stomach of control rat (lower right) with intact antrum (a), compared with stomachs of three antrectomdzed rats 8 weeks after surgery . . . . . . . . . . . . . . . . . . . . . . . . . 101 Weekly live body weight, and daily feed consumption and fecal output : SE of rats infected orally with 1000 eggs of Taenia taeniaefbrmis . . . . . . . . . . . . 108 Cellular transfer of immunity to T. taeniaeformis: diagrammatic summary of experimental procedure. . . . . . 123 viii LITERATURE REVIEW Cysticercosis and hydatidosis are cyclozoonotic infections caused by metacestodes of the genera Taenia and Echinococcus, members of the family Taeniidae, order Cyclophyllidea. Development of these larval cestodes within the tissues of man and domesticated food animals results in serious human disease, as well as economic losses due to condemnation of infected animal organs or carcasses, and embargoes on export trade of infected animals. Renewed interest in possible immunological control of this stage of the cestode life cycle has focused attention on the basic aspects of host-parasite interaction, and their relationship to immune responses mounted by the host. Of particular interest are the means whereby established metacestodes successfully evade both non-specific and specific host rejection systems in animals that are highly immune to challenge infection. Taenia taeniaeformis infection in the rat is a convenient experi- mental system for examining interaction of an established metacestode with its natural host. In this literature review the first 2 sections deal with the morphologic tissue, and protective immune responses of the host to taeniid metacestodes, and in particular T. taeniaeformis. The third section concerns gastrointestinal hormones and their possible involve- ment in host-parasite interaction. 2 Morphologic Response of the Host to Taeniid Metacestodes Smyth and Heath (1970) reviewed the histologic pathogenesis of taeniid metacestode infections of medical, veterinary and economic importance. Discussion will therefore be limited to a general review of the infection process, with brief reference to distinctive features of specific infections, and a more detailed account of the histopatho- logic response of the rat to infection by T. taeniaeformis. General The taeniid egg is composed of an outer layer of keratinized embryophoral blocks which surround a series of membranes which envelop the hexacanth (6-hooked) embryo. The intermediate host becomes infected by ingesting eggs passed in the feces of the defini— tive host infected with the adult tapeworm. During passage of the egg through the stomach and small intestine of the intermediate host there is swelling of the cement-like substance binding the embryophoral blocks which are then dispersed from the surface of the parasite. In the small intestine the embryo is activated and initiates intense movements, tearing the oncospheral membrane and moving to the villi of the small intestine (Silverman and Maneely, 1955; Banerjee and Singh, 1969a,b). Penetration of the villi occurs within 30 minutes (Banerjee and Singh, 1969a) and involves enzymic lysis of host tissue and physical disruption by the hooks (Silverman and Maneely, 1955; Banerjee and Singh, l969a,b; Heath, 1971). The embryo migrates within the mucosa until it reaches a venule of adequate size, after which it is carried passively in the blood stream to a site of predilection which varies with the species involved (Heath, 1971). In the cases of infection by Taenia pisiformis in rabbits (WOrley, 1974; Flatt and Moses, 1975) and Taenia hydatigena in sheep and goats (Sweatman and Plummer, 1957), a period of extensive migra- tion by the parasite in the liver precedes final localization in the peritoneal cavity. The histopathologic responses to larvae have been described to some extent for most taeniid parasites, especially for Taenia taeniaefbrmis (Bullock and Curtis, 1924; Orihara, 1962; Singh and Rao, 1967), Taenia saginata (Silverman and Hulland, 1961), Taenia ovis (Sweatman and Henshall, 1962), Taenia solium (Viljoen, 1937) and Echinococcus multilocularis (Rausch, 1954; Ali-Khan, 1978a,b). Regardless of the parasite involved, its survival at the predilec- tion site in the intermediate host is usually associated with a decline in cellular reaction within the adjacent host tissue which undergoes fibrosis to form a connective tissue capsule. Degeneration of the parasite, as is usual for T. saginata and T. ovis (Smyth and Heath, 1970) and is associated with onset of clinical signs in brain infections with T. solium in man (MacArthur, 1934; Menon and Veliath, 1940) provokes a marked inflammatory re5ponse with necrosis, caseation and calcification of the lesion (Silverman and Hulland, 1961). Taenia taeniaefbrmis The earliest larval stage of T. taeniaeformis observed in the liver by Bullock and Curtis (1924) was a multicellular organism 15 um in diameter and seen 21 hours after infection. Well-defined larvae of similar size were observed by Lewert and Lee (1955) and Banerjee and Singh (1969c) in hepatic sinusoids 24 hours after infection, with the former group identifying embryonic hooks on the larvae. Pathologic changes were not seen up to 72 hours after infection (Lewert and Lee, 4 1955; Banerjee and Singh, 1969c) and at 4 days after infection (DAI) when the larvae measured 35-55 pm in diameter there was only mild degeneration of hepatic cells (Banerjee and Singh, 1969c). Bullock and Curtis (1924) characterized the reaction around the parasite during the first week as exudative with associated degenera— tion or necrosis of hepatic tissue near the parasite. At 5 DAI they described hyaline degeneration of individual hepatic cells around a "coarse fibrinous network" which surrounded the parasite and was partially condensed on its surface. This amorphous zone has been considered to consist of a microvillar layer, and was shown to decrease progressively in size during the first 7 DAI. This layer has been credited with protecting the larvae from lymphocyte invasion during early development (Heath and Pavloff, 1975). The larvae grew rapidly to reach 500 pm in diameter by 7 DAI (Hutchison, 1958), by 10 DAI (Banerjee and Singh, 1969c) or by 13 DAI (Lewert and Lee, 1955). Hepatic necrosis,which increased until the commencement of cell proliferation around the parasite 9 DAI, was either localized or involved all hepatic tissue adjacent to the parasite. Some larvae simply compressed adjacent hepatic cells and had no associated necrotic zone (Bullock and Curtis, 1924). A distinctive proliferative cellular reaction developed around the parasite from 9 DAI, reaching a peak 15 to 20 DAI, after which the active process subsided. During the active phase there was intense sarcomatous-like proliferation of fibroblastic cells which had hyperchromatic nuclei and numerous mitotic figures, and this host capsular zone was infiltrated by eosin0phils, lymphocytes and plasma cells (Bullock and Curtis, 1924). Byram (1974), using light and 5 electron microsc0py, observed that the host capsule was composed of connective tissue continuous with Glisson's capsule, fibroblasts, lymphocytes, plasma cells, eosinophils, mast cells, neutrophils, mononuclear phagocytes and vascular elements. He suggested, on morphologic grounds, that the highly modified cells lining the luminal boundary appeared to be derived from mononuclear phagocytes. He recognized a loose inner connective tissue layer, an inflammatory cell layer and a dense, highly vascularized outer connective tissue layer. There were variations in cellular composition and extent of these layers within and between capsules. Mast cell infiltration of the middle cellular zone of the host capsule has been documented (Coleman and De Salva, 1963; Varute, 1971). The hepatic cells surrounding the parasite were depleted of glycogen until after formation of the host capsule (Lewert and Lee, 1955). IntracytOplasmic hepatic inclusions containing lipofuscin were observed within a narrow zone of hepatocytes 200 to 400 um from the surface of T. taeniaeformis in 22/30 infected rats but not in hepatocytes of non-infected animals (Varute and More, 1971). Increases in numbers of myeloid cells were observed in the spleen of rats, mice and hamsters infected with T. taeniaeformis (Hoeppli and Feng, 1933). Bullock and Curtis (1930) mentioned "hypertrOphic gastritis" as a common finding in rats heavily infected with T. taeniaeformis, although the time of development and extent of the change were not specified. Blumberg and Gardner (1940) briefly described the change in 8 rats infected for 235 to 303 days with 61 to 200 strobilocerci. Stomachs were enlarged "up to more than twice normal size." 6 Bullock and Curtis (1924) first reported the development of sarcomas within the host capsule of T. taeniaefbrmis during chronic infections. They described 3 histologic forms of the sarcomas as polymorphous cell, Spindle cell and mixed cell types (Bullock and Curtis, 1925). Rats given a single intraperitoneal injection of washed ground T. taeniaefbrmis larvae deve10ped multiple peritoneal sarcomas within 23 to 410 days (Dunning and Curtis, 1946). Immupitypto TaeniidLMetacestodes Immunity to cestodes has been extensively reviewed by Weinmann (1966, 1970) and Gemmell and MacNamara (1972). The following section deals mainly with T. taeniaefbrmis and recent findings regarding protective immune responses. Humoral Immun i121 Resistance to superinfection has been demonstrated for the larval metacestodes of T. taeniaeformis in rats (Miller, 1931), Hymenolepis nana in mice (Hunninen, 1935), T. pisiformis in rabbits (Kerr, 1935), T. saginata in cattle (Penfold et a1., 1936), and T. hydatigena in sheep (Sweatman, 1957). The early work of Miller and Gardiner (1932) and Campbell (1938) established conclusively the participation of antibody in protection against T. taeniaefbrmis. Immunity to this larval metacestode was first demonstrated by the successful passive immunization of rats with serum collected 28 days after experimental infection (Miller and Gardiner, 1932; Campbell, 1938). Similar studies demonstrated passive immunization against T. pisifbrmis in rabbits (Kerr, 1935) and Hymenolepis nana in mice (Hearin, 1941). Blundell-Hasell et al. 7 (1968) transferred immunity to T. hydatigena with serum from arti- ficially immunized lambs. Leid and Williams (1974a) extended these findings by demonstrat— ing that in the rat, antibodies of a single immunoglobulin class (7572a) were primarily responsible for passive transfer of resistance to T. taeniaeformis. In the mouse also, antibodies of one immuno- globulin class (7Syl) were shown to mediate resistance (Musoke and Williams, 1975a). Rat 7SY2a and mouse 7SY1 normally share the analogous function as short term skin sensitizing antibodies. How- ever, in neither species did antibodies of these classes exhibit short term skin sensitizing activity against T. taeniaeformis (Leid and Williams, 1974a; Musoke and Williams, 1975a). The protective effect of immune serum was abolished by decomple- mentation of recipients using cobra venom factor in the rat (Musoke and Williams, 1975b) and in the mouse (Mitchell et al., 1977). This observation suggested that complement-fixing antibodies were involved in resistance to establishment of larvae. Later during infection in the rat, protective antibodies of class 7Sy1, but not of class yM, were found (Musoke and Williams, 1975b). Serum from rats immunized by intraperitoneal implantation of mature cysticerci had protective antibody in the 7Sy1 and yM fractions. This finding indicated that mode of antigen presentation as well as stage of larval development affected the type of the antibody response (Musoke and Williams, 1976). Anticomplementary activities of larval metacestodes have been demonstrated in the case of T. taeniaefbrmis (Musoke and Williams, .1975b; Hammerberg et al., 1976) and Echinococcus granulosus (Herd, 1976; Kassis and Tanner, 1976; Hammerberg et al., 1977). 