Mechanistic specificity of various anti-parasitics

The immunorestorative or immunopotentiating effects of various anti-parasitics may be occur via the various mechanisms illustrated in Table 1.

Levamisole

Levamisole (6-phenyl 2,3,5,6-tetrahydroimidazo (2,1-b) thiazole hydrochloride), a levo-isomer of tetramisole and member of thioimidazoles, was initially developed as an anthelmintic (Thienpont et al. 1966). It has been used for many years for the treatment of gastrointestinal nematodes and lungworms. Later, the drug received considerable attention as an immunomodulator (Renoux and Renoux, 1974; Jansen, 1976; Symoens and Rosenthal, 1977; Symoens, Cree and Van Bever, 1979; Brunner and Muscoplat, 1980; Lowe, 1980; Webster, 1985). It was the first chemically defined agent shown to have an immunomodulatory effect (Renoux, 1986).

Levamisole modulated the immune function at a dose of 2–5 mg kg⁻¹ body weight (Rojas et al. 1976; Brunner and Muscoplat, 1980; Babiuk and Misra, 1981) or a total dose of 150 mg day⁻¹ for a week in mice (Renoux and Renoux, 1977). Intermittent treatment was reported to be more effective than continuous treatment in restoring the immune response of bovines (Lejan and Asso, 1981). In vitro studies have demonstrated that various concentrations of levamisole increased the blastogenic activity of bovine lymphocytes stimulated with pokeweed mitogen (PWM) (Babiuk and Misra, 1981). Levamisole has got a beneficial effect due to an immunopotentiating activity in cows during the dry period by reducing the incidence of mastitis from 9·6% to 3·7% (Flesh et al. 1982). According to Alex, Alikutty and Mathew (1995), levamisole failed to control mastitis in cows when administered during the dry period. Levamisole, when administered in combination with canine parvovirus (CPV) vaccine, enhanced antibody production against parvo-virus, increased phagocytic activity and stimulated the proliferation activity of lymphocytes (Mojzisova et al. 2004). It was also shown that, in vitro, levamisole potentiated Brucella abortus-induced blastogenesis in lymphocytes from sterile cattle (Kaneene et al. 1981). Antibody titres in heifers given levamisole and Brucella abortus strain 19 vaccine were moderately higher than those given vaccine only (Chukwu, 1985). Research on cattle and swine shows that levamisole can influence both the primary and secondary immune responses and the ability of lymphocytes to respond to mitogen (Kehrli and Roth, 1990; Quinn, 1990). In turkeys, levamisole has been reported to be an immunorestorative agent under immunosuppressive conditions caused by antibiotics or X-irradiation (Panigraphy et al. 1979).

There are contradictory reports concerning the effect of levamisole on serum immunoglobulin levels of chickens, some indicating decreased antibody titres after using levamisole with Newcastle disease virus (NDV) vaccination, while others reporting no effects on antibody production (Guerrero, 1977). Six-week-old birds subjected to levamisole hydrochloride (I/M) at the dose of 8 mg kg⁻¹ for 3 successive days before NDV (LaSota strain intraocular) vaccination showed a significant increase in the haemagglutination inhibition antibody titres after 1 week of vaccination (Hassan et al. 1989). The immunomodulatory effect of levamisole hydrochloride in Mycobacterium paratuberculosis-infected rabbits was demonstrated through leukocyte migration following levamisole treatment (Mondal et al. 1993). Giambrone and Klesius (1985) reported the effect of levamisole on the response of commercial broilers to coccidiosis vaccination and levamisole was found to improve the development of immunity to coccidiosis. It has been observed that immunity against Eimeria tenella developed in chickens treated with levamisole prior to infection within 2 weeks post-infection, reached its peak within 3–4 weeks and showed a gradual decline from the fifth week onwards (Rehman et al. 1989).

Levamisole is able to restore normal functions of effector cells of the cell-mediated immune response. Evidence is available that the maturation of granulocytes and functioning of T lymphocytes is induced in vivo but not in vitro. The potential effects of levamisole in combination with vaccination or the increase in resistance to viral challenge are rather variable depending on the method used for the evaluation of the trial (Lejan and Asso, 1981). In cattle experiments involving levamisole as immunomodulator with infectious bovine rhinotracheitis, Herpes, foot and mouth disease virus and brucellosis, favourable results have been reported (Schimied and Rosenbusch, 1973; Kaneene et al. 1981) and its use in cattle during the last stage of pregnancy resulted in the prevention or reduction of the disease conditions (Espinasse, 1980).

