Immunotoxicological risk of mycotoxins for domestic animals

Abstract

Mycotoxins are a group of structurally diverse fungal secondary metabolites that elicit a wide spectrum of toxicological effects. Of particular interest is the capacity of some mycotoxins to alter normal immune function when present in food at levels below observable overt toxicity. The sensitivity of the immune system to mycotoxin–induced immunosuppression arises from the vulnerability of the continually proliferating and differentiating cells that participate in immune–mediated activities and regulate the complex communication network between cellular and humoral components. Mycotoxin–induced immunosuppression may be manifested as depressed T– or B–lymphocyte activity, suppressed antibody production and impaired macrophage/neutrophil–effector functions. The immune system is primarily responsible for defence against invading organisms. Suppressed immune function by mycotoxins may eventually decrease resistance to infectious diseases, reactivate chronic infections and/or decrease vaccine and drug efficacy.

Keywords:

• Mycotoxins
• immunosuppression
• lymphocytes
• macrophages
• neutrophils
• infectious diseases
• farm animals

Introduction

Mycotoxins are structurally diverse secondary metabolites of fungi that grow on a variety of feed and food consumed by animals and man respectively. The clinical toxicological syndromes caused by ingestion of moderate to high amounts of mycotoxins have been well characterized. The effects range from acute mortality, to slow growth and reduced reproductive efficiency (Berry 1988). Consumption of lesser amounts of fungal toxins may result in impaired immunity and decreased resistance to infectious diseases. Indeed, it has long been recognized by veterinary clinicians that marked immunosuppression is observed in livestock ingesting mycotoxins at levels below those that cause overt toxicity (Richard et al. 1978). Mycotoxin-induced immunomodulation of farm animals is significant for several reasons. First, from an agricultural standpoint, it is conceivable that altered immune function may contribute mechanistically to the symptoms of some animal mycotoxicoses. Mycotoxins could also predispose livestock to infectious diseases and reduce productivity. Second, from a public health perspective, increased infections in animals may well result in increased animal-to-human transmission of pathogens and/or increased antibiotic concentrations in meat or milk, as a consequence of animal treatment. In addition, ingestion or inhalation of mycotoxins by humans may contribute aetiologically to immune dysfunction diseases or to an increased susceptibility to infectious agents.

The sensitivity of the immune system to mycotoxin-induced immunosuppression arises from the vulnerability of the continually proliferating and differentiating cells that participate in immune-mediated activities and regulate the complex communication network between cellular and humoral components. Mycotoxin-induced immunosuppression may manifest as depressed T- or B-lymphocyte activity, suppressed antibody production and impaired macrophage/neutrophil-effector functions. Several reviews have detailed the effects of mycotoxin on the immune response in laboratory animals (Corrier 1991; Bondy and Pestka 2000). The present paper will present examples concerning the effects of mycotoxins on different aspects of the immune system: inflammation, cellular response and the humoral response. As the immune system is primarily responsible for defence against invading organisms, the second part of this review will focus on the significance of mycotoxin intoxication in terms of animal health with special emphasis to poultry and pigs. Indeed, suppressed immune function by mycotoxins may eventually decrease resistance to infectious diseases, reactivate chronic infection or reduce vaccine and therapeutic efficacy.

Complexity of the immune response

In animals and humans, the immune response is a major defence mechanism against microbial pathogens or against any disruption of the organism integrity (burning, cutting, etc.). Two different mechanisms are involved in the immune response: the inflammatory response and the immune response associated with memory (also named acquired immunity). Inflammation is a non-specific response that occurs very rapidly and leads to the activation of phagocytes (macrophages and neutrophils). The activated phagocytes secrete many different molecules such as cytokines (involved in the recruitment and the activation of other cells), metabolites of arachidonic acid (postaglandines and leucotrienes), but also active compounds of oxygen and nitrogen (H₂O₂, O₂ ⁻, NO, etc.). The acquired immune response involves lymphocytes. It occurs after a second contact with the foreign antigen and is characterized by a rapid and specific response. Two cell types participate in this response: (1) B-lymphocytes that secrete antibodies and induce the humoral immune response; and (2) T-lymphocytes that participate in the cell mediated immune response by developing a cytotoxic activity and by producing cytokines. Two different subsets of lymphocytes (Th1 and Th2) can be distinguished by the cytokines they produce. These subsets orientate the immune response towards the cellular or the humoral immune response respectively. The domination of a Th1 or a Th2 response has been shown to have a particular relevance in response to many pathogens (Sher et al. 1992).