8 Anticomplementary substances produced by mature cysticerci of T. taeniaeformis (Hammerberg and Williams, 1978a) have recently been characterized chemically (Hammerberg and Williams, 1978b). Activity of these substances in vivo may in part explain the insusceptibility of established metacestodes to the lethal effects of protective, complement-dependent antibody. IE. Reagins constitute one of the most striking antibody responses to parasitic infections (Ogilvie, 1964; Leid and Williams, 1974b, 1975). These antibodies were first described for animals by Ogilvie (1964), who used passive cutaneous anaphylaxis (PCA) tests to demon- strate reagins in rats, monkeys and sheep infected with Nippostrongylus brasiliensis, Schistosoma mansoni and Trichostrongylus colubrifbrmis, respectively. Reaginic antibody in rats was defined by Stechschulte, Orange and Austen (1970) to be associated with rat immunoglobulin yE, similar to the immunoglobulin class designated yE in man (Ishizaka and Ishizaka, 1967). yE has the capacity to persist at a skin site in the rat for weeks, as assessed by PCA, and the ability to prepare mast cells passively in vitro for antigen-involved release of histamine and serotonin (Becker and Austen, 1966). yE antibody was a prominent feature of T. taeniaefbrmis infection in the rat (Leid and Williams, 1974b) and of T. pisifbrmis in the rabbit (Leid and Williams, 1975). Reaginic antibody and an allergen associated with T. taeniaeformis were characterized by Leid and Williams (1974b). The functional importance of yE antibody in parasitic infections has not been established. Leid and Williams (1974a) postulated a 9 collaborative effect between 7SY2a and YB antibodies in the destruc- tion of invasive embryos of T. taeniaefbrmis in the rat. Musoke et a1. (1978) demonstrated an increased rate of killing of T. taeniaefbrmis in rats passively immunized with reagin-rich immune serum. yE anti- body to T. taeniaefbrmis might sensitize mast cells in the intestinal mucosa for antigen-induced release of inflammatory mediators. These could alter local vascular permeability and enhance the influx of circulating protective antibodies, as has been suggested by Murrell et a1. (1975) for S. mansoni, or directly affect the survival of the parasite at the site of invasion, as has been demonstrated in T. colubrifbrmis infection in guinea pigs (Rothwell et al., 1974). The feasibility of the second mechanism is supported by the finding that the infection rate following infusion of activated oncospheres of T. taeniaefbrmis into an occluded loop of small intestine is reduced by simultaneous infusion of histamine cu: slow-releasing substance of anaphylaxis (Musoke et al., 1978). Leid, Williams and Austen (1975) have demonstrated antigen— induced release of histamine in vitro by peritoneal cells and lung fragments from rats infected with T. taeniaeformis. Indirect evidence of possible involvement of reaginic antibody in immune rejection of N. brasiliensis in the rat was a marked increase in mast cell and globule leukocyte numbers in the small intestine (Jarrett et al., 1968; Miller and Jarrett, 1971). Kelly and Ogilvie (1972) noted that the intestinal mastocytosis occurred from 20 days after infection and followed immune rejection. They concluded that mast cells could only be implicated in worm rejection be postulating a 3-step mechanism involving antibody, lymphocyte and, finally, amine action. 10 Increase in basophils in the bone marrow and blood, and of baso- phils and mast cells in the intestinal mucosa, have been observed during T. colubriformis infection in the guinea pig (Rothwell and Dineen, 1972; Rothwell, 1975). Degranulation of eosinophils and baso- phils at the site of infection by this nematode was observed by Rothwell, Dineen and Love (1971), who proposed participation of pharmacologically active amines produced by these cells in rejection of the parasite. Cell Mediated Immunity Cell mediated immune (CMI) reSponses have been shown to occur in natural infections with taeniid larvae (Kagan et al., 1966), and Rickard and Outteridge (1974) demonstrated CMI responses in rabbits experimentally infected with or vaccinated against T. pisifbrmis using skin tests and in vitro blast transformation of lymphocytes. Rickard (1974) proposed that the established parasite evades CMI responses by becoming coated with blocking antibody. Kwa and Liew (1977) elicited delayed type hypersensitivity skin responses in rats vaccinated with both excretory-secretory and somatic antigens. This response was transferred to recipient rats of the same inbred strain by peritoneal cells from vaccinated rats. In vitro and in Vivo measurements of CMI responses may not correlate with cell mediated protection. This appeared to be the case for T. pisifbrmis infection, where CMI pro- ducing antigens did not seem to be the same as those that induced protection in the rabbit (Rickard and Katiyar, 1976). There is no conclusive evidence of direct cellular involvement in protective immunity to metacestodes. Nemeth (1970) transferred only partial immunity to T. pisiformis infection in rabbits with ll allogeneic lymphoid cells, and Blundell et a1. (1969) were not success- ful in transferring immunity to T. hydatigena or T. ovis in sheep with allogeneic cells from infected animals. Mitchell et al. (1977) were unable to protect athymic ("nude") mice from infection with T. taeniae- fOrmis by transfer of a p0pu1ation of purified T cells from infected mice, but demonstrated T cell-dependence of protective antibody production. Gastrointestinal Hormones and Parasitic Disease General In the gastrointestinal tract 2 main groups or families of hormonal peptides can be distinguished on the basis of similarities in amino acid sequence (Dockray, 1977). Gastrin and cholecystokinin (CCK) are related, as are secretin, glucagon, vasoactive intestinal peptide (VIP) and gastric inhibitory polypeptide (GIP) (Rayford et al., 1976a). Other polypeptides recently identified and which may also be involved with initiation and modulation of the normal digestive process include bombesin, motilin, chymodenin, bulbogastrone, entero-oxyntin and pancreatic polypeptide (Rayford et al., 1976b). While each gastrointestinal hormone has a wide spectrum of biological actions, a physiological role has been established for only gastrin, CCK, secretin and GIP (Grossman, 1977). Secretin and gastrin were discovered at the beginning of the century, but their biochemical isolation, purification, structural analysis and synthesis did not occur until the 1960's. Since then the availability of pure gastrointestinal hormones has greatly facili- tated study of their dramatic effects on secretion and motility of the digestive tract (Debas, 1977) and research in this area has greatly 12 expanded. It has permitted studies of mechanisms of action, showing that like other peptide hormones the gastrointestinal hormones alter cyclic nucleotide metabolism (Thompson et al., 1977). Two new categories of action have been described which make it clear that gastrointestinal hormones as a group are not simply digestive hormones, but that they play an important integrated role in the endocrine regulation of growth and metabolism (Johnson, 1977). Many gastrointestinal hormones affect the release of other hormones, and gastrin and CCK have potent trOphic effects, considered to be physiological, on the gastrointestinal mucosa and exocrine pancreas (Johnson, 1976; Enochs and Johnson, 1977). Gastrin will be considered in further detail, as it is the gastrointestinal hormone most studied, and has been implicated in pathogenic processes in man (Walsh and Grossman, l975a,b) and animals (Titchen and Anderson, 1977). Gastrin Gregory and Tracy (1964) extracted pure gastrin from the mucosa of 600 pig antrums, after which it was chemically identified and synthesized. This put an end to doubts about the claim by Edkins (1905) that a specific stimulant of acid secretion that he called gastrin could be extracted from antral mucosa, and about whether applications of stimulants to antral mucosa elicited acid production. The compounds isolated from pig antral mucosa were heptadecapeptides named gastrin I and II, which were identical except for the presence of sulphate on the tyrosine of gastrin II. Gastrin heptadecapeptides purified from antral mucosa of man, pig, dog, cat, sheep and cow differed by only one or two amino acid substitutions in the middle 13 of the linear peptide chain (Kenner and Sheppard, 1973). Sensitive radioimmunoassays for gastrin developed in the late 1960's have been used extensively to study the molecular forms of gastrin in plasma and tissues. The chemistry and physiology of gastrin have been reviewed by Walsh and Grossman (l975a). It appears that, like other peptide hormones, gastrin is synthesized as a larger polypeptide, which undergoes trypsin-like cleavage to form a biologically active carboxy- terminal fragment, big gastrin (G34) containing 34 amino acids, little gastrin (617), the heptadecapeptide, or mihigastrin (G13), the tri- decapeptide. The carboxy-terminal portion of the gastrin molecule has all the biological activity of the whole molecule (Tracy and Gregory, 1964). The G cells that contain gastrin are found in the pyloric glands of the antrum and in the proximal duodenum. Conversion of G34 to G17 is assumed to occur in the G cell as most of the stored hormone is in the G17 form. Stimulants of gastrin release include peptides and amino acids within the gut lumen, gastric distention, vagal stimulation and, on occasions, elevated levels of blood calcium and epinephrine. Release of gastrin is modulated by simple negative feedback in which gastric acid secreted from parietal cells in response to gastrin bathes the antral mucosa and inhibits further release of gastrin from G cells. All stimulants for gastrin release are inhibited by acid. The hormones secretin, GIP, VIP, glucagon and calcitonin are also inhibitory (Walsh and Grossman, l975a). The only physiological functions of gastrins are stimulation of gastric acid secretion by parietal cells and antral contraction, and 14 trophic effects on the gastrointestinal tract and pancreas (Grossman, 1977). Although gastrin has been shown to modify every major secre- tory, absorptive and smooth muscle activity of the digestive tract, these additional effects are considered to be pharmacological (Grossman, 1977). Gastrin exerts a dominant control of acid secretion by gastric parietal cells, which it is proposed have 3 major stimulatory receptors: for gastrin, acetylcholine and histamine (Grossman, 1975). Blockade of one type of receptor, as is possible for acetylcholine with atropine and histamine with the H -histamine receptor blocking agents (Black 2 et al., 1972), reduces or abolishes the response to the other stimu- lants. While this model opposes the hypothesis that histamine is the final common mediator at the parietal cell (Code, 1965), it does include a role for histamine in regulation of acid secretion (Code, 1977; Debas, 1977). Trophic Action of Gastrointestinal Hormones The trophic action of gastrointestinal hormones has been compre- hensively reviewed by Johnson (1976) and Enochs and Johnson (1977). Gastrin stimulates RNA, DNA and protein synthesis in the pancreas and mucosa along the entire length of the gut, with the exception of the esophagus and antrum. CCK exerts slight trOphic effect on the duodenum but mainly affects the pancreas. Secretin inhibits trOphic responses to gastrin. Clinical observations in man suggested that gastrin exerted trOphic action. Moderate to complete atrOphy of the gastric mucosa remaining after antrectomy (Lees and Grandjean, 1968; Gjurldsen et al., 1968) was attributed to lack of gastrin, as the decrease in acid 15 production following antrectomy in man could be partially prevented by infusing pentagastrin continuously during the first week after antrectomy (Olbe, 1974). Conversely, gastric mucosal hyperplasia characterized by an increased parietal cell count occurs in patients with hypergastrinemia due to the Zollinger-Ellison syndrome (Gregory et al., 1967; Neuburger et al., 1972). Johnson et al. (1969), working with the rat, concluded that gastrin stimulated protein synthesis in vitro. They found that this effect was specific for certain tissues of the digestive tract and was independent of secretory phenomena. They hypothesized that gastrin was a trophic hormone and regulated the growth of gastrointestinal tract mucosa. Injection of pharmacological amounts of pentagastrin in vivo produced marked parietal hyperplasia in rats (Crean et al., 1969) and pancreatic hypertrOphy and hyperplasia (Mayston and Barrowman, 1973). Martin et a1. (1970) hypothesized that removal of endogenous gastrin was responsible for the atrOphy of the fundic mucosa after antrectomy in the rat. Johnson and Chandler (1973) almost completely prevented the 40% decreases in gastric and duodenal mucosa RNA and DNA during the month following antrectomy in the rat by a series of pentagastrin injections. These experiments were strong evidence that endogenous gastrin has a physiological role as a trOphic hormone. Further support for a