The effect of levamisole on cell-mediated immunity is mainly on anergic or depressed thymus-dependent lymphocytes, macrophages and polymorphonuclear leukocytes (Guerrero, 1977). Levamisole enhances macrophage and T-lymphocyte function and reduces suppressor T-cell function (Hersey, Ho and Werkmeister, 1981). Modulations of T-cell maturation as well as cytokine production are potential mechanisms. Both enhancement of the production of IL-1 in a macrophage cell line (Kimball et al. 1991) and a shift to Th1 cytokine production are the clues to its mode of action (Sceto, Gillespie and Mathieson, 2000).

Levamisole is a drug extensively used to boost immunity in a number of human diseases as well, including leprosy, some cancers (Sceto et al. 2000), Hodgkin's disease, rheumatoid arthritis, and in adjuvanted therapy of colorectal cancer (Chirigos, 1992; Kumran, 1993). Moreover, it has also been found to speed the recovery of malnourished children suffering from various infections (Prakash, Rao and Reddy, 1998). It also appears to possess immunomodulatory properties and be capable of disrupting the interaction of ferritin with T lymphocytes and, therefore, is therapeutically useful in conditions of ferritin excess, such as progressive human immunodeficiency virus (HIV) infection and its associated opportunistic complications (Wigginton, 1995).

Agencies worldwide, with the hopes of demonstrating anticancer activity, have sponsored numerous pre-clinical evaluations and clinical trials with levamisole in the cancer arena. Trials in advanced breast cancer, lung cancer, colorectal cancer, melanoma, and lymphoproliferative diseases have generally been negative or inconclusive (Stevenson et al. 1991). However, adjuvanted use of levamisole has shown to enhance delayed-type hypersensitivity (DTH) (Hadden, 1994); for example, it has a synergistic effect in conjunction with 5-fluorouracil (5-FU) in the post-surgical treatment of adenocarcinoma of the colon (Moertel, Fleming and Macdonald, 1990, 1995; Holcombe et al. 2001; Tall, Van Tinteren and Zoetmulder, 2001). Levamisole has been shown to increase natural killer (NK) cells and activated T-cells in this adjuvanted treatment (Holcombe, Li and Stewart, 1998). Later, the studies of Gwilt et al. (2000) concluded that there is no evidence that the pharmacokinetics of levamisole are altered by 5-FU administered immediately prior to levamisole administration and levamisole has been approved for use in patients with colon cancer.

Levamisole treatment has also been found to enhance protective antibody response to hepatitis B vaccination in haemodialysis patients (Kayatas, 2002). Usually, it is believed that levamisole does not affect B-lymphocytes directly, but can still influence humoral responses indirectly by affecting macrophages and T-lymphocytes (Pelletier, Willoughby and Giroud, 1978) and this immunopotentiation is pronounced in immunologically compromised hosts. In vivo studies on humans show that imidothiazoles might enhance the serum levels of thymic hormone-like factor (Hadden, 1994). However, in vitro studies on the immunological effects of levamisole showed that it is not a potent modulator of immune parameters including effects on monocyte and lymphocyte cytotoxicity, activation, proliferation, induction of cytokine-induced proteins, and the expression of tumor-associated antigens (Schiller et al. 1991).

Not all workers have been able to demonstrate that levamisole has an immunostimulatory effect. For example, Irwin et al. (1976) found that levamisole depressed the primary humoral response of calves vaccinated with infectious bovine rhinotracheitis virus. Further, Van Der Maaten et al. (1983) found that levamisole did not affect the virological and serological responses of bovine leukaemia virus-infected cattle and sheep. In contrast to the previously mentioned work of Chukwu (1985), Saini et al. (1991) found that levamisole treatment resulted in a significant decrease in agglutination antibody titre against Brucella abortus strain 19. Levamisole had no consistent enhancing effect on the blastogenic responses to Con A or PWM, in one-way mixed lymphocyte reaction or the antibody-dependent cellular cytotoxicity of lymphocytes from dexamethasone-treated cattle (Roth, Kaeberle and Hubbard, 1984).

