Regulation of immunity to Ostertagia ostertagi

Veterinary Parasitology, 46 (1993) 63-79 Elsevier Science Publishers B.V., Amsterdam

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Regulation of immunity to Ostertagia ostertagi Phillip H. Klesius USDA, ARS, Animal Parasite Research Laboratory, P.O. Box 952, Auburn, AL 36830-0952, USA

ABSTRACT Klesius, P.H., 1993. Regulation of immunity to Ostertagia ostertagi. Vet. ParasitoL, 46: 63-79. Knowledge of bovine immune response to ostertagiasis is important to understanding the mechanisms of innate and acquired immunity to this economically important helminth parasite that infects cattle worldwide. Infection causes both antibody and cellular immune responses. Evidence shows that Ostertagia possesses excretory-secretory (ES) molecules that may regulate immune cell responses that affect acquired immunity and pathophysiological changes to infection. Ostertagia can down-regulate antibody and cellular immune responses. One of these ES regulatory molecules is a lectin that causes eosinophil chemotaxis. In addition to its antigenicity, this regulatory molecule serves as a means of communication between the parasite and cells of the host immune system. It is suggested that, lacking this type of communication, Ostertagia infection may not be readily recognized by the host immune cells. A hypothesis is proposed for the mechanisms of acquired immunity to Type I ostertagiasis. Regulatory molecules of Ostertagia ES are suggested as suitable vaccine candidates.

INTRODUCTION

It is now widely accepted that acquired immunity to ostertagiasis develops slowly and is most evident after the second grazing season or experimentally, after prolonged or repeated infections (Michel et al., 1973; Holtenius et al., 1983; Entrocasso et al., 1986; review by Klesius, 1988 ). Herlich and Tromba (1982 ) found no evidence that single monospecific experimental infection provided protection against subsequent challenge infection with Ostertagia ostertagi. The major manifestations of acquired immunity are reduced pathophysiological reactions, worm burdens, egg output, size of adult worms and absence of vulval flaps (reviewed by Klesius, 1988). These manifestations are less evident in younger cattle. Infection with Ostertagia ostertagi causes both antibody and cellular immunity responses (Klesius, 1988 ). The survival of Ostertagia ostertagi is presumably facilitated by modulation of the host immune system. Evidence is presented that Ostertagia ostertagi possesses excretory-secretory (ES) subCorrespondence to: P.H. Klesius, USDA, ARS, Animal Parasite Research Laboratory, P.O. Box 952, Auburn, AL 36830-0952, USA.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4017/93/$06.00

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stances that can communicate with host immune cells and their accessory cells to regulate their activity independent of host-derived cytokines and other regulatory molecules. Host immune cells respond to Ostertagia mediators as they would to host mediators because the former mimic the latter in their ability to be recognized by host cell receptors. The commonality of these mediators and their complementary cellular receptors allows this helminth to communicate with the host. It is possible that, lacking this means of communication, the host would not recognize the presence of the parasite except by stimulation of its immune system by structural and metabolic (ES) antigens. This antigen-independent Ostertagia-host cell communication network may open new approaches to the development of vaccines that would disrupt ES mediators that regulate immune cell function. This type of vaccine would not stimulate 'sterile immunity', but would negate the adverse pathophysiological changes caused by host cells in response to Ostertagia mediators. This different vaccine approach is suggested because attempts to produce 'sterile' vaccines with exoantigens, cultured larval stages (Herlich and Douvres, 1979), X-ray (Burger et al., 1968; Armour, 1976) and ultraviolet attenuated larvae (Herlich and Tromba, 1982), and infectious larvae administered intravenously (Williams et al., 1974) have not been successful. In addition to the small numbers of immunogens in third-stage larval (L 3 ) extracts and irradiated larvae (Mansour et al., 1990), other reasons for these failures may include low concentrations or absence of specific protective antigens in fourthstage larval (L4) and adult extracts, non-enrichment of ES regulatory molecules and non-use of adjuvants. This article describes the immune responses to ostertagiasis and the role Ostertagia mediators play in these responses through their regulatory effects on host immune cells. Further, our current understanding of Ostertagia ostertagi antigens is reviewed. ANTIGENS