trophic role for gastrin came from obser- vations of the growth of the gastrointestinal tract after natural alterations of serum and antral gastrin levels. In the rat, relative intestinal weight and nucleic content increased during the third week of life, while antral gastrin levels rose from low levels during days 1 to 18 to adult levels on day 21 16 (Lichtenberger and Johnson, 1974). By delaying weaning, these changes were prevented, while injection of pentagastrin into rats prevented from weaning increased gut, RNA and protein to body weight ratios compared to those of weaned control animals. Lichtenberger and Johnson (1974) concluded that some aspects of gut development are dependent on weaning and that the mediator between these 2 events may be gastrin. Starvation in the rat was associated with a dramatic increase in weights of pancreas, fundic stomach and small intestine, in prOportion to body weight, a marked decrease in mucosal RNA, DNA and protein after 3 to 6 days of starvation (Lichtenberger et al., 1976b) and a decrease in antral serum gastrin levels (Lichtenberger et al., 1975). Similar effects were produced in parenterally fed rats (Johnson et al., 1975a) but were prevented in parenterally fed rats that were given infusions of pentagastrin at physiological levels (Johnson et al., 1974, l975b). These results were interpreted to indicate that the oral ingestion of food and its presence in the gastrointestinal tract are necessary to maintain endogenous gastrin levels, and that the trophic effect of endogenous gastrin is essential for the day to day maintenance of the structural and functional integrity of the gut. Gastrointestinal hyperplasia in the lactating rat was associated with hyperphagia (Campbell and Fell, 1964), but it has not been determined whether or not the hypergastrinemia observed by Lichtenberger et al. (1976a) in lactating rats was a mediator. Gastrin and Disease Since radioimmunoassay of gastrin became available, disease con- ditions that result in hypergastrinemia have now been recognized in 17 man (Walsh and Grossman, 1975b). One category includes those gastric diseases such as pernicious anemia and gastric carcinoma where atrophic gastritis develops. If there is insufficient acid secretion to reduce the pH of antral contents below pH 3.0, there will be no acid inhibi- tion of gastrin secretion by antral G cells. Hypergastrinemia therefore results from failure of the negative feedback 100p as the gastric mucosa is unable to acidify gastric contents in response to hypergastrinemia (Titchen and Anderson, 1977). The other category of conditions includes gastrinoma (Gregory et al., 1967) and some cases of renal disease with failure of gastrin excretion, and plasma gastrin levels are independent of antral control. There is persistent gastric hypersecretion in response to hyper- gastrinemia and gastric mucosal disease usually results. The Zollinger-Ellison syndrome (Neuburger et al., 1972) with pyloric ulceration is an example of such an effect associated with a gastrinoma, located usually in the pancreas. Gastrin and Parasitism In view of the wide range of both pharmacologic and physiologic effects of the gastrointestinal hormones, and in particular gastrin, it was logical to suspect that some of the effects of gastroenteric parasitism were mediated by this system. McLeay et a1. (1973) described increased secretory activity in the surgically prepared and parasite-free abomasal fundic pouch of sheep from 4 days after infection of the abomasum with 150,000 or 100,000 larvae of Ostertagia circumcincta. Secretory activity in the infected abomasum was decreased. Ultrastructural examination of parietal cells in the pouch and abomasum indicated increased and 18 decreased secretory activity, re5pectively. They postulated that the pouch changes might be due to hypergastrinemia. Hypersecretion also occurred within the abomasal pouch when sheep were infected with 20,000 larvae 3 times weekly for 6 weeks (Anderson et al., 1974, 1976a). Hypergastrinemia was confirmed in 3 of these sheep which exhibited a lO-fold increase in serum gastrin to a peak volume 20-35 days after the first dose of larvae (Anderson et al., 1976b). Gastrin levels returned towards the pre-infection levels after treatment with an anthel- mintic but rose again after reinfection. The hypergastrinemia was of antral origin, as it did not develop in antrectomized sheep (Anderson et al., 1976b). As the hypergastrinemia and the hypersecretion it produced in abomasal pouches occurred while the pH of abomasal contents was still within the normal range, the increase in gastrin levels was attributed to the parasite and not to the increase in pH of abomasal contents which develOped later (Anderson et al., 1976a). Castro et a1. (1976) observed an increase in mean serum gastrin level from 80 to 115 fmole/ml in rats during infection with Trichinella spiralis, which induced inflammatory changes in the small intestine. 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In The Biology of Parasites. (Ed. by E. J. L. Soulsby). Academic Press, New York: 301-320. Weinmann, C. J. 1970. Cestodes and acanthocephala. In Immunity of Parasitic Animals. (Ed. by C. J. Jackson, R. Herman and I. Singer). Vol. 2. Appleton, New York: 1021-1059. Worley, D. E. 1974. Quantitative studies on the migration and development of Taenia pisifbrmis larvae in laboratory rabbits. ARTICLE 1 PATHOLOGY OF TAENIA TAENIAEFORMIS INFECTION IN THE RAT R. W. Cook and A. L. Trapp 29 30 Summary During a sequential clinicopathologic study of Taenia taeniae- formis infection in the rat, several changes were observed which have not been described previously. A minimal amount of hepatic migration was indicated by microsc0pic tracks of necrosis adjacent to the parasite 4 to 10 days after infection (DAI). Serum levels of alanine aminotransferase and sorbitol dehydrogenase rose sharply 6 to 7 DAI when necrotic tracks were most obvious and before a distinctive fibroblastic response developed around the parasite at about 10 DAI. Enzyme levels peaked again 13 to 14 DAI. There was enlargement of hepatic lymph nodes with marked medullary plasmacytosis, which was also observed within mediastinal and mesenteric nodes. Splenomegaly was associated with expansion of the red pulp with myelopoiesis and plasmacytosis. Thymic atrophy was observed microscopically from 44 DAI and grossly from 52 DAI. Introduction A number of histopathologic studies have been made of the develop- ment of Taenia taeniaeformis in the rat liver (Bullock and Curtis, 1924; Banerjee and Singh, 1969; Singh and Rao, 1967; Lewert and Lee, 1955). We have reexamined the sequential histOpathologic reaction to this parasite because of our interest in the immunological response of the rat to the establishment and growth of T. taeniaefOrmis (Cook and Trapp, 1979; Leid and Williams, 1974: Musoke and Williams, 1975). We report here on the morphologic and clinicopathologic evidence of an early migratory phase, and on lymph node and thymic changes which occur during the course of infection. 31 Materials and Methods Ninety-six 28-day-old female Spartan (Spb [SD] BR) rats were infected orally with 1000 or 2000 eggs of Taenia taeniaeformis. After a 20 hour fast, groups of 3-4 rats were killed 4, 7, 10, 13, 16, 19, 22, 25, 28, 32, 34, 38, 61, 67 and 75 days after infection (DAI), while a total of 39 rats were killed 33, 41 or 52 DAI. Sixty- seven control rats of the same age, sex and strain were killed in series, usually on alternate days when infected rats were killed. A range of tissue samples from all rats were fixed in 10% buffered formalin, dehydrated, embedded in paraffin and cut into 6 pm sections for staining with hematoxylin and eosin and Giemsa stains. In a second experiment serum levels of the enzymes alanine aminotransferase and sorbitol dehydrogenase were measured during infection. One milliliter of blood was collected from the infra— orbital sinus in 32 female rats, 28 days old, the day before 24 were infected orally with 1000 eggs of T. taeniaeformis. The infected rats were divided equally into 3 groups, A, B and C, and the 8 unin- fected controls formed Group D. Blood samples were taken from Group A, l, 4, 7, 10 and 13 DAI, from Group B, 2, 5, 8, 11 and 14 DAI and from Groups C and D, 3, 6, 9, 12 and 15 DAI. Blood was also taken from 6 additional infected rats (Group E) 28 and 75 DAI with 1000 eggs of T. taeniaefbrmis. At the 28 day sampling time (Group E), 2 age-matched non-infected rats (Group F) were also sampled. The blood was allowed to clot and serum was collected and stored at -30 C until enzyme estimations were made using an automatic kinetic enzyme analyzer.a The results for infected and control groups were compared statistically using the Student's t-test. aGemsaec Analyzer, Electro-Nucleonics, Fairfield, NJ 00706. 32 Results Gross findings The effect of T. taeniaeformis infection on body weight and weights of stomach, intestine and liver have been reported, together with a description of pathologic changes in the gastrointestinal tract (Cook and Trapp, 1979). In the liver there was progressive growth of T. taeniaefbrmis larvae, which were first detected macroscoPically 7 DAI. By 75 DAI each larva had deve10ped into a strobilocercus within a cyst up to 5 mm in diameter. Enlargement of hepatic lymph nodes and spleno- megaly were marked by 22 DAI. Spleens weighed 500 to 700 mg in control animals and were enlarged to between 1 and 2 grams in infected rats. Thymic atrophy (Figure l) was first observed macroscopically 52 DAI. Microsc0pic findings Changes were observed in the gastrointestinal tract (Cook and Trapp: 1979), thymus, spleen, lymph nodes and liver. Thymus. The thymus of infected rats contained microscopic evi- dence of atrophy from 44 DAI. There was multifocal lysis of cortical thymocytes with accumulation of nuclear debris within macrophages. By 52 DAI there was narrowing and severe thymocyte depletion of the cortex (Figure 2). Spleen. Increased numbers of eosinophils and megakaryocytes were observed in the red pulp of the Spleen from 10 DAI. Between 13 and 22 DAI there was marked expansion of the red pulp where dense clusters of lymphoplasmacytoid cells, eosinOphilic myeloid cells and 33 Figure l. Thymic atrophy 75 DAI with T. taeniaeformis (above); thymuses from contrOls (below). Figure 2. A — Thymic atrophy characterized by marked thymocyte depletion and narrowing of cortex due to cell lysis and phagocytosis. B - Thymus from control rat. H & E stain; x45. 34 E as;- PMWMHU J+llulllliTLlllplil+lllgfnl Figure 1 Figure 2 35 megakaryocytes formed extensive confluent cell cords. These changes were maintained, in association with an increase in plasma cells in the marginal zone of the white pulp until 61 DAI, when cellularity of the red pulp was decreased. Lymph nodes. The following changes observed in hepatic lymph nodes deve10ped to a lesser degree in mediastinal nodes adjacent to the thymus and were least obvious in the mesenteric nodes. At 10 DAI eosinophils were scattered in small numbers through hepatic nodes. Plasmacytosis within the medulla was first observed 13 DAI, after which the nodes rapidly increased in size and medullary cords packed with plasma cells and eosinophils became prominent and tortuous. Medullary sinuses were distended by histiocytes, eosinophils and fluid, and the paracortical zone appeared expanded but depleted of cells. Cortical follicles were enlarged and active. By 28 DAI plasma cells extended into the paracortical area and in some cases into the outer cortex, leaving a narrow rim of lymphocytes and follicles. At 32 to 34 DAI large numbers of Russell bodies containing plasma cells 'were observed. Thereafter the latter were less numerous but the level of plasmacytosis was maintained until 61 DAI, when medullary cords appeared contracted and separated by wide medullary sinuses and plasma cell density was decreased. Lizgr, Larvae observed in liver sections 4 DAI were 60 to 100 um in diameter (Figure 3). All were surrounded by a clear zone, and beside many there was a small focus of hepatic cell necrosis (Figure 3A,B,C). Some portal triads were infiltrated by eosinOphils (Figure 3D). but the area surrounding the parasite was free of inflammatory cells at this stage. 36 Figure 3. Taenia taeniaeformis in the rat liver 4 DAI. A clear zone surrounds the parasite. A, B, C - The focus of paren— chymal degeneration (arrows) adjacent to the parasite indicates migration. D - Inflammatory cells, including eosinophils, have infiltrated the portal triad but not the area around the parasite. H & E stain; X300. 