Fenvalerate

Fenvalerate is a synthetic pyrethroid insecticide extensively used for the control of insect pests in agriculture and ectoparasites in veterinary practice. Oral administration of fenvalerate at a dose of 15 mg kg⁻¹ body weight daily for 270 days and vaccination of goats with Brucella abortis strain 19 after 90 days of dosing, resulted in the reduction of both the humoral and cell-mediated immune response as assessed by standard tube agglutination test and the delayed hypersensitivity test, respectively (Singh and Jha, 1996). Oral administration to sheep of fenvalerate at 1·25 ppm, along with other pesticides including lindane at 1·25 ppm, monocrotophos at 0·025 ppm and carbofuran at 2·5 ppm, resulted in suppression of cell-mediated immune response as indicated by significant reduction in the DTH reaction to dinitrofluorobenzene (DNFB) (Khurana, Mahipal and Chauhan, 1999). Immunosuppressive effects of the drug at 1·25 mg kg⁻¹ body weight were also observed against Brucella antigen in lambs (Khurana and Chauhan, 2000). Studies on the fenvalerate-induced cell mediated alterations in chicken showed that the birds fed with 20 ppm fenvalerate in feed daily for a period of 6 months resulted in significant reduction in total leukocyte count, absolute lymphocyte count and DTH reaction. This confirmed the suppression of CMI responses in fenvalerate-treated birds (Singh et al. 2001). A significant reduction of haemagglutination titres against sheep red blood cells (SRBC) and skin thickness in DTH response of fenvalerate-treated calves demonstrated that fenvalerate causes immunosuppression following a single administration (0·6% dermal spray) in buffalo calves (Singh et al. 2003). Fenvalerate at the dose rate of 1·25 mg kg⁻¹ body weight in lambs showed significant suppression in serum globulins, gammaglobulins and specific Brucella melitensis Rev. 1 antibodies in comparison to controls (Khurana and Chauhan, 2003).

Dieldrin, carbofuran and aminocarb

Dieldrin, a non-aromatic organo-chlorinated insecticide has been shown to be a potent immunomodulator. It was observed that cell-mediated immunity can be a potential target for adverse effects of this pesticide. Studies concerning the effects of agricultural pesticides on the immune system of northern leopard frog (Rana pipiens) and its resistance to parasitic infection concluded that these pesticides could alter the immune response of frogs and affect their ability to deal with parasitic infection (Christin et al. 2003). The effect of single, sublethal intraperitoneal (i.p.) injection of dieldrin on the primary response to T-cell-dependent (SRBC) and T-cell-independent (lipopolysaccharides, LPS) antigens, investigated in inbred C571B1/6 mice showed significant suppression of the anti-SRBC IgM, IgG and anti-LPS IgM response at 7–24 days and at 4–14 days, respectively after exposure to 0.6 LD50 dieldrin (Bernier et al. 1987). A decrease in the anti-mouse hepatitis virus 3 (MHV3) IgM serum antibody titre by aminocarb was found to be comparatively less marked than in the dieldrin group (Bernier et al. 1988).

Carbofuran (2,3-dihydro-2, 2-dimethyl-7-benzofuranyl methylcarbamate) is a carbamate pesticide. The primary IgM antibody response to SRBC antigen and macrophage phagocytosis returned to control levels indicating a lack of any synergistic or additive effect of dieldrin in combination with a carbofuran (Flipo et al. 1992). Suppression of in vitro phagocytosis and killing of bacteria by peritoneal exudate cells and significant lowering of antibody response to Salmonella typhimurium in vivo after sublethal i.p. administration of dieldrin, confirms that dieldrin inhibits the natural resistance of mice to bacterial infection (Jolicoeur, Fournier and Krzystyniak, 1988). A marked decrease in the phagocytosis of fluorescein-labelled microspheres and Salmonella typhimurium has also been documented (Krzystyniak et al. 1989) and a transient inhibition of the mixed lymphocyte reactivity (MLR) was noted at 7 days after i.p. exposure to 0.6 LD50 dieldrin (Hugo et al. 1988a). Similar results were demonstrated following assessment of lymphoid cells from mice injected i.p. 7 days earlier with 36 mg kg⁻¹ b.w. (0.6 LD50) dieldrin for their ability to recognize a foreign antigen and to proliferate in a MLR at 4, 7, and 24 days post-treatment (Hugo et al. 1988b). Dieldrin-induced immunosuppression of the cellular immune response has been seen in mice infected with the MHV3 virus (Fournier et al. 1988).