Limited information is available on the nature of Ostertagia ostertagi antigens. Antigens are both structural and metabolic substances derived from parasitic larval and adult stages. No information is available on antigens from eggs or free-living first (Ll) and second (L2) larval stages. Most antigens from Ostertagia stages are cross-reactive with other gastrointestinal parasites of cattle; however, Ostertagia-specific antigens do exist, but are poorly defined. There is a lack of solid evidence of species-specific antigens; however, extracts of L3, L4 and adults are the best described antigen sources. Predominant antigens from L3 extracts of Cooperia spp. (Keus et al., 1981; Kloosterman et al., 1984a), Dictyocaulus viviparus, Haemonchus contortus and Trichostrongylus retortaeformis (Sinclair, 1964) cross-react with Ostertagia ostertagi. Antigen extracts from either Ostertagia ostertagi or Cooperia oncophora adult

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worms cross-react with antibodies in cattle sera infected with Ostertagia ostertagi and C. oncophora (Kloosterman et al., 1984a). Previously, genus specificity was reported between Ostertagia ostertagi and Cooperia sp. L4 antigens, but not between their L 3 antigens (Keus et al., 1981 ). However, Mansour et al. (1990) showed that Ostertagia ostertagi L 3 possess two specific antigens with molecular masses of 17 and 43 kDa under reducing conditions using Type I ostertagiasis sera. Two cross-reacting antigens of 67 and 81 kDa were also identified. These two specific 17 and 43 kDa antigens reacted with both pre- and post-infection sera and it was proposed that this cross-reactivity may be due to a naturally occurring gut antigen. Extract fractionation by Sepharose 12 gel filtration showed nine peaks, while DEAE ion-exchange chromatography yielded six peaks. The antigens in these peaks stimulated antibody and lymphocyte recognition and were most likely cross-reactive. Canals and Gasbarre (1990) characterized two somatic antigens from adults, one L4 molting antigen and one La/adult ES antigen for their role in immunogenicity and specificity for Type I ostertagiasis. The adult somatic antigens had the smallest number of antigens and the weakest reactivity in Type I infections. These antigens cross-reacted with antibodies from Oesophagostomum radiatum infections. The antigen from molting L4 contained components with molecular masses from 16 to 22 kDa that reacted with sera from infected animals, and some of these molting antigens were stage-specific. The ES antigens with molecular masses from 16 to 22 kDa had the strongest specific reactivity for Ostertagia ostertagi antibodies. In the only study using sera from pre-Type II cattle, Cross et al. (1988) detected seven ES antigens from L 3 which cross-reacted with Fasioloides magna infected sera from cattle. Fasciola hepatica and C. oncophora infected cattle sera reacted with two antigens present in the ES. A 32 kDa ES antigen had the highest level of species specificity. The adult antigen extract contained about 27 antigens, none of which showed specificity when tested with sera from cattle infected with Fascioloides magna, C. oncophora and Fasciola

hepatica. Lymphocyte proliferative responses to Ostertagia antigens also lack species specificity. Klesius et al. ( 1984) showed that antigens of L3 stimulated proliferation of lymphocytes from calves infected with Ostertagia ostertagi or Cooperia punctata. Dermal hypersensitivity responses to L3 antigens showed non-specific reactivity (Snider et al., 1985 ). It is apparent that without purification, cross-reactive antigens are present in crude structural and metabolic antigen preparations. The antibody and cellular responses to Ostertagia ostertagi reported to date are not species-specific. There is a strong need to find species- and stage-specific antigens for the characterization of immune responses against both types of ostertagiasis.