37 By 7 DAI the thin-walled parasite was larger, with a distinct zone of hepatic necrosis on one side, evidence of limited migration through the liver (Figure 4). Inflammatory cells, including large numbers of eosinophils, had invaded this necrotic zone. There was no reaction around the parasite itself, which was now in close contact with hepatic tissue. In the portal areas a moderate lymphoplasma- cytoid response was accompanied by marked eosinophil infiltration. At 10 DAI the parasite was surrounded by a narrow fibroblastic zone as first evidence of a host capsule, and the adjacent migratory track was undergoing fibrosis (Figure 5). Rapid expansion of the highly vascular host capsule continued until 22 DAI. There was intense proliferation of fibroblastic cells with vesicular, hyper- chromatic nuclei of variable shapes, many mitotic figures and indistinct cell boundaries (Figures 6 and 7). The densely cellular host capsule was up to 1 mm in width and clearly outlined the parasite to the naked eye during this period. The capsule was infiltrated by lymphocytes, eosinophils and mast cells by 16 to 22 DAI and contained prominent aggregates of plasma cells 22 DAI (Figures 8 and 9). By this stage there was often a prominent zone of eosinophils at the surface of the parasite, most frequently adjacent to the area of scolex formation (Figure 8). Portal triads contained large numbers of plasma cells and eosinophils from 10 DAI. Although the parasite continued to grow, the thickness of its host capsule did not increase from 22 DAI. There was progressive fibrosis of the capsule from its periphery as seen 42 DAI (Figure 10). By 75 DAI it consisted of mature fibrous connective tissue, frequently with a single row of hepatocytes lying beneath its outer edge (Figure 11). The inner zone adjacent to the parasite comprised a single 38 Figure 4. Taenia taeniaeformis 7 DAI. Inflammatory response is limited to the periphery of the necrotic migra- tory track to the left of the parasite. H & E stain; X54. Figure 5. Taenia taeniaefbrmis 10 DAI. Fibroblastic proliferation adjacent to the parasite and fibrosis of the migratory tract (arrows). H & E stain; x54. 39 Figure 6. Taenia taeniaefOrmis l6 DAI. The zone of fibro- blastic reaction around the parasite has widened to form a host capsule. H & E stain; X54. Figure 7. A high power view of the host capsule l6 DAI. The fibroblastic reaction appears sarcomatous with many mitotic figures (arrows) and hyperchromatism of nuclei. H & E stain; x450. 40 ..'\ I 9: r i‘;§§“fl 3 Figure 6 Figure 7 41 Figure 8. Taenia taeniaeformis 22 DAI. There is prominent accumulation of plasma cells at the periphery of the host capsule and eosinophil infiltration at the surface of the parasite near the invaginated scolex. H 5 E stain; X54. Figure 9. A higher pOWer view of the cyst wall shown in Figure 8. Peripheral zone of plasma cells (arrows). H & E stain; X135. 42 Figure 10. Taenia taeniaeformis 42 DAI. There is moderate cellularity and increased fibrosis of the host capsule. H & E stain; X54. Figure 11. Taenia taeniaefbrmis 75 DAI. A - Marked fibrosis of the host capsule. H & E stain; x54. B - A higher power view of the host capsule shown in A, illustrating the layer of polygonal cells adjacent to the parasite and the single cord of hepatocytes near the periphery. H & E stain; X135. 43 Figure 10 Figure 11 44 layer of epithelioid cells which resembled the polygonal, histiocytic- like cells with abundant cytOplasm scattered through the fibrous capsule. Occasional foci of plasma cells and smaller numbers of eosinophils were also observed in the capsule. Cellular infiltra- tion of the portal triads had resolved by this stage and in many cases there was residual portal fibrosis. Enzymology The results of serum enzyme estimations are shown in Figure 12. A similar pattern of changes occurred for both enzymes during the course of infection. Control levels of alanine aminotransferase fluctuated more than those of sorbitol dehydrogenase, and increases during infection were more marked for the latter. Levels of sorbitol dehydrogenase in infected rats were significantly greater than in control animals 6, 7, 8, ll, 12, 13, 14 and 15 DAI (p<0.05), while alanine aminotransferase was significantly increased 6, 7, 10 and 13 DAI (p<0.05). Peaks in enzyme levels occurred 6 to 7 DAI and 13 to Discussion Hepatic tissue changes up to 10 DAI were of interest as the developing parasite becomes progressively more resistant to the lethal effects of passively transferred immune serum during this period (Musoke and Williams, 1975). This may be due to changes in structure of the parasite or factors it produces. There was distinct morpho- logic evidence of change in host response to the parasite during this period. During the early stages of infection when the parasite was Susceptible to immune serum, it did not provoke a local tissue reaction despite its rapid growth and limited migration. The clear zone Alanine Sorbitol 4S ‘1’ (0 5:5: <9 35353 ‘- :;:;: .93. (I) 33333 c < g 3 Ii 0 éfi E E :fi < g 15.4 I (U 5 _l C) e 3 . 13 ._ >~ .: Q) (3 5 “ I O 5 10 15 28 75 Days after infection Figure 12. Serum levels of enzymes alanine aminotransferase (above) and sorbitol dehydrogenase (below) following oral infection with 1000 eggs of T. taeniaeformis. Group A - black; Group B - vertical lines; Group C - light stipple; Group D (controls) - plain; Group E - dark stipple; Group F (controls) - diagonal lines. 46 surrounding the parasite 4 DAI was probably due to the microvillar system on the surface of the parasite (Picone, 1978). The interpretation that the small area of necrosis adjacent to the organism 4 DAI represented the site from which the parasite had migrated was supported by the presence of a more advanced lesion 7 DAI. At this time the elongated necrotic zone was clearly a para— sitic tract, although there was no evidence of degeneration or necrosis of hepatocytes adjacent to the advancing surface of the organism. Variation in the extent to which the necrotic zone sur- rounded the parasite in tissue sections was determined by the plane in which the organism was out. Although Singh and Rao (1967) observed "extensive hemorrhagic tracts" which they thought suggested migration of parasites in the liver parenchyma at 4 and 7 days after infection, Bullock and Curtis (1924) and Banerjee and Singh (1969) did not associate these necrotic zones with movement by the parasite. From 10 to 22 DAI there was a distinctive fibroblastic reaction around the parasite to form the host capsule. This coincided with but was not resPonsible for the parasite becoming refractory to the lethal effects of immune serum (Musoke and Williams, 1975). Both phenomena appear to be independent evidence of change in the develop- ing parasite. The sarcomatous nature of the fibroblastic response may have reflected mitogenic stimulation by parasite products, as has been noted previously (Bullock and Curtis, 1924; Singh and Rao, 1967). This suggestion is further supported by the observation in the rat that sarcomas deve10p in the host capsule of T. taeniaefbrmis in chronic infections (Bullock and Curtis, 1924, 1925) and in the peritoneal cavity following intraperitoneal injection of washed, ground larvae of T. taeniaeformis (Dunning and Curtis, 1946). 47 The peak in serum levels of both enzymes measured 6 and 7 DAI corresponded to the period of hepatic migration by the parasite prior to proliferative host tissue response. The reason for the second peak 13 and 14 DAI was not clear, but it coincided with the most rapid fibroblastic expansion of the host capsule. Plasma cell hyperplasia within hepatic lymph nodes and spleen, and within the host capsule around the parasite from 13 DAI, occurred at the time when levels of humoral protective activity were rapidly rising towards a peak at 28 DAI (Leid and Williams, 1974). Hyper- plasia of myeloid cells of the eosinOphilic series during T. taeniae- fbrmis infection in rats and mice has previously been observed (Hoeppli and Feng, 1933) and in the rat is associated with eosinophilia within peripheral blood (Ansari and Williams, 1976; Cook and Trapp, 1979). Mast cells have previously been noted in the host capsule of T. taeniaeformis (Varute, 1971), and we have recently observed marked increases in mast cell numbers in the small intestine of infected rats (Cook and Trapp, unpublished). We have also detected many of these cells in hepatic sarcomas associated with chronic T. taeniae- fbrmis infection (Cook and Trapp, unpublished). The immunologic relationship between this parasite and the mast cell response warrants further examination. AtrOphy or acute involution of the thymus associated with mal- nutrition, severe systemic disease or trauma has been termed stress involution and appears to be steroid-mediated (Rosai and Levine, 1976). Ionizing radiation, cytotoxic agents, corticosteroids and some viruses exert a direct cytolytic effect on thymocytes. Thymic atrophy produced by T. taeniaeformis infection in this experiment 48 appeared to be an example of stress involution, rather than a direct or specific effect of the parasite on this arm of the immune system. Acknowledgement This work was supported by NIH Grant number AI-10842 from the United States Public Health Service. References Ansari, A., and Williams, J. F. 1976. The eosinophilic response of the rat to infection with Taenia taeniaeformis. J. Parasit. 62; 728-736. Banerjee, D., and Singh, K. S. 1969. Studies on Cysticercus fasciolaris. III. HistOpathology and histochemistry of rat liver in cysticercosis. Ind. J. Anim. Sci. 32; 242-249. Bullock, F. D., and Curtis, M. R. 1924. A study of the reactions of the tissues of the rat's liver to larvae of Taenia crassicollis and the histogenesis of cysticercus sarcoma. J. Cancer Res. 8; 446—481. Bullock, F. D., and Curtis, M. R. 1925. Types of cysticercus tumors. J. Cancer Res. 2; 425-443. Cook, R. W., and Trapp, A. L. 1979. Pathology of Taenia taeniae- formis infection in the rat: Gastrointestinal changes. (Submitted for publication) Dunning, W. F., and Curtis, M. R. 1946. Multiple peritoneal sarcoma (in rats from intraperitoneal injection of washed ground Taenia larvae. Cancer Res. 6: 668-670. 49 Hoeppli, R., and Feng, L. C. 1933. Myeloid changes in the spleen of experimental animals due to infections with Cysticercus fasciolaris and to emulsions prepared from tapeworms. Chinese Med. J. 51: 1146-1153. Leid, R. W., and Williams, J. F. 1974. Immunological response of the rat to infection with Taenia taeniaeformis. I. Immuno- globulin classes involved in passive transfer of resistance. Immunology 22: 195-208. Lewert, R. M., and Lee, C. L. 1955. Studies on the passage of helminth larvae through host tissues. III. The effects of Taenia taeniaefbrmis on the rat liver as shown by histochemical techniques. J. Infect. Dis. 22; 177-186. Musoke, A. J., and Williams, J. F. 1975. Immunological response of the rat to infection with Taenia taeniaefbrmis. V. Sequence of appearance of protective immunoglobulins and the mechanism of action of 7SY2a antibodies. Immunology 22; 855-866. Picone, J. 1978. Ultrastructure of post-oncospheral stages of Taenia taeniaefbrmis and uptake of ferritin by metacestodes of Taenia taeniaefbrmis. M.S. Thesis. 61p. Michigan State University. Rosai, J., and Levin, G. D. 1976. In Tamors of the Thymus. Atlas of Tumor PathOlOgy, Second Series, Fascicle 13. Armed Forces Institute of Pathology, Washington, p. 17-18. Singh, B. B., and Rao, B. V. 1967. On the development of Cysticercus fasciolaris in albino rat liver and its reaction on the host tissue. Ceylon Vet. J. 12: 121-129. 