Effect of Mycotoxins in Swine

Mycotoxins are structurally diverse secondary metabolites of fungi that grow on a variety of feeds and foods 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; Neldon-Ortiz and Quereshi 1991). 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 is significant for several reasons. First of all, 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. Secondly, 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 etiologically 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 mycotoxins on immune response in laboratory animals (Bondy and Pestka, 2000; Corrier 1991). In this paper we will present examples concerning the effects of mycotoxins on different aspects of the pig immune system: inflammation, cellular response and the humoral response. As the immune system is primarily responsible for defense against invading organisms, we will conclude this article by looking at the significance of mycotoxin intoxication in terms of pig health. Indeed, suppressed immune function by mycotoxins may eventually decrease resistance to infectious diseases, reactivate chronic infection or reduce vaccine and therapeutic efficacy.

In pigs as in all mammals, the immune response is a major defense 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 (prostaglandins and leucotrienes), but also active metabolites of oxygen and nitrogen (H2O2, O2
-, NO and others). 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: (i) B lymphocytes that secrete antibodies and induce the humoral immune response, (ii) T lymphocytes that participate in the cell mediated immune response by developing cytotoxic activity and by producing cytokines. Two different subsets of lymphocytes (Th1 and Th2) are distinguished by the cytokines they produce. These subsets orientate the immune response toward 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 mycotoxins can act on all immune cell types and at different levels of the immune
response.

Several reports show that mycotoxins, such as aflatoxin, 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 inhibits in vitro phagocytosis, intracellular killing and the spontaneous production of oxygen radicals of rat peritoneal macrophages (Cusumano et al., 1990). It also decreases phagocytosis and causes cytoplasmic blebbing and/or nuclear disintegration when tested in vitro on cultured chicken and turkey macrophages (Neldon-Ortiz and Qureshi, 1991; 1992). In addition, ingestion of aflatoxin B1 reduces the number of rat and chick macrophages and decreases their functional properties (Ghosh et al., 1991; Michael et al., 1973; Raisuddin Singh et al., 1990). Aflatoxin 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; Moon et al., 1999). In vitro studies have demonstrated a suppressive effect of aflatoxin on inflammatory cytokine levels in mice (Moon et al., 1999), humans (Rossano et al., 1999) and cattle (Kurtz and Czuprynscki, 1992). In pigs, several recent papers have reported an alteration of the inflammatory response by aflatoxin. In utero exposure of piglets to aflatoxin (through exposure of sows) inhibited the oxidative burst of monocyte-derived macrophages but did not affect their phagocytic function. Neutrophil functions, including motility and chemotaxis, were also inhibited in piglets from aflatoxin-treated sows (Silvotti et al., 1997). A feeding trial conducted on weanling piglets for 4 weeks also indicated that low doses of aflatoxin (140 and 280 ppb) decreased pro-inflammatory (IL-1ß, TNF-a) and increased anti-inflammatory (IL-10) cytokine mRNA expression by PHA-stimulated blood cells (Marin et al., 2002). In vitro exposure of swine alveolar macrophages to aflatoxin B1 leads to a time- and dose-dependent decreased viability of the primary cultures and to a reduction in phagocytic ability of the cells. Aflatoxin B1 exposure also induced the expression of apoptosis-related heat shock protein 72 (HSP-72), but in this experiment it did not affect the expression of IL-1ß and TNF-amRNA (Liu et al., 2002). Ochratoxin A was reported to inhibit in vitro chemotactic activity of murine peritoneal macrophages (Klinkert et al., 1981). Likewise, diets containing 4 ppm ochratoxin impaired the motility and phagocytosis of neutrophils in chicks (Chang and Hamilton, 1979). Moreover, when injected into the mice peritoneum at 10-20 mg/kg, it increased macrophage capacity to kill tumor cells but provoked myelotoxicity of the bone marrow progenitors of macrophage granulocytes (Boorman et al., 1984).

Other studies concerning trichothecenes also show a decrease of chemotaxis and phagocytosis of diverse neutrophils and macrophages (Buening et al., 1982; Corrier et al., 1987; Gerberick and Sorenson 1984; Yarom et al., 1984). The underlying mechanism might be a superinduction 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 reduced cell viability to 80% of the control level (Qureshi and Hagler 1992). Similarly, incubation of swine alveolar macrophages with fumonisin B1 led to a significant reduction of the number of viable cells, their phagocytic activity and their expression of IL-1ß and TNF-amRNA. Fumonisin B1 induced apoptosis of the cells with evidence of DNA laddering and nuclear fragmentation (Liu et al., 2002).

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. In mice, 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 (Rotter et al., 1996). The threshold for this inductive effect is 2 ppm in mouse feed, with a maximal effect occurring in the 10-25 ppm range. Increases in serum IgA appear concomitantly with elevated IgA immune complexes 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 in IgA-secreting cells in the Peyer’s patches and that this affects the systemic compartment (Pestka et al., 1990). In mice, the immunopathology associated with DON consumption, which also includes glomerular IgA accumulation and hematuria, is very similar to human IgA nephropathy (Bondy and Pestka, 2000). These effects can persist long after the withdrawal of DON from the mouse diet (Dong and Pestka, 1993), but intermittent exposure is less effective at increasing IgA levels than continuous exposure (Banotai et al., 1999).