Aminocarb (4-dimethylamino-3-methylphenyl N-methylcarbamate 4-dimethylamino-m-tolyl N-methylcarbamate) is another carbamate pesticide. Comparative immunotoxic studies of sublethal exposure to aminocarb and dieldrin in mice revealed that virus-induced cytopathic effects in peritoneal macrophages were augmented to a lesser extent in the aminocarb as compared to dieldrin group (Bernier et al. 1988). In addition, macrophage antigen processing of a single protein, avidin, was significantly suppressed in dieldrin-treated animals (Bernier et al. 1988; Krzystyniak et al. 1989), while the avidin processing in macrophages was unaffected by aminocarb (Bernier et al. 1988). Dieldrin markedly affected the presentation of avidin on the macrophage surface and release of processed antigen. Thus, antigen processing could be a sensitive target for dieldrin-related injury of macrophage functional activities which, in consequence, could produce suppression of the humoral immune response (Krzystyniak et al. 1989). In vitro studies on the effect of dieldrin along with other environmental contaminants showed adverse effects on the immune function and reproductive physiology in mice (Wade et al. 2002). Dieldrin, which has pro-inflammatory properties in vivo, could not alter the ability of human neutrophils to phagocytose opsonized SRBC at non-necrotic concentrations (0·1, 1, 10, and 50 μM) but did increase human neutrophil superoxide production, RNA synthesis, and the pro-inflammatory cytokine (interleukin-8) production (Pelletier et al. 2001).

Thiabendazole

Thiabendazole (TBZ), a relatively non-toxic thiazole derivative, appears to be an immunorestorative agent, demonstrating maximum immunopotentiation in immunosuppressed hosts (Lundy and Lovett, 1978). Almost complete restoration of the delayed hypersensitivity responses (90% of control) in animals, immunosuppressed by sublethal exposure of radiation (450 Rads) has been observed when the irradiated animals were treated with TBZ. The immunosuppressive effects of adriamycin could also be reversed in a similar fashion (Lundy and Lovett, 1976). Initial in vivo and in vitro immune studies indicated that the drug was most effective when given 24 h prior to, or at the time as, administration of antigen. Single doses are more effective than multiple daily doses. One cell population potentiated by TBZ is the macrophage, either by direct activation or secondary to increased lymphokine production (Lundy and Lovett, 1978). However, oral administration of TBZ (16 mg kg⁻¹ day⁻¹ for 6 days and 20 mg kg⁻¹ day⁻¹ for 5 days) beginning 24 h prior to antigen and dexamethasone administration failed to prevent the dexamethasone-induced suppression of lymphocyte blastogenic or antibody responses and was associated with a significantly lowered antibody response to Brucella abortus (Roth et al. 1984). TBZ may have a role as an adjunct in cancer therapy (Lundy and Lovett, 1976). The use of TBZ as an immunorestorative drug in the peri-operative period resulted in an improved cytotoxic response and a significant decrease in pulmonary metastases of tumor growth. Peri-operative immunotherapy can be an effective adjunct to surgery in preventing the growth of micrometastatic foci (Lundy and Lovett, 1979). TBZ has significant effects on the development and differentiation of both lymphoid and haemopoietic cells. In vivo administration of TBZ along with the thymus-dependent neoantigen, dinitrofluorobenzene (DNFB) caused changes in expansion of the T-lymphocyte compartment and an increase in extramedullary haemopoiesis of lymph nodes and/or spleens, while the effect on B-cells appeared to be antigen independent (Donskaya et al. 1982). Smaller increases in the numbers of plasmacytes in the spleen, lymph glands, thymus and bone marrow were observed in hamsters treated with TBZ (10 mg/100 g body weight) either before or after infection with Ascaris suum, than in untreated, infected animals (Maiboroda and Shevchenko, 1978). Studies on the effect of TBZ on lung granuloma formation around Schistosoma mansoni eggs and delayed footpad oedema in response to Schistosoma egg antigen showed that the drug caused significant reduction in these responses in unsensitized animals when given as a single dose and in sensitized animals using a multiple dose regimen. These findings support the hypothesis that some of the clinical activities of TBZ may be mediated by interference with host response to antigenic stimuli (Hewlett et al. 1981). Contrary to what has been seen with fenbendazole (22·2% Panacur TM granules at 0·02 g/kg), TBZ (0·1 g/kg) has been found to raise the immune status in sheep having natural infection of nematodes (Movsesyan et al. 1988).