50 Varute, A. T. 1971. Mast cells in cyst wall of hydatid cyst of Taenia taeniaefbrmis (Batsch). Ind.J. Exp. Biol. 2; 200—203. ARTICLE 2 PATHOLOGY OF TAENIA TRENIAEFORMIS INFECTION IN THE RAT: GASTROINTESTINAL CHANGES R. W. Cook and A. L. Trapp 51 52 SUMMARY The develOpment of morphologic changes in the stomach and small intestine of rats was observed for 86 days after infection (DAI) orally with 1000 or 2000 eggs of Taenia taeniaeformis. Increases in size of both organs, due to mucosal hyperplasia, occurred earlier and were most marked in rats with the heaviest hepatic metacestode infections. Stomach weight increased up to 20-fold. Irregular pro- liferation of mucous epithelium and stroma of the lamina propria produced dramatic papillary and cystic thickening of the gastric mucosa with excessive mucus production. Weight and mucosal thickness of the small intestine doubled with little change in architecture or villus-crypt ratio of 2:1. Intestinal mast cell numbers increased throughout the observa- tion period and were highest in the duodenum of heavily infected rats. In contrast, intestinal eosinophil numbers reached a peak between 30 and 40 DAI and then declined, in parallel with peripheral blood eosinophil counts. The implications of these gastrointestinal changes caused by a hepatic metacestode are discussed. Introduction HistOpathologic studies have been made of the reSponse of the rat to hepatic infection by Taenia taeniaefbrmis (Bullock and Curtis, 1924; Banerjee and Singh, 1969; Lewert and Lee, 1955; Heath and Pavloff, 1975). In addition to host capsule formation around the parasite, pathologic changes have included deve10pment of hepatic sarcoma (Bullock and Curtis, 1925) and "hypertrophic gastritis." Bullock and Curtis (1930) mentioned the latter as a common finding 53 in rats heavily infected with T. taeniaefbrmis, although the time of develOpment and extent of the change were not specified. Blumberg and Gardner (1940) briefly described this change in 8 rats infected for 235 to 303 days with 61 to 200 strobilocerci. Stomachs were enlarged "up to more than twice the normal size." In this study of sequential gastrointestinal changes during infection with T. taeniaefbrmis in the rat we have measured the development of the hyperplastic gastropathy and of previously unre- corded small intestinal hyperplasia, mastocytosis and eosinophilia. The last two phenomena have been studied in relation to immune responses to gastrointestinal nematodes including Nippostrongylus brasiliensis in the rat (Jarrett et al., 1968; Miller and Jarrett, 1971; Kelly and Ogilvie, 1972), Trichostrongylus colubriformis in the guinea pig (Rothwell and Dineen, 1972; Rothwell, 1975) and Haemonchus contortus and T. colubrifbrmis in sheep (O'Sullivan and Donald, 1973). Materials and Methods Experiment 1 A total of 163, 28-day-old female Spartan (Spb [SD] BR) rats purchased from a commercial suppliera were studied. Group 1A (63 rats) and Group 1B (33 rats) were infected orally with 1000 and 2000 eggs of T. taeniaeformis, respectively, while Group 1C consisted of 67 uninfected controls. From Group 1A, 4 rats were killed in a C02 chamber after 20 hours' fast 4, 7, 10, l3, l6, 19, 22, 25, 28, 61, 67 and 75 days after infection (DAI) and 3 were killed 32, 34, 38, a . Spartan Research Animals, Inc., Haslett, MI. 54 41 and 52 DAI. Four control animals from Group 1C were killed 4, 10, 16, 22, 28, 52, 61, 67 and 75 DAI and 3 were killed 32, 34, 38 and 41 DAI. The killing of infected rats in Group 18 and of 19 more controls from Group 1C was divided over 3 days: 33, 41 and 52 DAI. Sequential peripheral blood eosinophil counts were made on 7 infected rats from Group 1A, 32, 33, 34, 35, 36, 38, 41, 46, 61 and 75 DAI, with only 4 alive for sampling on the last date. Eight control rats from Group 1C were similarly sampled excepting 38 and 46 DAI. Total body weight and weights of liver, stomach and small intes- tine were recorded for rats from Groups 1A and 1C killed 61, 67 and 75 DAI, and for rats in Group 1B and 1C killed 33, 41 and 52 DAI. Net body weight was measured after removal of liver, stomach, small intestine, spleen and thoracic contents. The small intestine, measuring approximately 1 meter, was folded into 3 equal lengths, corresponding approximately to duodenum, jejunum and ileum, and a 2 cm segment was removed from the proximal end of each length. All segments were incised longitudinally to expose the mucosa and were then transected. The proximal piece was placed in Carnoy's fixative and the distal one in 10% phosphate- buffered formalin, accompanied in each case by sections of stomach wall. After 12 to 24 hours all tissues were dehydrated, embedded in paraffin and sectioned longitudinally at 6 um. Carnoy-fixed tissue was stained for mast cell counts with Astra-blue at pH 0.3 (Bloom and Kelly, 1960; Enerback, 1966) and counterstained with 0.05% acid fuchsin in 1% acetic acid. The formalin-fixed sections were stained with hematoxylin and eosin for routine histopathologic study and with Giemsa for eosinophil differentiation. Mucosal mast cell and 55 eosinophil counts. were made on 6 groups of 10 "villus-crypt units" (VCU) in each section of intestine and the results expressed as the mean number of cells per 10 VCU (Jarrett et al., 1968; Kelly and Ogilvie, 1972). Following the above sampling, the stomachs and small intestines of rats killed 41 and 52 DAI in Groups 1B and 1C were dried for 12 hours in an oven at 98 C, to determine the difference in dry weight of these organs between infected and control groups. The portal venous pressure was determined 52 to 75 DAI in a series of rats anesthetized with sodium pentabarbitone (5 mg/lOO gm of body weight). A 25-gauge needle connected by catheter to a pressure transducerb and amplifier was inserted into the portal vein and the blood pressure was recorded on a multitrace oscilloscope.C The pressure transducer was calibrated each day against a water manometer. Experiment 2 Twenty female Spartan rats (Group 2A) were infected at 28 days of age with 1000 eggs of T. taeniaeformis and divided amongst 4 cages. Daily feed consumption and fecal output were measured for each cage of 5 rats from 56 to 84 DAI and eXpressed as grams per rat. During this period all animals were weighed at weekly intervals. Each rat was killed 86 DAI following 20 hours' fast. Total and net body weights and weights of liver, stomach, small intestine and cecum were recorded. bModel P23Db, Statham Co., Hato Rey, PR. quodel VR6, Electronics for Medicine, White Plains, NY. 56 Control groups consisted of 20 age-matched rats (Group 28) similarly housed, and 18 rats, 1 week older, divided amongst 3 cages (Group 2C). Statistical analysis Group means in both experiments were compared using the Student's t-test. Results Weights and feed consumption Body and organ weights for Experiment 1 are presented in Figure l and for Experiment 2 in Table 1. Two infected rats in Experiment 2 died, 60 and 80 DAI. Hepatic parasite burdens as indicated by liver weights were heaviest in Group 1B, and were greater in Group 2A than in 1A. Net body weight of infected rats decreased from 41 DAI in Group 1B, and in Groups 1A and 2A at the end of the observation periods, 75 and 86 DAI respectively, was 80 to 90 grams less than that of control animals. Total body weight of infected rats exceeded that of controls up to 52 DAI in Experiment 1 and to 63 DAI in Experiment 2. Thereafter there was no significant difference, as the weight advantage due to hepatic and gastrointestinal enlargement in infected rats was offset by loss of body condition. Increase in stomach weight was first observed 52 DAI in Experi- ment 1. The order of increase in stomach size was remarkable, reaching a mean of ll-fold (to 18.0 grams) by 86 DAI in Experiment 2. Indi- vidual rats with heavy infections had up to 20-fold increases in stomach weight. In Experiment 1, enlargement of small intestine was 57 Stomach Small intestine { B b -i ‘6 a r- I]; ---- I ‘112 r §” - 8 _ — §’§\§ "" 8 .— * o/6\O ‘ / 4 — 1’ -4 4 A [II E bid-:1), o 0—0 0 fl ‘3 3 .— L . .C . 9: Liver Body ( net ) i g 80 - %\ / “200 _ ,l l 60— I)" § 4 “180 x l i- 0-0 0 "‘ ° 1° 1° 1 l l l l 1 L Days after infection Figure 1. Effect of T. taeniaefbrmis infection in Experiment 1 on mean stomach, small intestine, liver and net body weight i SE. Net body weight was assessed after removal of liver, stomach, small intestine, spleen and thoracic organs. The SE symbol is omitted where SE is less than radius of the circular symbol. Group 1A (1000 T. taeniaeformis) = o—o ; Group 1B (2000 T. taeniaeformis) = o———o ; Group 1C (control) = o—o. 58 Table 1. Body and organ weights of rats 86 days after oral infection with 1000 eggs of Taenia taeniaefbrmis in Experiment 2 Body Net Body Small Group Weight Weight Stomach Intestine Cecum Liver 2A(18)a 23815.2b 142i5.6c 18.02213C 15.7io.6C 1,210.04c 51.2:2.7C 28(20) 240:3.1 220:2.9 1.6:0.05 6.510.2 O.8i0.03 6.7iO.2 2C(18) 251:5.2 230i5.0 1.6i0.04 6.5i0.2 --- 7.1i0.2 aNumber of rats per group. bGroup mean (grams) i standard error. cThe mean of infected Group 2A is significantly different from means of both control Groups 2B and 2C (p<.001) for all parameters except total body weight. There is no significant difference between Groups 2B and 2C for any parameter. first detected 33 DAI (the start of the recording period in Group 1B) and in both experiments was approximately 2-fold. Cecal weight of infected rats was slightly increased in Experiment 2. The percentage of dry weight of stomachs from infected and control animals was 17% and 26%, and of small intestines 22% and 26%, respectively. Despite the increased water content of the enlarged organs from infected rats, dry weights still exceeded those of control animals. In Experiment 2 there was no significant difference amongst groups in mean daily feed consumption calculated from 56 to 84 DAI. However, daily feed consumption in Group 2A first exceeded that of control groups 66 DAI, and did so on a total of 11/19 days to the end of the observation period (p<0.05). Between 66 and 84 DAI mean daily feed consumption of Group 2A was 18.510.l (SE) grams per rat and 59 exceeded that of both control Groups 2B and 2C, l6.8i0.3 and 16.2: 0.4 grams, respectively (p<0.01). For the same period mean daily fecal output of Group 2A, 6.010.02 grams per rat, was greater than that of Groups ZB and 2C, 5.0:0.03 and 5.0i0.1 grams, respectively (p<0.05). The difference was attributed mainly to increased fecal water content as feces from infected rats were very soft and not formed into pellets as in Groups 28 and 2C. Macrosc0pic changes Gastric and small intestinal enlargement (Figures 2A,B) was greatest and occurred earlier in the heaviest infections. In the stomach, changes were restricted to the glandular mucosa (Figure 2C) and were associated with excessive mucus production. Focal lesions comprised raised white plaques or nodules, located most frequently along the limiting ridge adjacent to the forestomach. The most common lesion was of diffuse nodular mucosal hyperplasia (Figures 2B,C,D). In these cases the irregularly thickened mucosa was pale pink and opaque. Its surface was uneven and covered by a large amount of viscid mucus into which there was petechial hemorrhage (Figure 2D). The pH of the mucoid gastric contents was between 5 and 9, in con- trast with a pH of less than 3 in control animals. The mucosa of the small intestine was grossly thickened, particu- larly in the duodenum and proximal jejunum. Intestinal contents were more mucoid than usual. The cecum appeared slightly edematous. Microscopic changes Gastrointestinal changes were limited to the mucosa. In the stomach, marked hyperplasia of mucus-secreting epithelium produced irregular papillary to cystic changes which extended into the gastric 6O .mwouofifiucmo on ma mflmom .msocum “my .Esuucw Amy «H49 me Aumma QOpV mommnwuoaor ananoouom one coa909©onm moose o>flmmooxo nuH3 nonmaoommm mmoose oeuummo unauocmHm mo nHmmHmuwm>2 unanooz i o .mEmum m.a mommam3 “unmau Hw3oav nomaoum Howucou .mfimum w.oa nonmwm3 Aumoa uozoflv someoum unwound .Hmo mm .mH mdouw ca mung Eoum mnomeoum HMHflUGMHm onu mo wannamummwn mummmwv on Hooch i U .Asoaonv Hmeacm Houucoo m Eoum omonu rues Aw>onmv mcmmuo pmuoommm mo cowaummeou i m .uo>«a on» :w wanwmfl> meHommmwgmmu .5 mo HonoooHHQouum nufl3 .Huomu3m Houmm mxoo3 m mums couaeouomuucm mourn mo wromEOum rufl3 monomfioo .Amv assess uomucfl :ue3 Aunmfiw nosoav you Houucoo mo romeoum i o .Escoposc may sues memoeoumocm now coaumummwum cw couscou sceuomm oeuummo mCeCmemu can co>0€ou coon mm: Eouucm i O .Uomomxw we A3ouumv woven oCfluMEHH mo caom ooecumo com cwumHOmH coon mm: Adv Esuuc< .pouoamsoo coon mm: coHuommcmuu oeuummu i m .Am3ouum mansoni msomrmowo on» mcumzou musum>uso noumoum one Scum come edema we coflmwocw ocoomm use .A3owum oaocfimv Houocwnmm canonm on» no mouoomcmuu coon mm: .mmoowom an paws .Escoposo .pmmomxo Amv Bounce cwuoHooiurmfiH on» £uw3 romaoum on» NO ecumwsm uofiuoucm i 4 .mm musmflm 101 Figure 2A 102 During surgery the rats were kept warm on a 37 C heating pad, and afterwards they were returned to cages where heat lamps maintained an ambient temperature of 28 C for 24 hours. Access to water and food was not permitted until 2 and 3 days, respectively, after surgery. Results and Discussion Of 31 rats which had been fasted overnight and antrectomized at 3 weeks of age, 27 (87%) survived without incident. Three died within 6 hours of surgery and 1 died 2 days later due to torsion of the small intestine. Less satisfactory results were obtained when rats were not fasted overnight before surgery. In this group, only 8 of 14 (57%) survived, and the remaining 6 died within 12 hours of surgery. Death was attributed to the excessive amount of trauma and resultant shock induced by manipulations necessary during surgery to remove food from the distended stomach. ' By eliminating the tedious and difficult multiple ligations of the tiny gastroepiploic arterial branches in these weanling rats, the time required for surgical antrectomy was considerably shortened and the procedure simplified. Whatever disadvantage accrued from hemorrhage caused by our rather drastic and rapid excision was apparently compensated for by the postoperative administration of blood and fluids. The survival rates in the fasted rats were acceptable for the production of antrectomized animals on a large scale for pathophysiologic experimentation. At necrOpsy 8 weeks later, there were adhesions of the left lateral lobe of the liver adjacent to the site of anastomosis, but the surgical site was always intact and free of infection. Anatomic ablation of the pyloric antrum was complete in all cases (Figure 2D). 