Thus the immune response is highly complex and various cells interact with one another to produce the desired effect. The examples presented below will show that mycotoxin can act on all immune cell types and at different levels of the immune response.

Mycotoxins and inflammation

Several reports show that mycotoxins, such as aflatoxin (AF), ochratoxin, patulin or fumonisin are able to affect the inflammatory response. They can act at different levels. They can directly affect the viability of phagocytes (macrophages and neutrophils), alternatively they can impair the activity or the secretory functions of these cells.

Aflatoxin B₁ (AFB₁) inhibits in vitro phagocytosis, intracellular killing and the spontaneous production of oxygen radicals of rat, chicken or turkey macrophages (Cusumano et al. 1990; Neldon-Ortiz and Qureshi 1991; 1992). In addition, ingestion of AFB₁ reduces the number of rat and chick macrophages and decreases their functional properties (Michael et al. 1973; Raisuddin Singh et al. 1990; Ghosh et al. 1991). AF also modifies the synthesis of inflammatory cytokines. Indeed, inhibition of inflammatory cytokines has been observed in rodents during respiratory aflatoxicosis (Jakab et al. 1994) or after oral intoxication (Dugyala and Sharma 1996). In vitro studies have demonstrated a suppressive effect of AF on inflammatory cytokine levels in mice (Moon et al. 1999), humans (Rossano et al. 1999) and cattle (Kurtz and Czuprynski 1992). In pigs, in utero exposure to AF (through exposure of sows), inhibited the functions of neutrophils and macrophages (Silvotti et al. 1997). Similarly, in vitro exposure of swine alveolar macrophage to this toxin leads to a time- and dose-dependent decreased viability and phagocytic activity of primary cultures cells (Liu et al. 2002). A feeding trial conducted on weanling piglets for 4 weeks also indicated that low doses of AF decreased pro-inflammatory (IL-1β, TNF-α) and increased anti-inflammatory (IL-10) cytokine mRNA expression by PHA stimulated blood cells (Marin et al. 2002).

Ochratoxin A was reported to inhibit in vitro chemotactic activity of murine peritoneal macrophages (Klinkert et al. 1981). Likewise, feeding chicks with 4 mg kg⁻¹ of ochratoxin impaired the motricity and phagocytosis of neutrophils (Chang and Hamilton 1979). Intraperitoneal injection of the toxin is myelotoxic as demonstrated by the decrease of the progenitors of macrophage-granulocytes in the bone marrow (Boorman et al. 1984).

Other studies concerning trichothecenes also show a decrease of chemotaxy and phagocytosis of bovine and murine neutrophils or macrophages (Buening et al. 1982; Gerberick and Sorenson 1984; Corrier et al. 1987). The underlying mechanism might be a super induction of the gene encoding for IL-2 and IL-1 in lymphocytes and macrophages respectively (Holtz et al. 1988).

Recent studies have also provided in vitro evidence that fumonisins influence the inflammatory response (Qureshi and Hagler 1992; Liu et al. 2002). The exposure of chicken peritoneal macrophages to fumonisin B₁ (FB₁) reduced cell viability to 80% of the control level (Qureshi and Hagler 1992). Similarly, incubation of swine alveolar macrophages with FB₁ led to a significant reduction of the number of viable cells, their phagocytic activity and their expression of IL-1β and TNF-α mRNA. FB₁, induced apoptosis of the cells with evidence of DNA laddering, and nuclear fragmentation (Liu et al. 2002).