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A C Q U I R E D A N T I B O D Y RESPONSES

Most evidence strongly suggests that antibody responses develop slowly against a variety of somatic and metabolic antigens from different stages of Ostertagia ostertagi (reviewed by Klesius, 1988). Cattle respond with relatively low serum antibody levels (Keus et al., 1981; Klesius et al., 1986a). Sequential infections induce serum IgG, IgM and IgA antibody responses to L 3 antigens in calves (Klesius et al., 1986a). The IgG antibody response to L3 increases slowly with multiple monospecific infections. An anamnestic IgG antibody response occurs after challenge infection. The IgM and IgA antibody responses are weaker and less pronounced in comparison with IgG responses. Jensen and Nansen ( 1978 ) showed that immunoglobulin levels were significantly different between calves with heavy and light Ostertagia ostertagi infections. The IgG 1, IgM and IgA levels were significantly greater in the heavily infected animals. The study concluded that increased immunoglobulin levels other than IgG2 and decreased serum albumin occur in calves that acquired ostertagiasis from grazing. The greatest antibody responses appear to be stimulated by the development of t 4 to adult worms (Kloosterman et al., 1984b; Entrocasso et al., 1986). Mansour et al. (1990) demonstrated that infections induced IgG1 antibody responses to two distinct L 3 antigens. IgG 1 was the predominant reactive subclass in calves that were sequentially infected either two or three times. Canals and Gasbarre (1990) also reported that the predominant reactive subclass was IgG 1 to structural and metabolic antigens. The kinetics of the IgG1 responses showed a peak at 35 days post-infection and then an anamnestic response at 65-77 days post-challenge infection. Serum IgM and IgG2 responses were not detected. The kinetics of the serum IgA antibody response were similar to IgG 1, but levels were lower. In a study by Thatcher et al. ( 1989 ), serum IgE levels increased in grazing cattle infected with both Ostertagia ostertagi and Cooperia sp.; however, the IgE decreased with increasing worm burdens. An association between worm burden and seasonal change to total IgE responses was found in this study, although no relationship was found between acquired IgE antibody and protection against Ostertagia ostertagi. The results of a more recent study on the nature of specific IgE responses to Ostertagia ostertagi and their relationship to acquired antibody immunity are presented elsewhere in this issue (see Baker and Gershwin, pp. 93-102 ). Kloosterman et al. (1984a) reported that calves with antibody titers against Ostertagia ostertagi and C. oncophora acquired from either monospecific or mixed infections had female worms with smaller vulval flaps after challenge infection. After primary infections, calves with high antibody titers had fewer and shorter worms with less ova per female and smaller vulval flaps. The study concluded that a negative interaction, mediated by the immune response, oc-

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curs between these two species. Cross-reactivity of antigens shared between the two species was believed to be responsible for this acquired immunity. Antibody levels to Ostertagia ostertagi were measured in cattle over two grazing seasons using adult worm extracts to detect the antibody responses (Entrocasso et al., 1986). The results showed that adult worms caused marked increases in antibody levels. Adult worm stimulation of antibody responses occurred after heavy infection or the development of arrested L4 in these animals. Antibody levels dropped when the animals were housed between the two grazing seasons. The study reported that the cattle had acquired immunity at the end of the second grazing season. However, these calves had relatively high concentrations of antibodies to adult antigens at the beginning of the first grazing season. The study reported that the calves were reared indoors under helminth-free conditions before their first grazing season. No explanations were given for their high antibody titers. Antibody responses to adult Ostertagia ostertagi extracts were found to have a significant positive correlation with age-adjusted body weights at the end of the second grazing season (Ploeger et al., 1990). This suggested that antibody responses initiated in the first grazing season had a positive effect on the yearlings' growth performance in the second grazing season, but these antibodies did not correlate with reduced weight gains caused by parasitic infection in the second grazing season. In these studies, the cattle were housed between grazing seasons. These and other findings of Ploeger are presented in more detail elsewhere in this issue (see Ploeger and Kloosterman, pp. 223-241 ). In summary, because of conflicting findings, additional research is needed to provide more information on the kinetics and the role of antibodies in acquired immunity against Ostertagia ostertagi, especially in ostertagiasis acquired by grazing cattle. Passive transfer of immune antibody or a specific antibody isotype may be the only means to demonstrate the nature and role of antibodies to the observed anti-parasitic effects. The nature of the antigen would also be learned, if passive antibody transfer experiments were successful, thus opening the door to the development of vaccines. ACQUIREDCELLULARRESPONSES Type I ostertagiasis causes inflammatory lesions in the abomasum (Osborne et al., 1960). The eosinophil is one of the predominant cell types present in this inflammatory response (Ross and Dow, 1964; Ritchie et al., 1966; Snider et al., 1988). Lymphocyte, globule leukocyte and plasma cell accumulations also occur in cattle with Type I infections (Ritchie et al., 1966), along with elevated levels of blood eosinophils (Wiggin and Gibbs, 1987). Although defining the exact role of the eosinophil in pathology is difficult, evidence suggests there is a strong connection between pathophysiological changes and eosinophil accumulations in ostertagiasis. Eosinophils are effec-