103 Acknowledgement This work was supported by Grant A110842 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. References 1. Johnson L R: Gut hormones and growth of gastrointestinal mucosa. In Endocrinology of the Gut, p 163-177. Chevy W Y, Brooks 8, Ed, Charles B Slack, Thorofare, 1974. Lambertfh Surgery of the Digestive System in the Rat, Charles C Thomas, Springfield, 1965. Martin P, MacLeod I B, Sircus W: Effects of antrectomy on the fundic mucosa of the rat. Gastroenterology 59:437-444, 1970. Williams C S F: Practical Guide to Laboratory Animals, C V Mosby, St Louis, 1976. APPENDIX B EFFECT OF INFECTION WITH TAENIA TAENIAEFORMIS ON BODY AND ORGAN WEIGHTS, FEED CONSUMPTION AND FECAL OUTPUT IN THE RAT 104 105 Introduction This study was carried out following our observation that gastric and small intestinal weights increased during hepatic infection with Taenia taeniaefOrmis. Hyperphagia has been implicated as a cause of gastrointestinal hyperplasia in rats during lactation (Campbell and Fell, 1964), alloxan-induced diabetes (Nakabou et al., 1974), and intermittent starvation (Holeckova and Fabry, 1959), and in associa- tion with hypothalamic lesions (Brobeck et al., 1943). Rats infected with T. taeniaeformis were studied during a 4-week period when gastrointestinal hyperplasia was known to be present. The objective was to determine if there were measurable changes in food intake in parasitized animals at this phase of infection. Materials and Methods Twenty female Spartan (Spb [SD] Br) rats (Group A) were dosed orally at 28 days of age with 1000 eggs of T. taeniaeformis and divided amongst 4 cages. Control groups consisted of 20 age—matched normal female rats (Group B) similarly housed and 18 normal female rats 1 week older, divided amongst 3 cages (Group C). Starting 56 days after infection (DAI), daily feed consumption and fecal output were measured for 3 cages of rats from each of Groups A, 8 and C, the results for each cage being expressed in grams of feed and feces per rat. A week later, 68 DAI, the fourth cage of rats from Groups A and B were included in the observations which continued until 84 DAI. Body weights of individual rats were measured at weekly intervals and expressed as the mean of weights recorded on 2 consecutive days. 106 All rats were killed 86 DAI, after being starved for 20 hours. Weights of whole body, liver, stomach, small intestine and cecum were recorded together with the net body weight, determined after removal of the above organs, spleen and thoracic contents. The cecum was not weighed in Group C. Group means were compared using the Student's t-test. Results At the start of the experiment, an enlarged stomach could be readily palpated in most affected rats. Two rats in Group A died during the observation period, 60 and 80 DAI. Mean weekly live body weight, and mean daily feed consumption and fecal output of rats within each group are shown in Figure 1. The daily measurements were carried out between 2 and 5 p.m. This variation in the sampling period and failure to maintain a strict lZ-hour lighting schedule may have contributed to the daily fluctua- tions observed. The mean live body weight of Group A was significantly greater than that of Groups 8 (p<0.001) and C (p<0.05) 56 DAI and of Group B (p<0.01) 63 DAI. This was due to the increased weight of parasitized livers and gastrointestinal organs in Group A. The weight differences between Groups 8 and C were significant 56, 63 and 70 DAI (p<0.05, <0.01 and <0.02, respectively). Progressive loss of condition of infected rats during the observation period offset the initial weight advantage of Group A, whose mean liver weight did not differ signifi- cantly from that of Groups 8 or C at 70, 77 or 84 DAI (Figure 18). Mean daily feed consumptions calculated for the entire period 56 to 84 DAI for Groups A, 8 and C were l7.4i0.2 (SE), l6.7iO.3 and 107 Figure 18. Weekly live body weight, and daily feed consumption and fecal output 1 SE of rats infected orally with 1000 eggs of Taenia taeniaeformis. The SE symbol is omitted where 88 is less than the radius of the circular symbol. Group A (infected) = o—-—o; Group 8 (control) = O o ; Group C (control) = 0-----0 . 108 HESS £903 >com $59.8 coon. 3888 modem 85 80 75 70 65 60 55 Days after infection Figure 18 109 l6.6:0.4 grams per rat, respectively. These were not significantly different from one another. Until 66 DAI mean feed consumption calculated daily for Group A was the same as or less than that for Groups 8 and C. Thereafter, rats in Group A ate slightly more than at least one control group (p<0.05) on 11/19 days to the end of the observation period. Mean daily feed consumption between 66 and 84 DAI for Group A was 18.5:0.l grams per rat, and exceeded that for Groups 8 and C, 16.8:0.3 and 16.2: 0.4 grams, respectively (p<0.01). From 56 to 84 DAI mean daily fecal output for Group A was 5.8: 0.2 grams per rat, exceeding that for Group B, 4.9:0.2 grams (p<0.05), but was not significantly greater than that for Group C, 5.1:O.2 grams. Fecal output for Group A exceeded that for one or both of the control groups on 7/29 days (p<0.05). Between 66 and 84 DAI daily fecal output for Group A, 6.0:0.2 grams per rat, was greater than that for Groups 8 and C, 5.0i0.3 and 5.0:0.l grams per rat, respectively (p<0.05). Feces of infected rats were very soft and not formed into pellets, except during most of the last week of the experiment. Feces of control animals were dry and pelleted throughout the observation period. Increased water content of feces from infected rats accounted for their higher fecal output. The fasted body and organ weights of rats killed 86 DAI are presented in Tablele There was no significant difference in total body weight between any 2 groups (as with unfasted live weight at 84 DAI). In Group A, net body weight was less (p<0.001) and weights of all organs measured were higher (p<0.001) than those of Groups 8 and C, which did not differ from one another. 110 Table 18. Body and organ weights of rats 86 days after oral infec- tion with 1000 eggs of Taenia taeniaefbrmis Body Net Body Small Group Weight Weight Stomach Intestine Cecum Liver A(18)a 238:5.2b 142:5.6C 18.0:1.BC 15.7:O.6C 1.2:0.04C 51.2:2.7C 8(20) 240:3.1 220:2.9 l.6i0.05 6.5:0.2 0.8:0.03 6.7:0.2 C(18) 251:5.2 230:5.0 1.6:0.04 6.5:0.2 --— 7.1:O.2 aNumber of rats per group. bGroup mean (grams) i standard error. CThe mean of infected Group It is significantly different from means of both control groups B and C (p<0.001) for all parameters except total body weight. There is no significant difference between Groups 8 and C for any parameter. Discussion The detection of gastric enlargement, by palpation, in most infected rats at the start of the experiment was consistent with our previous observations in rats with infections of similar size and duration. The small intestine of infected rats would also have been enlarged at this time, and both organs were markedly enlarged 86 DAI (Table 18). However, it was not until 66 DAI that there was signifi- cant increase in feed consumption by the infected group. Thereafter infected rats each ate approximately 2 grams more feed per day than control animals. If expressed per unit of net body weight, feed consumption by infected rats was almost twice that of controls by 84 DAI. It is of interest to compare the feed intake anui increases in weights of gastrointestinal organs in T. taeniaefbrmis infection with 111 those seen in other circumstances of gastrointestinal hyperplasia in the rat. Brobeck et a1. (1943) observed that rats with surgically-induced hypothalamic lesions ate 2 to 3 times the normal amount of food when fed ad libitum. They became obese and their gastrointestinal tracts were dilated and hypertroPhied, weighing twice that of control animals. In rats subjected to intermittent starvation for 6 to 17 weeks, total stomach weight increased by 20% compared with controls, with forestomach and glandular portion increasing by 40% and 10%, respec— tively (Holeckova and Fabry, 1959). Although intermittently starved rats ate more at each feeding, their mean daily feed intake was 5 to 8 grams less than that of controls, which consumed 15 to 20 grams of feed per day. After 17 weeks, mean body weights of intermittently starved and control rats were 173 and 234 grams, reSpectively (similar to net body weights of T. taeniaeformis-infected and control rats 86 DAI in Experiment 2). The size of the small intestine increased by 40% under similar circumstances (Fabry and Kujalova, 1958). The small increases in size of stomach (mainly forestomach) and small intestine occurred despite a decrease in body weight. Lactating rats ate 2 to 3 times more feed than non-lactating rats, while weights of stomach, small intestine and cecum were increased by 59%, 108% and 64%, respectively, over control values (Fell, Smith and Campbell, 1963). During pregnancy, weights of stomach, small intestine and cecum increased by 17%, 12% and 26%. Gross changes were not seen in the stomach, although parietal cell hypertrOphy was noted. Crean and Rumsey (1971) also observed a similar degree of gastric hyperplasia with stomach weights increased by 20% and 40% during pregnancy and lactation, respectively. There 112 was an increase in parietal and peptic cell numbers but no clear temporal relationship between this increase and the main rise in food consumption which occurred during lactation after gastric and small intestinal mucosal hyperplasia had commenced. Body weights of lactating rats fed ad libitum increased, while those fed 20 grams per day lost weight and had only slight gastrointestinal tract enlarge- ment (Campbell and Fell, 1964). In alloxan-induced diabetes of 6 to 12 months' duration in rats, food intake increased 2- to 3-fold, and there was increase in dry weight of stomach and small intestine of 15% and 95%, respectively (Jervis and Levin, 1966). Increase in cecal weight was not significant. Mean body weights of diabetic and non-diabetic rats were 249 and 340 grams, respectively. In a 4-month study Schedl and Wilson (1971) observed 2-, 3- and 3-fold increases in weights of stomach, small intestine and cecum, respectively. Despite the increase in wet weight of the stomach in diabetics, dry weight was not significantly greater than that of controls. Diabetic rats weighed half as much as controls, and this was also seen after 3 weeks by Nakabou et al. (1974), who observed 2-fold increases in weight of small intestine. Stomach weight was not increased unless expressed per unit of body weight. Body weights of diabetic and control rats at the end of their experi- ment were approximately 275 and 160 grams. At its plateau, daily food intake by diabetic rats was 40 to 50% more than that of controls (200 to 260% when eXpressed in grams per 100 grams body weight). The small intestinal enlargement did not occur in the diabetic rats when food intake was restricted. Seelig et a1. (1977) demonstrated that after 50% resection of the proximal small intestine, rats temporarily lost and then recovered 113 body weight compared with controls. At 3, 6 and 9 months following resection, the residual small intestine was increased in thickness, while the stomach was not grossly altered. However, microscopic evidence of increased thickness of gastric mucosa and an increase in epithelial cell populations of gastric glands were observed at