Mycotoxins and humoral immune response

Many mycotoxins have been found to affect humoral immunity (review in Oswald and Comera 1998; Bondy and Pestka 2000). Of particular interest is the effect of deoxynivalenol (DON), also called vomitoxin, on antibody synthesis (review in Rotter et al. 1996; Pestka 2003). One of the most dramatic effects of this toxin is a pronounced elevation in serum immunoglobulin A (IgA) and concurrent depression in IgM and IgG. The threshold for this inductive effect is 2 mg kg⁻¹ in mice feed, with a maximal effect occurring in the 10–25 mg kg⁻¹ range. Increases of serum IgA appears concomitantly with elevated IgA immune complex and polymeric IgA. Lymphocytes of the Peyer's patches and, to a lesser extent, splenic lymphocytes isolated from DON-fed mice produced significantly more IgA than cultures isolated from control mice. This suggests that DON enhances differentiation to IgA-secreting cells in the Peyer's patches and that this impacts on the systemic compartment. In mice, the immunopathology associated with DON consumption, which also includes glomerular IgA accumulation and haematuria, is very similar to human IgA nephropathy. These effects can persist a long time after the withdrawal of DON from the mouse diet but intermittent exposure is less effective at increasing IgA levels than continuous exposure (Pestka 2003).

DON-induced increased IgA production may be mediated by T-lymphocytes and macrophages and especially through the superinduction of cytokine genes such as IL-2, IL-5 and IL-6. The specific mechanism for cytokine superinduction by the mycotoxin is incompletely understood but might involve increased cytokine mRNA stability and other transcriptional mechanisms (Pestka 2003).

In pigs, we and other workers have also demonstrated an increase of IgA in the serum of animals receiving DON contaminated feed (Bergsjo et al. 1993; Grosjean et al. 2002; Swamy et al. 2002; Pinton et al. 2004). However, in these experiments the level of IgG in the serum was not influenced by the diet (Grosjean et al. 2002; Swamy et al. 2002) as well as the levels of expression of several cytokines (IL-6, IL-10, IFN-γ and TNF-α) (Grosjean et al. 2002).

Mycotoxins and cellular immune response

Immunomodulatory effects of mycotoxins have been most extensively studied with AF. The greatest effect of AF is focused on cell mediated immunity, its effect on humoral immunity requiring higher toxin concentration and being inconsistent across different species (Pier 1992).

In mice orally exposed to AFB₁, there is a dose-related suppression of delayed-type hypersensitivity (DTH) to keyhole limpet haemocyanin (Reddy and Sharma 1989). Intoxicated mice also exhibit a decrease in splenic CD4 + T cell number as well as in IL-2 production by splenocytes (Hatori et al. 1991; Dugyala and Sharma 1996). In chickens AFB₁ also suppresses cell-mediated immunity as measured by DTH, graft versus host response, leukocytes migration and lymphoblastogenesis (Kadian et al. 1988; Ghosh et al. 1991). In pigs, the effects of AF on the cellular immune response have lead to conflicting results. Several investigators demonstrate a reduction in lymphocyte proliferation stimulation in animals receiving contaminated feed, whereas others have not observed any suppression of proliferative response and cytokines expression by lymphocyte (review in Oswald et al. 2003a).

A genetic component seems to be involved in AFB₁-related cell-mediated immune suppression. Human peripheral blood lymphocytes bearing the leukocyte antigen HLA-A3 are more sensitive to suppression of PHA-stimulated blastogenesis by AFB₁ than lymphocytes negative for HLAA3 (Wang et al. 1987). Studies conducted on two lines of chicken selected for high and low plasma protein concentrations in response to AFB₁ indicate that animals also differ for T-cell and thymocyte proliferation. Indeed, oral administration of AFB₁ to chicks from the high line results in lower proliferation response of peripheral blood lymphocytes to a T cell mitogen compared to chicks from the low line in response to AFB₁ (Scott et al. 1991).