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tor cells in acquired immune responses to parasites; Grove et al. (1977) showed that infectious larvae of Trichinella spiralis are killed by eosinophils in the presence of antibody and complement. Washburn ( 1984 ) showed that eosinophils bind to infectious larvae of Ostertagia ostertagi; however, it was not determined whether these larvae were damaged by the eosinophils. Mast cells and eosinophils are closely associated in Type I hypersensitivity reactions in that mast cells possess eosinophil mediators and histamine, while eosinophils possess inactivators of mediators from mast cells (Wasserman, 1979). Hypersensitivity is most likely the nature of the cellular response to Type I ostertagiasis (Snider et al., 1985; Wiggin and Gibbs, 1987, 1990; Cross et al., 1987; Klesius, 1988 ). Soluble extracts from L 3 of Ostertagia ostertagi show Type II hypersensitivity reactions in the dermis of Ostertagia ostertagi infected calves, but not in Cooperia infected calves (Snider et al., 1985 ). Type I hypersensitivity reactions occur in helminth-free and Ostertagia ostertagi infected calves, but the greatest intensity occurs in infected calves (Cross et al., 1987 ). Infected calves showed the greatest number of infiltrating eosinophils which occurred within 4 h of antigen challenge. Mast cell numbers decrease in infected calves in response to antigen challenge. Basophil numbers increase in the antigen challenge sites of infected calves. Perivascular accumulation of mononuclear and some polymorphonuclear cells in the deep dermis of infected animals was observed (Cross et al., 1987 ). Wiggin and Gibbs (1990) showed evidence of Type I hypersensitivity reactions in the abomasal mucosa caused by soluble L 3 products of Ostertagia ostertagi. Infected animals became sensitized and the number of globule leukocytes and mast cells were increased in their abomasal mucosa, and furthermore the eosinophil counts were highest in the infected animals. This hypersensitive reaction was associated with adverse immune reactions and pathogenesis of Type I ostertagiasis. Wiggin and Gibbs ( 1987 ) demonstrated dermal hypersensitivity to larval antigens in infected calves. The infected animals had mucosal changes consistent with hypersensitive reactions. It may follow that the cellular responses cause mucosal hypersensitivity reactions that are responsible in part for the pathogenesis and initiation of acquired immunity to ostertagiasis. Evidence for this concept is provided by the reduced pathogenesis seen after sequential infections. Older hosts are less affected and their adult Ostertagia ostertagi cause greater damage. Additional experimental evidence is needed to support this concept. In summary, evidence to date strongly suggests that acquired immunity is a combination of both antibody and cellular reactions because antibodies specific for somatic and metabolic Ostertagia ostertagi antigens are produced and cellular responses associated with hypersensitivity are initiated by the same antigens, especially metabolic antigens. Passive immune cell transfer experiments are needed to better define the nature and role of antibodies and immune cells in acquired immunity.