these times, but not at 12 months in the rats with small intestinal resection. Slight enlargement has been seen in the small intestine of rats fed a diet rich in lactose (Fischer, 1957) or corn starch (Wierda, 1950), and in the gastrointestinal tract of rats fed a high fat diet (Slabochova and Placer, 1962). Gastrointestinal hyperplasia has occurred in association with body weight increase (hypothalamic lesion and lactation), sustained body weight loss (alloxan-induced diabetes and intermittent starvation) or temporary body weight loss (following resection of small intestine). For the first three, the order of small intestinal enlargement was consistently 2- to 3-fold and feed consumption usually similarly increased. However, increases in stomach weight were never more than 2-fold. In T. taeniaeformis infection the increases in weights of small intestine and cecum were similar to that seen in the above circum- stances but preceded the small increase in feed consumption by infected rats. The mean ll-fold increase in stomach weights in rats with T. taeniaefbrmis in this experiment (up to 20-fold in individual rats) has not been approached in any other circumstances where gastroin- testinal hyperplasia develops in the rat. Even in the 12-month studies of alloxan-induced diabetes in which feed consumption by rats was 114 markedly increased, there was no disproportionate increase in stomach weight. Therefore, the dramatic degree of gastric hyperplasia in T. taeniaefbrmis infection appeared to be related specifically to the presence of this parasite and not a result of altered food intake. References Brobeck, J. R., Tepperman, J., and Lang, C. N. H. 1943. Experimental hypothalamic hyperphagia in the albino rat. Yale J. Biol. Med. 1%: 831-853. Campbell, R. M., and Fell, B. F. 1964. Gastrointestinal hypertrOphy in the lactating rat and its relationship to food intake. J. Physiol. 121; 90~97. Crean, G. P., and Rumsey, R. D. E. 1971. Hyperplasia of the gastric mucosa during pregnancy and lactation in the rat. J. Physiol. 115; 181-197. Fabry, P., and Kujalova, V. 1958. Wachstum des Dfinndarmes bei intermillierend hungernden Ratten. Naturwissenschaften 45; 373. Fell, B. F., Smith, K. A., and Campbell, R. M. 1963. Hypertrophic and hyperplastic changes in the alimentary canal of the lac- tating rat. J. Path. Bact. 85: 179-188. Fischer, J. E. 1957. Effects of feeding a diet containing lactose upon 8 D galactosidase activity and organ development in the rat digestive tract. Am. J. Physiol. 188; 49-53. Holeckova, E., and Fabry, P. 1959. Hyperphagia and gastric hyper- trophy in rats adapted to intermittent starvation. Brit. J. Nutr. 12; 260-266. 115 Jervis, E. L., and Levin, R. J. 1966. Anatomic adaptation of the alimentary tract of the rat to the hyperphagia of chronic alloxan-diabetes. Nature 119; 391-393. Nakabou, Y., Okita, C., Takano, Y., and Hagihiri, H. 1974. Hyper- plastic and hypertrOphic changes of the small intestine in alloxan diabetic rats. J. Nutr. Sci. Vitaminol. 19; 227-234. Schedl, H. P., and Wilson, H. D. 1971. Effects of diabetes on intestinal growth in the rat. J. exp. Z001. 116; 487-496. Seelig, L. L., Winborn, W. B., and Weser, E. 1977. Effect of small bowel resection on the gastric mucosa in the rat. Gastro- enterology 22; 421-428. Slabochova, 2., and Placer, Z. 1962. Adaptation of the small intestine to a high fat diet containing saturated and unsatu- rated fatty acids. Nature 125; 380-381. Wierda, J. L. 1950. A comparison of the weight of the intestine with the body and kidney weights in rats which were fed artificial unbalanced diets. Anat. Rec. 107: 221-233. APPENDIX C CELLULAR TRANSFER OF IMMUNITY TO THENIA TAENIAEFORMIS IN THE RAT 116 117 Introduction The antibody-mediated component of the protective immune response to Taenia taeniaeformis infection in the rat has been studied (Miller, 1931; Campbell, 1938; Leid and Williams, 1974), and humoral effector mechanisms are known to occur in acquired resistance to other metacestodes (Gemmell and Macnamara, 1972). Cell-mediated immune (CMI) reSponses have been detected in natural infections with taeniid larvae (Kagan et al., 1966), and Rickard and Outteridge (1974) demonstrated CMI responses in rabbits eXperimentally infected with, or vaccinated against, Taenia pisifbrmis using skin tests and in vitro blast transformation of lymphocytes. Kwa and Liew (1977) elicited delayed type hypersensitivity skin responses in rats vac- cinated with both excretory-secretory and somatic antigens. This response was transferred to recipient rats of the same inbred strain by peritoneal cells from vaccinated rats. Rickard (1974) has pro- posed that the established parasite evades CMI responses by becoming coated with blocking antibody. However, there is no conclusive evidence of direct cellular involvement in immunity to metacestodes. Nemeth (1970) transferred partial immunity to T. pisifbrmis infection in rabbits with allogeneic lymphoid cells, and Blundell et a1. (1969) were not successful in transferring immunity to Taenia hydatigena or Taenia ovis in sheep with allogeneic lymphoid cells from infected animals. Mitchell et a1. (1977) were unable to protect athymic (nude) mice from infection with T. taeniaefbrmis by injection with a popu- lation of purified T cells from infected mice, but demonstrated T cell dependence of protective antibody production. If a protective CMI response is evoked by taeniid metacestodes, it then becomes necessary to determine how the established organism 118 of the primary infection evades rejection by this mechanism as well as by the antibody-mediated response that it provokes. Difficulties arise in the interpretation of adoptive transfer of immunity with cells in taeniid metacestode systems because of the problem of separat- ing cell—mediated from humoral effects on challenge organisms. We have therefore studied the protective effect of adoptively transferred syngeneic cells during the first 12 hours of primary infection in an attempt to demonstrate CMI effects before transferred cells might be exPected to produce protective amounts of antibody. Also, previous work (Musoke and Williams, 1975) had indicated that protective anti- body did not exert its maximum effect during this period, suggesting that the contribution of transferred antibody-producing cells would be minimal in this system. Materials and Methods Experimental Animals Spartan (Spb [SD] BR) and inbred (Fischer 344 strain) female rats were purchased from Spartan Research Animals, Haslett, Michigan, and A.R.S. Sprague-Dawley, Madison, Wisconsin, respectively, and were given proprietary brand food and water ad libitum. Preparation of immune rat serum and mixed spleen and hepatic lymph node cell suspensions For the preparation of suspensions of immune cells (IC), 28-day- old female Fischer strain rats were given 500 to 1000 eggs of T. taeniaeformis orally and killed using CO vapor 28 to 56 days later 2 on the day-of adeptive cell transfer. Blood was collected from the thoracic cavity after severing the vessels anterior to the heart, and allowed to clot. Serum from those donors which had been infected 119 for 28 days was used in subsequent experiments and identified as immune rat serum (IRS). Spleen and hepatic lymph nodes were removed aseptically and dispersed by mincing with fine forceps and iris scissors in cold (4 C) Eagle's minimal essential mediuma containing 5% fetal calf seruma (MEM-FCS). Extraneous fibrous connective tissue was discarded. The remaining tissue suspension was aspirated into a syringe and through needles progressing from 19 through 22 to 26 gauge before being washed 3 times with MEM-FCS. Cell viability was estimated by trypan blue dye exclusion and the cell suspensions adjusted so that the desired number of viable cells was contained in 1 ml for injection. Suspensions of normal cells (NC) were prepared from normal female Fischer strain rats of the same age as the infected rats. Preparation of T- and B-cell-rich suspensions Separation of T- and B-cell-rich fractions from cells of immune rats was made using nylon wool columns as described by Trizio and Cudkowicz (1974), with the exception that columns of larger volume were used. The cells obtained from the spleens and hepatic lymph nodes of 2 rats (total approximately 8 x 108 cells) were suspended in 8 m1 of MEM-FCS and poured over a glass woo1 column. This was prepared by loosely packing fine glass woolb to the 32 m1 mark in a 50 m1 plastic syringe and washing with 200 m1 of phosphate-buffered saline (PBS) and 150 m1 of MEM-FCS before use. The cells were eluted from the column with 200 ml of MEM-FCS, the eluate was centrifuged at 1000 rpm for aGrand Island Biological Co., New York. berex brand WOol: filtering fiber. Catalogue Number 3950. 120 10 minutes, and the cells were then resuspended in 8 ml of MEM-FCS. The nylon wool column was prepared by packing a 50 ml plastic syringe to the 25 ml mark with 2.5 grams of nylon wool.C It was washed with 200 ml of PBS and then 150 ml of MEM-FCS before being sealed with parafilm and incubated upright at 37 C for 1 hour. The column was then washed with 20 m1 of warm MEM-FCS to correct pH gradients. The cell suspension of the eluate from the glass wool column was added dropwise to this column, which was washed with 4 m1 of warm MEM-FCS, sealed and incubated upright at 37 C for 1 hour. The T-cell-rich fraction was then obtained by recovering the cells eluted from the column with 100 m1 of warm MEM-FCS. After this the column was washed rapidly with 300 ml of warm MEM-FCS before eluting the cells adhering to the nylon wool (the B-cell-rich fraction) by squeezing the nylon wool with stainless steel forceps and washing with 50 m1 of warm MEM-FCS. A yield of approximately 2 x 108 cells was obtained in each fraction from this column. An alternative method used to prepare the B-cell-rich fraction was by incubating spleen cells (5 x 107 per ml) with a 1:32 dilution of rabbit anti—brain—associated—theta serum in the presence of guinea pig serum as a source of complement. Anti-brain-associated-theta serum was prepared as described by Golub (1971) and absorbed at 4 C with a 30% volume of washed packed erythrocytes from Fischer strain rats, a similar volume of washed packed liver homogenate, and finally with 100 mg of agarose per 10 m1 of serum. The guinea pig serum was also absorbed with agarose. At a 1:32 dilution the anti-theta serum was cytotoxic for 95 to 100% of thymocytes and 20 to 30% of spleen cells. cScrubbed nylon fiber 3 denier 1.5 inch type 200. Fenwell Laboratories, Morton Grove, IL 60053. 121 Experimental Procedure The procedure is summarized diagrammatically in Figure 1C. Groups of 3 or 4 normal 28-day-old female inbred Fischer strain rats (Recipients) were given 6000 T. taeniaeformis eggs orally, and within 2 hours they received intravenous injection with 1-5 x 108 IC from rats of the same strain which had been infected with T. taeniaefbrmis for 28 to 56 days (Donors). Other groups were pre- treated with either similar numbers of NC from rats of the same strain and age, 1 ml of 28-day IRS, or 1—5 x 108 IC with 1 m1 of IRS. Twelve hours later the rats were killed. Their livers were removed, pooled within each treatment group, finely minced using razor blades, and then stirred at room temperature in 200 ml of 0.25% trypsin solution for 30 minutes. After the larger fragments of liver tissue had settled, the supernatant fluid was collected and centrifuged at 1000 g for 10 minutes and the sediment retained. The larger fragments were digested in a further 200 ml of trypsin solu- tion for 30 minutes and the resulting supernatant fluid treated as before. The sediments, consisting of free hepatic cells and para- sites, were washed twice in MEM, pooled within each group and resus- pended to a volume of 1 ml per liver digested. To determine the number of viable parasites within this digest (a function of the number surviving in the liver when the rats were killed 12 hours after infection), it was injected into mesenteric veins of normal female Sprague-Dawley rats (Assay Rats) and the number of hepatic cysticerci counted 21 days later. The ratio of Assay to Recipient Rats was usually 2:1. 