DON-induced increases in IgA production may be mediated by T lymphocytes (Warner et al., 1994) and macrophages (Yan et al., 1998), especially through the superinduction of cytokine genes such as IL-2, IL-5 and IL-6 (Yan et al., 1997). The specific mechanism for cytokine superinduction by the mycotoxin is incompletely understood but might involve increased cytokine mRNA stability and other transcriptional mechanisms (Bondy and Pestka, 2000).

In pigs, we and other workers have also demonstrated an increase in IgA in the serum of
animals receiving DON-contaminated feed (Bergsjo et al., 1993; Grosjean et al., 2002 ; Swamy et al., 2002). However, in these experiments serum IgG levels were not influenced by diet (Grosjean et al., 2002; Swamy et al., 2002), nor were the levels of expression of several cytokines (IL-6, IL-10, IFN-? and TNF-a) (Grosjean et al., 2002).

MYCOTOXINS AND CELLULAR IMMUNE RESPONSE

Immunomodulatory effects of mycotoxins have been most extensively studied with aflatoxins. The greatest effect of aflatoxin is focused on cell mediated immunity. Its effect on humoral immunity requires higher toxin concentrations and is inconsistent across species (review in Pier, 1992).

In mice orally exposed to aflatoxin B1, there is a dose-related suppression of delayed-type
hypersensitivity (DTH) to keyhole limpet hemocyanin (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 (Dugyala and Sharma, 1996; Hatori et al., 1991). In chickens aflatoxin B1 also suppresses cellmediated immunity as measured by DTH, graft versus host response, leukocyte migration and lymphoblastogenesis (Ghosh et al., 1991; Kadian et al., 1988).

In pigs, attempts to evaluate the effects of aflatoxin on the cellular immune response has led to conflicting results. Several papers have demonstrated a reduction in lymphocyte stimulation by mitogens in animals receiving feed contaminated with 280 to 2500 ppb aflatoxin (Miller et al., 1981; Harvey et al., 1995; van Heughten et al., 1994). In contrast, other investigators have not observed any suppression of the lymphoproliferative response to mitogens in pigs receiving feed contaminated with 500 to 800 ppb aflatoxin (Panangala et al., 1986; Silvoltti et al., 1995). Using lower doses of this mycotoxin (140 and 280 ppb) we also failed to detect any effect of aflatoxin on the expression of regulatory cytokines produced by either Th1 (IL-2) or Th2 (IL-4) lymphocyte subsets in PHAstimulated blood samples (Marin et al., 2002).

Recent results indicate that developing piglets might be especially susceptible to this toxin (Silvotti et al., 1997). Indeed, after exposure of sows to 800 ppb aflatoxin B1or G1 during gestation and lactation, the mycotoxins could be detected in the milk at levels up to 500 ppt. This led to the functional alteration of immunocompetent T-cells of the piglets. Their lymphoproliferative response to mitogens was reduced and monocyte-derived macrophages failed to efficiently produce superoxide anions after oxidative burst stimulation (Silvotti et al., 1997).

A genetic component seems to be involved in aflatoxin B1-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 aflatoxin B1 than lymphocytes negative for HLA-A3 (Wang et al., 1987). Studies conducted on two lines of chickens selected for high and low plasma protein concentrations in response to aflatoxin B1 indicate that animals also differ for T-cell and thymocyte proliferation. Indeed, oral administration of aflatoxin B1 to chicks from the high line resulted in lower peripheral blood lymphocyte proliferation response to a T-cell mitogen compared to chicks from the low line in response to aflatoxin B1 (Scott et al.,1991).

The molecular-cellular basis and general mechanism responsible for the broad immunosuppressive effects of aflatoxin B1 appear to be directly related to impaired protein synthesis. Aflatoxin B1 is transformed in vivo into active metabolites that bind to DNA and RNA, impair DNA-dependent RNA polymerase activity and inhibit 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 aflatoxin alters cytokine synthesis by macrophages and/or T-cells (Dugyala and Sharma, 1996; Marin et al., 2002). Ultrastructural studies show that aflatoxin B1 causes selective mitochondrial damage in murine lymphocytes and does not affect other cellular organelles and external structure of the lymphocytes (Rainbow et al., 1994).

SUSCEPTIBILITY TO INFECTIOUS DISEASES

The broad immunosuppressive effect of mycotoxins on cellular and humoral immune responses has been demonstrated to decrease host resistance to infectious diseases. This has been shown not only in mice (Tai and Pestka, 1988; Vidal, 1990) but also in rabbits (Niyo et al., 1988b) and chickens (Bekesi et al., 1997; Boochuvit et al., 1975). In pigs, consumption of feed contaminated with aflatoxin increased the severity of the Erysipelothrix rhusiopathiae infection as demonstrated by the analysis of macroscopic lesions performed after an experimental challenge (Cysewski et al., 1978).