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REGULATION OF ACQUIRED IMMUNITY

Immune responses are regulated by Ostertagia ostertagi. Their are four sets of evidence for this: ( 1 ) parasite-mediated suppression of lymphocyte reactivity; (2) suppression of antibody production; (3) eosinophil chemotaxis; (4) lymphocyte proliferation. Kinetic studies of lymphocyte reactivity to L 3 antigen and phytohemagglutinin (PHA) show that responses are suppressed to both of these stimuli in calves experimentally or naturally infected with Ostertagia ostertagi (Klesius et al., 1984). The parasite-mediated immunosuppression was demonstrated to be non-specific and transient suppression of cellular responses that occur during the prepatent periods of Type I ostertagiasis (Klesius et al., 1984). The transient suppression began on day 9 and ended on day 14 post infection. This pattern was repeated with a subsequent challenge infection. The timing of the immunosuppression appeared to correlate with the development of L4 to young adults (Ritchie et al., 1966 ). Cross et al. (1986) reported Ostertagia-mediated suppression of lymphocyte responses to concanavalin A (Con A), but not to PHA. The Con A lymphocyte response was repeatedly suppressed by serial infections and was suppressed between days 6 and 20 post challenge with a single infection. A difference in the sensitivity of extended culture assays may explain why PHA suppression was not significant in this study in comparison with previous results (Klesius et al., 1984). PHA lymphocyte responses were found to be suppressed by Ostertagia ostertagi infections by both Snider et al. ( 1986 ) and Wiggin and Gibbs (1990). Evidence that Ostertagia mediates suppression of antibody responses is both indirect and direct. Indirect evidence is the slow development of specific antibodies in infected cattle. Prolonged or repeated exposure to Ostertagia ostertagi appears to be necessary for enhanced antibody responses to structural and metabolic antigens. Direct evidence emerges from the findings of Cross and Klesius (1989 ) that soluble extracts from L 3 suppress the antibody responses of mice immunized with Ostertagia ostertagi antigen and keyhole limpet hemocyanin (KLH) or sheep erythrocytes (SR). A distinct fraction of the L 3 extract suppressed KLH antibody response and anti-SR IgM-secreting B cells; this fraction also depressed Con A responses of mouse splenic lymphocytes in vitro. Mansour et al. ( 1991 ) showed that IgG1 responses to Ostertagia ostertagi L 3 antigen were significantly greater from 6 weeks postinfection in Ostertagia ostertagi infected calves on a low protein and energy diet than in infected calves on a high protein and energy diet. The IgG 1 responses to KLH were decreased by infection and the low protein and energy diet compared with the high protein and energy diet. A single infection was used in this study. Washburn and Klesius (1984) showed that L 3 and their soluble extracts were chemokinetic for bovine eosinophils. In a later study (Klesius et al.,

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1985 ), the soluble extracts were shown to be chemotactic for eosinophils but not neutrophils in vitro and in vivo. Briefly, to identify Ostertagia substances that might contribute to the attraction and accumulation of eosinophils, chemotactic experiments were done in blind well chambers. A blind well chamber consists of two compartments separated by a 5/tm pore membrane filter. Eosinophils are placed in the uppermost compartment and their movement toward the lowermost compartment is measured by their accumulation on the underside of the filter. The number of eosinophils counted on the filter lower surface is used to determine the degree of attraction by Ostertagia substances. These substances are placed in various concentrations in the lowermost compartment, uppermost compartment or both compartments to determine their chemotactic properties for eosinophils. Typically, if the substance is placed in either the upper or the lower compartment and the eosinophils accumulate on the lower surface of the filter in greater numbers than in the complete absence of the substance then it would show that the substance is chemokinetic. If no migration occurs at equal concentrations in both compartments and migration occurs only when the concentration is higher in the lowermost compartment, then the results show that the substance is chemotactic. There is a checkerboard titration where a chemotactic gradient is established. To provide evidence that eosinophil chemotaxis to Ostertagia substances is dependent on its interaction with sugar receptors of the eosinophil ligands, various sugars were examined for their ability to inhibit the eosinophil chemotactic response to Ostertagia ES (Klesius, 1991 ). This was done by preincubating the eosinophils with a sugar at various concentrations for 30 min before measuring their chemotactic response. The sugar that caused the greatest inhibition of chemotaxis at the lowest concentration was identified as the sugar receptor of the eosinophil ligand. Table l shows eosinophil chemotactic activity in vitro. A ct'leckerboard titration of the eosinophil chemotactic factor (ECF) showed that eosinophil TABLE 1 Effect of varying the concentration gradient of ECF on eosinophil migration. Results are expressed as mean numbers of eosinophils for duplicate filters each read for 20 fields on high power (magnification X 1000) Concentration of ECF in lower compartment