122 Figure 1C. Cellular transfer of immunity to T. taeniaeformis: diagrammatic summary of experimental procedure. ]23 EGGS \A‘ DONORS U 2. DAYS SPLEEN AND LYMPH NODE CELLS ; RECIPIENTS ' 12 HOURS LIVERS DIGESTED WITH TRYPSIN ASSAY RATS 21 DAYS LIVER CYSTICERCI COUNTED Figure 1C 124 Results The results of 5 experiments are summarized in Table 1C. There was a consistent reduction in numbers of parasites develop- ing in the Assay Rats from groups receiving either IC or IRS compared with the control groups. In Experiment 1, IC were as effective as IRS and no improvement was obtained by giving both IC and IRS. In Experiment 2, the numbers of parasites surviving in the IC group were significantly less than for controls but greater than for the IRS group. In Experiment 3, where IC (T) and IC (B) were prepared by nylon wool column separation, both cell preparations reduced parasite numbers in Assay Rats to a similar extent compared with the control group. Likewise, 1J1 Experiment 4, in which nylon wool column and treatment with anti-theta serum was used in preparing T and B cell suspensions, activity against the organism was demonstrated with all 3 cell preparations, although the number of parasites in the group given IC (T) and IC (T+B) was significantly lower than in that given IC (B). Transfer of immunity was also successful using allogeneic cells (Experiment 5). It was necessary to determine whether antibody from Donors might have been passively carried over in the cell suSpension and been responsible for the protective effects observed. A suspension of NC in IRS was incubated for 30 minutes at 37 C and the cells were then washed in the usual manner before injection into Recipients. Cyst numbers in Assay Rats from this group were equal to those of the control group that received NC alone. 125 Table 1C. Protective capacity of immune cells (IC) and immune serum (IRS) during the first 12 hours of primary infection with Taenia taeniaeformis Number of Mean Number of Treatment Assay Rats Cysts t SE P Value Experiment 1 vs NC vs IRS IC 7 28.1 i 5.9 <0.01 NS IC + IRS 6 20.8 i 2.9 <0.001 NS IRS 6 19.3 i 2.9 <0.001 —-- NC 6 66 7 i 7.9 --- <0.001 Experiment 2 vs NC vs IRS IC 9 66.9 i 2.4 <0.001 <0.001 IRS 6 16.2 i 0.7 <0.001 --- NC 5 96.0 i 8.0 --- <0.001 Experiment 3 vs NC vs IC(B) IC (T) 7 20.1 i 6.5 <0.001 NS IC (B) 7 31.7 i 2.5 <0.001 --- NC 8 69.1 i 5.7 --- <0.001 Experiment 4 vs NC vs IC(B) IC (T) 8 69.3 i 4.4 <0.01 <0.01 IC (B) 8 88.5 i 3.4 <0.05 --- IC (T+B) 8 69 8 i 3.9 <0.01 <0.01 NC 8 136.0 i 18.9 --- <0.05 Experiment 5* vs NC IC 6 24.2 i 4.2 <0.05 NC 5 40.2 i 3.9 --- NS = not significant * Allogeneic cell transfer in Spartan rats. 126 An additional experiment was performed in order to show that the protective effect was actually due to the activity of transferred cells during the 12-hour infection of the Recipient rather than to continued activity in Assay Rats of immunocompetent cells that had survived tryptic liver digestion. Rats were challenged with 300 eggs of T. taeniaeformis after receiving an intravenous injection of liver digest from normal rats that had been given suspensions of IC intravenously. No activity of immunocompetent cells was demonstrated in these rats which developed the same numbers of parasites as control rats. Discussion The results of the eXperiments indicated that a rapid protective effect was consistently achieved by transfer of both IC and IRS. The effect of IRS proved to be more rapid than we had expected from previous work (Musoke and Williams, 1975). However, substantial numbers of organisms still survived when exposed to doses of IRS that are completely protective in rats challenged in the conventional manner. This indicated that a prOportion of the parasite population is either inaccessible or refractory to the immune effector system during the first 12 hours in the host. Our results showed that the washing procedure prior to cell transfer was sufficient to remove antibody passively associated with cells and we were unable to demon- strate protective activity by any immune cells remaining in the tryptic digest. These last two results indicated that the effects observed resulted from activity of transferred cells during the l2-hour period prior to liver digestion. 127 It seems unlikely that during this 12-hour period transferred cells would produce sufficient antibody to effect the degree of protection seen. However, this cannot be discounted, especially if the ”homing" of immune cells to the liver occurred and the effector system therefore became rapidly localized. It was even a particularly rapid transfer of a cell-mediated effect, although the number of cells transferred in these experiments was large. The effectiveness of allogeneic cells in this system was perhaps not surprising, as the period during which their effects were being measured would probably have preceded the onset of their recognition and immune rejection by the Recipients. The results of T- and B-cell separation were equivocal and consistent results have not been obtained in subsequent experiments. While characterization of T and B cells has not been achieved in the rat to the degree which it has in the mouse, subpopulations of rat lymphoid cell types have been identified (Goldsneider, 1976; Strober, 1976). However, it may prove difficult to implicate any subpopulation of cells in an immune process which is being assessed by in vivo survival of the parasite. This problem is compounded in systems like that of T. taeniaefbrmis where antibody protection is potent and must be suspected of contributing to responses even when the B cell proportion in cell preparations is very low. Despite these difficulties, working with whole organisms in vivo remains a necessity when cell-mediated protection is being studied. In vitro and in vivo measurements of CMI responses may not correlate with cell-mediated protection. This appeared to be the case for T. pisifbrmis infection, where the CMI-producing antigens did not 128 seem to be the same as those that induced protection in the rabbit (Rickard and Katiyar, 1976). References Blundell, S. K., Gemmell, M. A., and Macnamara, F. N. 1969. Immuno— logical responses of the mammalian host against tapeworm infections. VIII. Some evidence against cellular immunity induced in sheep by activated embryos of Taenia hydatigena and T. ovis. Exp. Parasit. 24; 291-298. Campbell, D. H. 1938. The specific protective property of serum from rats infected with Cysticercus crassicollis. J. Immunol. 15; 195-204. Gemmell, M. A., and Macnamara, F. N. 1972. Immune responses to tissue parasites. II. Cestodes. In Immunity to Animal Parasites. (Ed. by E. J. L. Soulsby). Academic Press, New York, pp. 236-272. Goldsneider, I. 1976. Antigenic relationship between bone marrow, lymphocytes, cortical thymocytes and a subpopulation of peripheral T cells in the rat: Description of a bone marrow lymphocyte antigen. Cell. Immunol. 14; 289-307. Golub, E. S. 1971. Brain-associated theta antigen, reactivity of rabbit anti-mouse brain with mouse lymphoid cells. Cell. Immunol. 1; 353-361. Kagan, I. G., Osimani, J. J., Varela, J. C., and Allain, D. S. 1966. Evaluation of intradermal and serologic test for diagnosis of hydatid disease. Am. J. TrOp. Med. Hyg. 15; 172-179. Kwa, B. H., and Liew, F. Y. 1977. Immunity to taeniasis—cysticercosis. I. Vaccination against Taenia taeniaefbrmis in rats using purified antigen. J. exp. Med. 146: 118-131. 129 Leid, R. W., and Williams, J. F. 1974. Immunological response of the rat to infection with Taenia taeniaeformis. I. Immunoglobulin classes involved in passive transfer of resistance. Immunology 22; 195-208. Miller, H. M. Jr. 1931. Immunity of the albino rat to superinfection with Cysticercus fasciolaris. J. Preventive Med. 5; 453-464. Mitchell, G. F., Goding, J. W., and Rickard, M. D. 1977. Studies on immune responses to larval cestodes in mice. Increased suscep- tibility of certain mouse strains and hypothymic mice to Taenia taeniaefOrmis and analysis of passive transfer of resistance with serum. Aust. J. exp. Biol. Med. Sci. 55; 165-186. Musoke, A. J., and Williams, J. F. 1975. The immunological response of the rat to infection with Taenia taeniaefbrmis. Sequence of appearance of protective immunoglobulins and the mechanism of actiOn of 7872a antibodies. Immunology 22; 855-865. Nemeth, I. 1970. Immunological study of rabbit cysticercosis. II. Transfer of immunity to Cysticercus pisiformis with parenterally administered immune serum or lymphoid cells. Acta vet. Acad. Sci. Hung. _2_(_)_: 69-79. Rickard, M. D. 1974. Hypothesis for the long term survival of Taenia pisifbrmis cysticerci in rabbits. Zeitschrift ffir Parasitenkunde 45; 203-209. Rickard, M. D., and Katiyar, J. C. 1976. Partial purification of antigens collected during in vitro cultivation of the larval stages of Taenia pisiformis. Parasitology 11; 269-279. 130 Rickard, M. D., and Outteridge, P. M. 1974. Antibody and cell- mediated immunity in rabbits infected with the larval stages of Taenia pisiformis. Zeitschrift fur Parasitenkunde 44; 187-201. Strober, S. 1976. Maturation of B lymphocytes in rats. III. Two subpopulations of memory B cells in the thoracic duct lymph differ by size, turnover rate and surface immunoglobulins. J. Immunol. 112; 1288-1294. Trizio, D., and Cudkowicz. 1974. Separation of T and B lymphocytes by nylon wool columns: Evaluation of efficacy by functional assays in vivo. J. Immunol. 113: 1093-1097. VI TA VITA Roger Wallace Cook was born in North Sydney, Australia, on February 4, 1945. He attended Willoughby Public Primary School and North Sydney Boys' High School and received his Bachelor of Veterinary Science degree from The University of Sydney in January 1967. Following graduation, he worked as a field veterinary officer with the Department of Agriculture at Goulburn, in the Southern Tablelands of New South Wales. In March 1972 he began work as a graduate assistant in veterinary clinical pathology at Michigan State University, where he completed a Master of Science degree in December 1974, on the subject "Iron Toxicosis in the Young Pig." Since then he has worked in association with Dr. Jeffrey Williams in the area of immunOparasitology. He married Ruth McKay on October 19, 1968, and their daughter Amanda Michelle was born in East Lansing, Michigan, on June 21, 1973. 131 ABSTRACT PATHOLOGY OF TAENIA TAENIAEFORMIS INFECTION IN THE RAT By Roger W. Cook Sequential morphologic changes were observed in female Spartan rats infected orally with 1000 eggs of Taenia taeniaefbrmis. A minimal amount of hepatic migration was indicated by microscopic tracks of necrosis adjacent to the parasite 4 to 10 days after infec- tion (DAI). Serum levels of alanine aminotransferase and sorbitol dehydrogenase rose sharply 6 to 7 DAI, when necrotic tracks were most obvious and before a distinctive fibroblastic reaction developed around the parasite at about 10 DAI. Enzyme levels peaked again 13 to 14 DAI. There was enlargement of hepatic lymph nodes with marked medullary plasmacytosis, which was also observed within mediastinal and mesenteric nodes. Splenomegaly was associated with expansion of the red pulp with myelopoiesis and plasmacytosis. Thymic atrophy was observed microsc0pically from 44 DAI and grossly from 52 DAI. Increases in size of stomach and small intestine, due to mucosal hyperplasia, occurred earlier and were most marked in rats with the heaviest hepatic metacestode infections. Stomach weight increased up to 20-fold. Irregular proliferation of mucous epithelium and stroma of the lamina propria produced dramatic papillary and cystic thickening of the gastric mucosa with excessive mucus production. Roger W. Cook Weight and mucosal thickness of the small intestine doubled with little change in architecture or villus-crypt ratio of 2:1. Intes- tinal mast cell numbers increased throughout the observation period and were highest in the duodenum of heavily infected rats. In contrast, intestinal eosinophil numbers reached a peak between 30 and 40 DAI and then declined, in parallel with peripheral blood eosinophil counts. Female Fischer 344 strain rats infected for 5 days with 2000 eggs of Taenia taeniaefbrmis were joined in parabiosis to syngeneic non-infected partners. Hyperplastic gastropathy deve10ped in both rats of the parabiotic pairs, indicating transfer of the stimulus for gastric hyperplasia to the non-infected partners. Duodenal masto- cytosis developed in both partners. Intact infected female Sprague-Dawley rats with hyperplastic gastropathy had markedly elevated serum levels of the gastrointestinal hormone, gastrin. When rats were antrectomized soon after infection, gastric hyperplasia still deve10ped but hypergastrinemia was prevented or was of only moderate degree. Hypergastrinemia was therefore not considered the primary stimulus for gastric hyperplasia. The increase in pH within the lumen of hyperplastic stomachs probably contributed to the elevation of serum gastrin levels.