Fenbendazole

Fenbendazole is a member of benzimidazole group of anthelmintics. Repeated use of anthelmintics of the benzimidazole group may interfere with the immune responsiveness in young sheep (Parish et al. 1996). The studies in mice as judged by Reiss, Herrman and Hopkins (1987) concluded that this type of anthelmintic treatment did not interfere with immune responses in mice as examined by induction of allospecific cytolytic T-lymphocytes (CTLs) in vitro, influenza-specific memory T-cells in vivo, influenza-specific antibody secretion in vivo, or influenza-specific helper T-cells and CTLs in vitro. Reductions in the primary and secondary humoral responses to bovine viral diarrhoea vaccination were documented in both parasitized and parasite-naïve fenbendazole-drenched (5 mg kg⁻¹ b.w.) lambs as measured by serum neutralization titre (Parish et al. 1996). Antibody responses were similar in the fenbendazole-drenched and control lambs against human erythrocytes and ovalbumin antigens. However, after the second injection, there was a significant reduction in antibody response to human erythrocytes in the fenbendazole-treated lambs (Cabaj et al. 1994). Administration of fenbendazole to healthy mice stimulated the proliferative response of T- and B-cells to non-specific polyclonal activators, but partially inhibited the percentage of CD4+ and CD8+T-lymphocytes and the production of superoxide anion (O⁻²) increased insignificantly. The treatment of infected mice considerably stimulated the proliferative response of B-cells in comparison with T-cells. The percentage of CD4+ cells in spleen was moderately reduced after treatment while that of CD8+ increased significantly. A considerable activation of the production of peritoneal macrophage superoxide anion (O₂) was also observed by day 28 after the last administration of fenbendazole (Dvoroznakova et al. 1998).

Oxfendazole

Oxfendazole, another benzimidazole, has since long been used in the treatment of gastrointestinal nematode infections (Campbell, 1990; Sangster et al. 1991; Ali and Chick, 1992). It has been shown that benzimidazole derivatives affect rat bone marrow cells by inducing a delay in division at the metaphase stage (Lapteva, 1988). Decreased lymphocyte blastogenesis, especially in the secondary response to human erythrocyte and ovalbumin antigen, confirmed that the immune response to nematode parasites is impaired by the use of oxfendazole (Vercruysse et al. 1987; Stankiewicz et al. 1994). However, high levels of acquired immunity to reinfection under natural and experimental conditions were achieved when animals were treated with oxfendazole (Jacobs et al. 1987; Borgsteede, deLeeuw and van de Burg, 1988; Downey, 1988; Eysker and Boersema, 1989; Eysker, Boersema and Kooyman, 1990). Oxfendazole is metabolized in the rumen to fenbendazole and both of these compounds are metabolized by the liver to fenbendazole sulphone (Marriner and Bogan, 1981; Gottschall, Theodorides and Wang, 1990). It is, therefore, not clear which of these compounds is responsible for the immunomodulatory and other effects seen as a consequence of drenching lambs with oxfendazole (Stankiewicz et al. 1994).

Ivermectin

Ivermectin (22,23-dihydroavermectin B₁a), a macrocyclic lactone derivative is a semi-synthetic analogue of avermectin B₁a (abamectin) originally isolated from the fermentation of Streptomyces avermitilis (Miller et al. 1979). Ivermectin paralyses and ultimately kills parasitic nematodes, arachinids and insects and is now extensively used to control and treat a wide variety of parasitic nematodes (roundworms) and arthropods (insects, ticks and mites) that plague livestock and other domestic animals. However, there is growing evidence that the activity of ivermectin may not be limited strictly to neurophysiology of parasites, but may also influence the immune system of the laboratory animals and humans (Rao et al. 1987).