Concentration of ECF in upper compartment (%)

(%)

0

20

40

60

0 20 40 60

1.7 3.0 9.4 8.4

1.2 4.3 3.3 8.7

5.0 6.4 5.7 11.1

2.0 2.9 5.3 2.5

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migration occurred only when the ECF was at a greater concentration in the lower compartment of the blind well chemotactic chamber. When equal concentrations of the ECF were present in the lower and upper compartments of the blind well chamber, no migration above the background level was found. Horton ( 1985 ) showed that ECF reacted directly with the eosinophil. In studies of the in vivo activity of ECF, eosinophil peak tissue accumulations could be found at 4 and 48 h after intradermal injection of ECF (Table 2 ). The ECF causes little neutrophil migration to the dermal sites. This ECF is an ES product of Ostertagia ostertagi larvae and adults (Klesius et al., 1986b). The relative concentration of ECF in ES is unknown. The ECF is localized predominantly in the intestinal cell and lateral hypodermal cords of d e v e l o p i n g t 4 larvae and is probably released by developing larvae into the abomasal tissue surrounding the parasitized gastric gland (Klesius et al., 1989 ). Illustrated in Fig. 1 is a cross-section of Ostertagia ostertagi L4 larvae stained using anti-ECF followed by avidin-biotin-peroxidase complex. The arrow shows the stained intestinal cells within the nematode. Results of recent studies show that the ECF is a lectin that appears to recognize a complementary surface receptor present on eosinophils that regulates cellular movement. After lectin-glycoconjugate reactions, the eosinophils migrate towards the positive ECF lectin gradient. These results strongly suggest that Ostertagia-secreted ECF lectin is responsible for the accumulation of eosinophils in the abomasal tissues of infected cattle. Table 3 shows the results of sugar inhibition of eosinophil chemotaxis mediated by ECF lectin. The parasite lectin is most specific for fucose (P.H. Klesius, unpublished data, 1991 ). Figure 2 illustrates how Ostertagia initiates eosinophil chemotaxis. The parasite secretes ES substances that contain the ECF lectin. The ECF lectin reacts with the fucose surface receptor. The activated cell surface receptor serves to alert the eosinophil of the presence of the parasite and the eosinophil TABLE 2 In vivo chemotactic activity of ECF for eosinophils. Results are expressed as mean numbers of eosinophils accumulating after intradermal injection of ECF. A total of 20 high-power fields were counted for eosinophils Time

Chemotactic activity

(h) 0.25 1

4 8 12 24 48

ECF

Saline

0.3 0.8 6.5 3.7 0.8 3.5 15.8

0.3 0.2 1.7 0.3 0.6 0.2 0.5

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Fig. 1. Cross-section (magnification X 300 ) of Ostertagia ostertagi L 4 larvae stained using antiES antibody followed by avidin-biotin-peroxidase complex (anti-mouse Ig) and 4-chloro-lnapthol substrate for color development. Arrow indicates stained intestinal cell within nematode. TABLE 3 Effect of sugars on eosinophil chemotaxis to ECF

Sugar

Percentage inhibition of chemotaxis

D-glucose D-galactose D-mannose N-acetyl galactosamine o-fucose L-fucose

12 2 10 2 70 65

responds by migrating towards the greatest concentration of the ECF-lectin (chemotaxis). The eosinophil and parasite cell-to-cell interactions continue with the eosinophil in close proximity to the parasitized gastric gland. Marom and Casale (1983 ) found that activation of mast cells causes the release of substances which include ECF because larval substances provoke Type I hypersensitivity reactions involving mast cells; mast cell derived ECF would also play a role in the accumulation of eosinophils at the site of infection and inflammation. Production of cytokines which regulate eosinophil

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Ostertagia regulation of chemotaxis Receptors