The molecular-cellular basis and general mechanism responsible for the broad immunosuppressive effect of AFB₁ appears to be directly related to impaired protein synthesis. AFB₁ is transformed in vivo into active metabolites that bind to DNA and RNA, impair DNA-dependent RNA polymerase activity and inhibits RNA and protein synthesis. Inhibition of DNA, RNA and protein synthesis directly and indirectly impairs the continual proliferation and differentiation of cells of the lymphoid system, and the synthesis of cytokines that regulate the communication network of the immune system. Indeed a recent study indicated that AF alters cytokine synthesis by macrophages and/or T cells (Dugyala and Sharma 1996; Marin et al. 2002). Ultrastructural studies show that AFB₁ causes selective mitochondrial damages in murine lymphocytes and does not affect other cellular organelles and external structures of the lymphocytes (Rainbow et al. 1994).

Significance to health of farm animals

Susceptibility to infectious diseases

The broad immunosuppressive effect of mycotoxins on cellular- and humoral-mediated immune responses has been demonstrated to decrease host resistance to infectious diseases. For example, in mice repetitive exposure to the trichothecene T-2 toxin decreases their resistance to Mycobacterium bovis, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus and herpes virus simplex type 1 infection (Tai and Pestka 1988; Vidal 1990). Similarly, the ingestion of this toxin reduces the resistance of rabbits to Aspergillus fumigatus infection (Niyo et al. 1988).

In poultry, T-2 toxin, ochratoxin and aflatoxin have been both described to increase the susceptibility to several infectious diseases. For example, T-2 toxin increases the susceptibility of chicken to infection by Salmonella species (Boochuvit et al. 1975; Ziprin and Elissalde 1990; Kubena et al. 2001) and Cryptosporidium baileyi (Bekesi et al. 1997). Similarly, the ingestion of AF increases the severity of infection of coccidiosis and salmonellosis in chicken and Japanese quail (Ruff 1978; Wyatt et al. 1975; Boochuvit and Hamilton 1975; Rao et al. 1995; Kubena et al. 2001). Ochratoxin A has also been described to increase the susceptibility of chicken to coccidiosis (Huff and Ruff 1975; Stoev et al. 2002), salmonellosis (Elissalde et al. 1994; Fukata et al. 1996) and colibacillosis (Kumar et al. 2003).

In pigs, consumption of feed contaminated with AF increased the severity of experimental Erysipelothrix rhusiopathiae infection (Cysewski et al. 1978). More recently, Stoev et al. (2000) demonstrated that ingestion of ochratoxin A contaminated feed increases the susceptibility of pigs to natural infection by Salmonella cholerasuis, Serpulina hyodysenteriae or Campylobacter coli. We have also demonstrated that oral administration of purified FB₁ significantly increases the susceptibility of piglet to experimental infection with a pathogenic strain of Escherichia coli (Oswald et al. 2003b). This increased susceptibility was associated with a decreased level of mRNA encoding for IL-8 in the ileum of FB₁ treated pigs (Bouhet and Oswald, unpublished results). Data obtained in vitro on a porcine epithelial intestinal cell, indicated that FB₁ blocks cell proliferation and division and impairs the ability of epithelial cells to form a monolayer (Bouhet et al. 2004). We hypothesize that (1) by decreasing IL-8 levels, FB₁ reduces the recruitment of inflammatory cells in the intestine and (2) by affecting the proliferation and the integrity of the epithelial cell monolayer FB₁ increases the translocation of bacteria across the epithelium. Both phenomenons may participate in the increased susceptibility of the animals to intestinal infections.

Reactivation of chronic infection

The effect of mycotoxin intoxication on the reactivation of chronic infection was also investigated, however the experiment was not performed with domestic animals but with rodents (Venturini et al. 1996).

In the immunocompetent host, Toxoplasma gondii infection progresses to a chronic phase characterized by the presence of encysted parasites, mainly within the central nervous system or skeletal muscle. Cyst rupture may occur, but infection remains latent and reactivation is prevented. In immunosupressed animals and human subjects, such as HIV infected patients, rupture is associated with the formation of new cysts and disease (Suzuki and Remington 1993). Venturini et al. (1996) demonstrated that low and repeated doses of either AFB₁ or T-2 toxin are able to accelerate Toxoplasma cyst rupture in previously infected mice. In fact, the percentage of ruptured cysts increased from 15% in infected non-intoxicated mice to 56 and 29% in infected mice that were treated for 6 weeks with AFB₁ and T-2 toxin respectively.