The immunomodulatory effects of ivermectin reported in the literature can be described as variable at best. Ivermectin has also been found to have immunomodulatory properties that are associated with altered function of T-lymphocytes (T-helper lymphocytes in particular). Antibody production against SRBC, a T-lymphocyte and macrophage-dependent response, was enhanced by ivermectin treatment in mice (Blakley and Rousseaux, 1991). At a therapeutic dose, ivermectin altered the lymphocyte count without affecting the total serum immunoglobulins, total serum proteins or phagocytic index of animals (Savanur et al. 1996). However, an increase in total immunoglobulins against the specific antigen and total serum proteins has been observed in ivermectin-treated rabbits (Savanur et al. 1995). Similar antibody responses were seen for the treated and control groups in response to intravenous injections of human erythrocytes and subcutaneous administration of ovalbumin antigens except that after the second injection, there was a significant reduction in antibody response to ovalbumin in ivermectin-treated lambs. There were no differences in serum complement or serum nitric oxide levels between the two groups at any stage, but insulin-like growth factors were significantly reduced in the ivermectin-treated groups 4 days after the treatment (Stankiewicz et al. 1995). In lambs, ivermectin has been found to decrease the blastogenic activity (Stankiewicz et al. 1995). In rabbits, the drug has been found to have a dose-dependent immunopotentiating response against Pasturella multocida type II (capsular polysaccharide extracted protective) antigen and SRBC, i.e. increase in the dose caused increase in immunopotentiating response and vice versa. The cell-mediated immunity (determined by macrophage phagocytic activity and dinitrochlorobenzene test) and the humoral immunity (determined by indirect haemagglutination assay and Jerne haemolytic plaque formation assay) were both found to be significant in the ivermectin-treated groups (Sajid, 2004).

Human patients with onchocerciasis are usually expected to receive parasitological and clinical relief from ivermectin (at 150 μg kg⁻¹ body weight) for at least 10 months after their initial treatment; suppression of pruritis usually occurs for at least 12 months (Brieger et al. 1998). The in vitro cellular reactivity of O. volvulus-derived antigen (Ov Ag) was reduced in ivermectin-untreated patients as compared with controls, and the lymphocyte blastogenic response improved up to 14 months after treatment (Soboslay et al. 1992). After ivermectin treatment, the Ov Ag-induced production of IL-1 beta and TNF-alpha increased significantly (P<0·05) at 1 and 14 months, and PHA-induced production of IL-2 and IL-4 increased 1 month after treatment. The results suggested that onchocerciasis-mediated immunosuppression is reversible following ivermectin-mediated permanent clearance of microfilariae from the skin. The parasite-specific cellular immunity and consistent production of IL-2 and IFN-gamma contribute to control the re-infection (Soboslay et al. 1994). In younger children (5–9 years), repeated treatment of ivermectin resulted in some enhancement of Onchocerca-specific responses measured 6 months after administration of the drug (Luty et al. 1992). Ivermectin has been reported to be central to the control of onchocerciasis through self-sustainable community-based treatment (Ali et al. 2002). Comparative study of the effects of diethylcarbamazine (DEC) and ivermectin on filarial antigen-specific immune responses concluded no differences between the two anti-parasitic drugs in terms of humoral and cellular reactivity to filarial antigen (Lammie et al. 1992). Although, ivermectin and DEC are believed to exert their anti-parasitic activity via different mechanisms, the same pattern of serological changes was observed in patients treated with either drug for the control of microfilaraemia (Zheng et al. 1991). However, ivermectin seemed to synergize host immune factors against infection (Rao et al. 1987).

Not all workers, however, have been able to demonstrate that ivermectin has an immunostimulatory effect. For example, Uhlir and Volf (1992) administered ivermectin subcutaneously to observe its influence on the specific immune response in rabbits infested with mites (Psoroptes cuniculi) and in rats infested with lice (Polyplax spinulosa). A pronounced specific antibody activity and a change in immunoblotting pattern were observed in rabbits after the ivermectin treatment. However, in rats the antibody activity decreased and the profile of specific antibodies, tested by immunoblotting remained the same as before the treatment. These investigators concluded that the enhanced immune response in the mite-infested rabbits was caused by the massive release of antigens associated with the synchronous death of the mites. Stankiewicz et al. (1995) reported that the effect of ivermectin on immunity in lambs seems to be limited to its depressive effect on lymphocytes and the secondary antibody response to ovalbumin.