I'a

Fucose

ECF-lectin

Cell barrier

.,.O

Eosinophil Chemol

Parasitized gastric gland Fig. 2. Scheme for the regulation of eosinophil chemotaxis by ECF lectin secreted by Ostertagia

ostertagi.

migration is most likely stimulated by Ostertagia secretions while the larvae are developing in the gastric glands. This activity would also cause eosinophil accumulation and activation in infected tissue. Evidence that Ostertagia mediates lymphocyte blastogenesis includes reports of in vitro antigen-induced lymphocyte proliferation and enlargement of lymph nodes regional to the abomasum in infected animals. Larval extracts stimulated blastogenic responses of lymphocytes from uninfected calves (Cross et al., 1986; Wiggin and Gibbs, 1990). Wiggin and Gibbs (1990) further confirmed the finding reported by Curtain and Anderson ( 1971 ) and Gasbarre ( 1986 ) that the abomasal lymph nodes from the fundic and pyloric regions of the abomasum are enlarged with increased mitotic activity in the germinal centers in parasitized versus uninfected calves. Lymphocyte responses to PHA were not suppressed as was previously reported by Gasbarre (1986) when lymphocytes were tested from the enlarged lymph nodes of infected calves. These results show that Ostertagia can stimulate non-specific lymphocyte proliferative responses. MECHANISMS OF OSTERTAGIA R E G U L A T I O N OF I M M U N E RESPONSES

The molecular relationship between Ostertagia ostertagi and its host is unique because the parasite is sequestered in a partial immunologically priv-

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ileged host site, where a cellular barrier exists between the developing larvae and the cells of the immune system. Recognition is a central event in biology and the first step in the processes of parasite-host relationships, which require intercellular communication. A variety of molecules are excreted or secreted by helminths into host tissue (Stirewalt, 1963 ). Some of the ES molecules, in addition to being antigens, are regulatory molecules that cause a variety of cellular responses by host cells (Lightowlers and Rickard, 1988 ). For additional information on how helminths other than Ostertagia affect the immune responses of a variety of hosts, the reader should refer to the review of Lightowlers and Rickard ( 1988 ). The results summarized here show that Ostertagia possesses ES molecules that are both antigens and regulators of the bovine immune system. There are strong indications that some of these ES regulator molecules are lectins - - a class of proteins of non-immunological origin that bind carbohydrates specifically and non-covalently. The lectin specifically recognizes glycoconjugate receptors of cells and forms a complementary bond that transfers information between the cells. More information on the properties and activities of various lectins are found in Hedo (1984), Lis and Sharon (1986) and Sharon and Lis ( 1972, 1989). These lectins, depending on the cell type that recognizes them, may cause a variety of cellular responses associated with ostertagiasis and acquired immunity. The lymphoproliferative responses associated with ostertagiasis (Snider et al., 1988 ) may thus be caused by Ostertagia lectins with properties similar to Con A or PHA that induce non-specific lymphocyte proliferation. In addition to antigenic recognition by the host immune system, these lectins constitute part of the parasite-immune cell communication network, similar to the cytokine network (Beck and Habicht, 1991 ) that the host uses for cellcell communication in regulation of its immune system. Lev et al. (1986) showed that Giardia lamblia secretes lectin that is activated by a host protease. The lectin is specific for mannose-6-phosphate that is the sugar component of the complementary cell membrane receptor. The parasite-cell interaction is mediated by this host-activated parasite lectin, resuiting in an affinity of the protozoan for the infection site. This is most likely the molecular basis of specific adherence of Giardia to host cells; however, host cell responses to this lectin were not examined in the study. Considerable evidence is presented from research with other parasites that supports some of the concepts made here on how Ostertagia regulates immunopathology and acquired immunity by its ES products, including lectins. A PROPOSED MECHANISM OF ACQUIRED IMMUNITY

The first step of acquired immunity may thus be the production of specific antibodies and their 'neutralization' that blocks the parasite ES lectin net-

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Infections Parasite regulatory molecules

Eosinophil

M~on ocy tes/mast cells

n s'" it ivitv~t~ Type l h y p e ~se "y ---~Down~e gulated cytok,nes " ~•

Suppressed antibody/cellular immune responses

Pathophysiological tissue and parasite damage

~

Enhanced antigen presentation Up regulation by cytokines?