Vaccination efficacy

Immunity acquired through vaccination is also impaired by mycotoxin ingestion. For example, AFB₁ interferes with the development of acquired immunity in swine following erysipelas vaccination and in rabbits vaccinated with an oil-adjuvant Bordetella bronchoseptica vaccine (Cysewski et al. 1978; Venturini et al. 1990). AFB₁ ingestion by chickens and/or turkeys does not affect vaccine efficacy for Newcastle disease but decreases vaccine efficacy for fowl cholera and Mareck disease (Batra et al. 1991; Hegazy et al. 1991). Indeed, feeding AFB₁ at 0.5 mg kg⁻¹ to young chicks does not result in the development of any clinical lesions, mortality or gross lesions specific to aflatoxicosis. However, in vaccinated chickens, the occurrence of Marek's disease lesions is more severe in those fed with AFB₁-mixed food than in those given control feed. Observations concerning the histopathological changes in various lymphoid organs showed that the spleen was more frequently and severely affected in AFB₁-fed than in normally fed animals. This could be attributed to a predisposition of lymphoid tissues to be damaged by AFB₁ (Batra et al. 1991).

In our laboratory, we have recently demonstrated that the ingestion of low doses of another mycotoxin, FB₁, decreases the specific antibody response during vaccination (Taranu et al. 2005). Indeed, a prolonged exposure (28 days) to feed contaminated with FB₁ does not modify the serum concentration of the three immunoglobulin subsets (IgG, IgA and IgM) but significantly decreases specific antibody response towards a model antigen. In vitro analysis of pig lymphocytes revealed that this toxin inhibits cell proliferation (Gouze and Oswald 2001) and alters cytokine production (Taranu et al. 2005). FB₁ increases the synthesis of IFN-γ, a Th1 cytokine involved in the cell mediated immune response and decreases IL-4 synthesis, a Th2 cytokine involved in the humoral response. This alteration of both lymphocyte proliferation and cytokine production might explain the failure in vaccination that we observed in vivo.

Therefore, the presence of mycotoxins in the feed may lead to a breakdown in vaccinal immunity and to the occurrence of disease even in properly vaccinated flocks (Pier 1992).

Drug efficacy

The effect of mycotoxin intoxication on drug efficacy has also been investigated (Varga and Vanyi 1992). Indeed, contamination of the feed with various doses of T-2 toxin significantly reduced the anticoccidial efficacy of lasalocid in chickens. This effect was dependent as observed by the mortality rate and the percentage of animal developing characteristic lesions upon an experimental challenge with Eimeria tenella or E. mitis (Varga and Vanyi 1992). These reactions are of considerable consequence in animals for which we rely on an effective therapeutic program for disease prevention.

Conclusion

The investigations described in this review clearly indicate that several mycotoxins alter immune-mediated activities in farm animals. Furthermore, mycotoxin-induced immunosuppression may result in decreased host resistance to infectious disease and decrease vaccine efficacy. However when analysing the immunosuppressive effect of mycotoxins, several other considerations may need to be taken into account. First, mycotoxin mixtures are likely to occur naturally and these may alter immunity in an additive or synergistic manner as it has been described for aflatoxin and T-2-toxin or for DON and fusaric acid (Pier 1992; Smith 1992). Second, nutritional effects associated with feed refusal may also contribute to observed alterations. Finally, while systemic immunity is the focus of most investigations, it is very probable that mycotoxins have their greatest effect on mucosal lymphoid tissue (particularly gut and bronchial) before they are absorbed and subsequently metabolized. Additional investigation of the immune effects of inhaled mycotoxins would also be of interest because of the risk of environmental exposure via grain dust or mould-contaminated air supplies.