More recently, Stoev and collaborators (2000) demonstrated that ingestion of ochratoxin A contaminated food increases susceptibility to natural infectious disease in pigs. In this experiment, salmonellosis arose spontaneously in all piglets receiving a diet contaminated with 3 ppm ochratoxin A and in 1/3 of the animals receiving a diet contaminated with 1 ppm of toxin. In contrast, none of the animals fed the control diet were affected.
In a further experiment, the authors vaccinated the animals against S. choleraesuis hemorrhagic diarrhea. In this case the mycotoxin contamination led to spontaneous Serpulina hyodysenteriae and Campylobacter coli infection (Stoev et al., 2000).

In our laboratory we have found that the mycotoxin fumonisin B1 is a predisposing factor to infectious disease (Fournout et al., 2000; Oswald et al., 2001). Weaned piglets received a daily intake of 0.5 mg/kg (body weight) of fumonisin B1 as a crude extract or as a purified toxin for seven days.

On the last day of the toxin treatment, pigs were orally inoculated with a pathogenic E. coli associated with extra-intestinal infection and the animals were sacrificed 24 hrs later. Our data indicated that the oral administration of fumonisin B1 significantly increased colonization of the small and the large intestine by the inoculated E. coli strain (Fournout et al., 2000; Oswald et al., 2001). We have further demonstrated that this increased susceptibility was associated with a decreased level of mRNA encoding for IL-8 in the ileum of fumonisin B1-treated pigs. In addition, we have obtained in vitro data on a porcine epithelial intestinal cell line indicating that fumonisin B1 decreases IL-8 synthesis. These experiments also show that fumonisin B1 blocks cell proliferation and division of porcine epithelial intestinal cells and impairs their ability to form a monolayer (Bouhet et al., unpublished results). We hypothesize that: (i) by decreasing IL-8 levels, fumonisin B1 reduces the recruitment of inflammatory cells in the intestine and (ii) by affecting the proliferation and the integrity of the epithelial cell monolayer this toxin increases the translocation of bacteria across the epithelium. Both phenomena may participate in the increased susceptibility of the animals to intestinal infections (Figure 1).

The effect of mycotoxin intoxication on the reactivation of chronic infection was also investigated. However the experiment was not performed with pigs, 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 immunosuppressed 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 aflatoxin B1 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 aflatoxin B1 and T-2 toxin, respectively.

Immunity acquired through vaccination is also impaired by mycotoxin ingestion. For example, aflatoxin B1 interferes with the development of acquired immunity in pigs following erysipelas vaccination (Cysewski et al., 1978). In this experiment, pigs fed either a normal diet or aflatoxincontaminated diet were vaccinated with erysipelas bacterin and challenged 21 days later with a virulent strain of Erysipelothrix rhusiopathiae (Cysewski et al., 1978). In the group of pigs receiving the normal diet (total of six animals), three animals could be considered immune and two were partially immune following vaccination and challenge. In contrast, in the group of pigs receiving aflatoxin, none of the animals were fully immune and only one was partially immune, indicating that aflatoxin consumption interfered with the development of acquired immunity (Cysewski et al., 1978).
In our laboratory we have recently demonstrated that ingestion of low doses of fumonisin B1 decreases the specific antibody response mounted during vaccination (Taranu et al., 2003). Indeed, a prolonged exposure (28 days) to feed contaminated with 8 ppm fumonisin B1 does not modify the serum concentration of the three immunoglobulin subsets (IgG, IgA and IgM) but significantly decreases specific antibody response to a model antigen. In vitro analysis of pig lymphocytes reveals that this toxin inhibits cell proliferation (Gouze and Oswald, 2001) and alters cytokine production (Taranu et al., 2003).



Figure 1. Possible mechanism for the increased susceptibility to Escherichia coli infection in piglets receiving fumonisin B1.

Fumonisin B1 increases the synthesis of IFNg, a Th1 cytokine involved in the cell mediated immune response and decreases synthesis of IL-4, a Th2 cytokine involved in humoral response. This alteration of both lymphocyte proliferation and cytokine production might explain the failure in vaccination that we observed in vivo (Figure 2).

Therefore, the presence of low levels of mycotoxins in the feed can lead to a breakdown in
vaccine-derived immunity and may lead to disease even in properly vaccinated flocks. These reactions are of considerable consequence in animal production as we rely on effective vaccination programs for disease prevention (Pier, 1992).

The investigations described in this review clearly indicate that several mycotoxins alter immunemediated activities in pigs. Furthermore, mycotoxin-induced immunosuppression may result in decreased host resistance to infectious disease and decreased vaccine efficacy. However, several considerations have not been taken into account.

First, mycotoxin mixtures are likely to occur naturally and these may alter immunity in an additive or synergistic manner as has been described for aflatoxin and T-2-toxin (Pier, 1992) or for DON and fusaric acid (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. Thus 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 mold-contaminated air supplies.



Figure 2. Possible mechanism for the impaired vaccine efficacy in piglets receiving fumonisin B1.

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