~/J

Antibodies to regulatory molecules ~ \ \~

// ~j/

Acqmred immunity Fig. 3. Schematic presentation of acquired immunity to ostertagiasis.

work, and thus prevents the adverse cellular hypersensitivity of ostertagiasis. The second step may be the production and action of specific antibody on the parasite itself, causing adverse reproductive changes in the female worms. The sequence of events (Fig. 3 ) leading to acquired immunity is slow because it requires prolonged infections. The initial Ostertagia ostertagi infection resuits in hypersensitivity responses mediated by immune ceils regulated by ES products, while the end of infection terminates ES cell communication and there is a return to normal abomasal function. The adverse hypersensitivity reactions destroy some of the parasitized gastric glands and cause the release of structural antigens from damaged L4 and L5 larvae, as well as ES products which stimulate specific antibody production. In subsequent infections, the adverse hypersensitivity is down-regulated by specific antibodies that block ES regulation of cellular responses. Host cytokines regain their regulation of immune cell functions that the Ostertagia ES regulators had competed for when they were present in higher concentrations after infection. Specific antibodies and cytokines cause larval damage that results in a loss of reproduction and acquired immunity is partially achieved. This acquired immunity is not 'sterile', but it lessens the parasite burden and regulation of host cell function and allows normal abomasal function and growth of the animal. CONCLUSIONS

Efforts to develop a hypothesis of acquired immunity requires a compromise between simplification and inclusion of most of the evidence of host

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immune responses to Ostertagia ostertagi. Alternative hypotheses to explain acquired immunity are also possible. This hypothesis attempts to give a molecular basis for a complex parasite-host relationship, and may be summarized as follows. Ostertagia produces regulatory molecules that are secreted into the surrounding tissues of the gastric glands. These regulatory molecules regulate host cellular responses, especially those of the immune system. The immune responses to Ostertagia ostertagi infection are most likely suppressed by the action of these regulatory ES molecules. Importantly, ostertagiasis may also cause depression of immune responses to cattle vaccines, especially for animals on poor protein diets (Mansour et al., 1991 ). These parasite-derived molecules are both antigenic and regulatory. The slow development of acquired immunity may be related to the relationships between the parasite regulatory molecules and the host cytokine network. In addition, antibody responses against the parasite regulatory molecules may act to inhibit this cellular communication network and allow acquired immunity to further develop in the older animals after prolonged exposures. A successful vaccine should therefore be composed of immunogenic peptides representing selected regulatory molecules. It is important to identify these regulatory molecules recognized by host cells and then to use them to induce protective immune responses. Young calves may have passively acquired antibodies and immunization should not be attempted with anti-regulatory vaccines until these antibodies are no longer present. The best criterion of successful vaccination should be the absence of clinical ostertagiasis and not an immediate effect on the parasite life cycle. Subsequent pasture exposure would be needed to stimulate acquired immune responses to affect Ostertagia fecundity. Ostertagia ES products offer promise as an ostertagiasis vaccine. A successful ostertagiasis vaccine would be the most cost-effective method for providing protective immune responses to primary and secondary infections of grazing cattle, especially in their first and second grazing seasons. A measure of this acquired immunity may be high antibody titers to ES regulatory molecules. ACKNOWLEDGEMENT

The author thanks Kathy Johnson for manuscript preparation.

REFERENCES Armour, J.A., 1976. Ostertagia ostertagi infections in the bovine: Field and experimental studies. Ph.D. Thesis, University of Glasgow. Beck, G. and Habicht, G.S., 1991. Primitive cytokines: Harbingers of vertebrate defense. Immunol. Today, 12: 180-183. Burger, H.J., Eckert, J., Chevalier, H.J., Rahman, M.S.A. and Konigsmann, G., 1968. Parasitic

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