Involvement of free radicals in parasitic infestations

Abstract

The amount of reactive oxygen species increased in cells of hosts infected with parasites as reported by a number of studies. The excess of free radicals in the parasitised cells depends on the nutritional status of the host, the degree of parasitic infestations and on the destructive effect on tissue, and reported associating blood parasites like Theileriosis, Babesiosis and Trypanosomosis. In addition, external parasites and endoparasites such as Fasciola sp., Trichostrongylus sp. and Eimeria sp. had been reported to be associated with lipid peroxidation. The current review throws light on parasitic infestations associated with oxidative stress and their harmful effect.

Keywords:

• free radicals
• parasite
• antioxidants

Introduction

Several studies have reported the presence of oxidative stress in human and animals infected with parasites (Stocker et al. 1986; Smith and Bryant 1989; Sarin et al. 1993; Selkirk et al. 1998; Upcroft and Upcroft 2001), as well as the antioxidant defence mechanism that exists between parasites and the mammalian host (Ueland et al. 1996). In human allergic inflammatory diseases, such as helminthic infections, the associated influx of eosinophils has been implicated as a primary source of tissue damage (Corrigan and Kay 1992), possibly via their potent reactive oxygen metabolites production (Petreccia et al. 1987).

The cells contain a variety of antioxidants mechanisms that play a central role in the protection against reactive oxygen species (Halliwell 1991). The antioxidant system consists of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX)], glutathione, ancillary enzymes [glutathione reductase, glutathione S-transferase and glucose 6-phosphate dehydrogenase (G6PD)], metal-binding proteins (tzransferrin, ceruloplasmin and albumin), vitamins (alpha-tocopherol, ascorbate and beta-carotene), flavonoids and urate (Halliwell 1994). Antioxidants may act by scavenging the radicals directly and sustaining the activity of antioxidant enzymes or inhibiting the activity of oxidising enzymes (Boczon et al. 1996; Dedra and Hadas 2000; Dedra et al. 2004; Abd Ellah 2010).

Lipid peroxidation is one of the best indicators of the level of reactive oxygen species (ROS) that induced systemic biological damage (Popova and Popov 2002). The antioxidant system has a cellular protective action against oxidative stress of cell, organs and tissue damage that result from parasitic invasion (Chuenkova et al. 1989; Smith and Bryant 1989; Das et al. 1996; Dede et al. 2002). It also plays a role in the protection of the phagocytic leukocytes against their own products and oxygen radicals. Reduced glutathione, an important antioxidant enzyme, reacts with peroxides to remove toxic substances and radicals, since it possesses active sulphydryl group (Novak et al. 1991). Although some investigations have revealed that parasitic infection causes change in lipid peroxidation parameters (Kaya et al. 2007; Cam et al. 2008).

Role of selenium

The nutritional status of the host can influence the rate of acquisition of immunity to parasitic infection (Coop and Holmes 1996), and a better understanding of the role of nutrition will be important if producers are to make better use of the host-acquired immunity and reduce dependence on anthelmintics (Van Houtert and Sykes 1996). The role of selenium is of particular relevance especially in selenium-deficient areas. Sheep produced and run in these areas are more susceptible to diseases and infection due to immunodeficiency. With escalating anthelmintic resistance, it is critical that more integrated approaches to parasite control are investigated.

A deficiency in selenium has been reported to cause delayed adult worm expulsion and increased egg production of established female worms in mice inoculated with the gastrointestinal parasite Heligmosomoides polygyrus, suggesting an impaired intestinal response in the mice (Smith et al. 2005). No differences in the overall immune response to Haemonchus contortus infection have been observed in sheep given an intraruminal selenium pellet (Jelinek et al. 1988). Selenium supplementation on helminthes burdens of marginally selenium deficient suckling Angora goat kids did not influence the level of parasitism (Fivaz et al. 1993). It seems then that selenium supplementation may not offer a useful additional means of controlling internal parasites of Angora goat kids. However, other studies have demonstrated that both selenium and vitamin E are required for specific IL-4-related changes in intestinal physiology that promote host protection against gastrointestinal nematode parasites in mice (Smith et al. 2005).

In selenium-supplemented sheep lower faecal egg counts than the non-supplemented counterparts have been observed, which suggests the selenium status of sheep may influence the rate of acquisition of resistance to parasitic infection (Celi et al. 2010). The observed negative correlation between faecal egg counts and GSH-PX suggests that an increase in GSH-PX activity may reduce parasitic infection. However, as the correlation was not very strong it is likely that selenium status is not the only factor responsible for the development of resistance to gastrointestinal parasites in Merino sheep (Celi et al. 2010). Indeed, there are a number of other factors that can also play a role in the development of resistance and resilience to gastrointestinal parasites such as age, climatic conditions, genetics, nutrition and grazing management (Van Houtert and Sykes 1996). Further evaluation of the contribution of selenium to worm expulsion should contribute to an understanding of the role of oxidative stress in the development of resistance to gastrointestinal parasites.

Free radicals and blood parasites

Theileriosis is a progressive lymphoproliferative disease of cattle caused by the protozoan parasite Theileria annulata. The parasite acts as a serious constraint to cattle production in endemic areas, causing lethal infections in exotic cattle and considerable mortality in indigenous and crossbred stocks (Glass and Jensen 2007; Morrison 2009). There is some evidence that oxidative stress and lipid peroxidation incorporate in pathogenesis of anaemia in theileriosis. An increase in oxidative stress and in lipid peroxidation in erythrocytes of cattle infected with T. annulata has been reported, and it seems that this might be the cause of increased erythrocyte fragility due to membrane lysis (Grewal et al. 2005). Intra-erythrocytic parasite, T. annulata (Sahoo et al. 2001; Grewal et al. 2005) and Theileria sergenti (Yagi et al. 1992; Shiono et al. 2003), infection in cattle metabolizes hemoglobin and produces superoxide (O₂ ^·−), which, in turn, causes increased oxidative stress as indicated by a significant increase in lipid peroxidation in erythrocytes (Ginsburg and Atamina 1994; Mishra et al. 1994).

The levels of methemoglobin, used as an index of erythrocytes oxidation, markedly increase at the onset of anaemia in experimental T. sergenti infection (Silanikove 2000), and an inverse relationship has been observed between methemoglobin levels and packed cell volume (PCV) (Shiono et al. 2001). An increase in protein oxidation in the membrane of erythrocytes has also been reported in T. sergenti-infected cattle (Yagi et al. 2002).

Lipid peroxidation in erythrocytes of theileria-affected cattle increases malondialdehyde (MDA) production (Grewal et al. 2005). Increased MDA concentration in erythrocytes of affected cattle may be an indication of elevated oxidative stress in theileriosis. High levels of MDA have been associated with erythrocyte infection rate with T. annulata and the severity of anaemia (Asri Rezaei and Dalir-Naghadeh 2006). It had been reported that in cattle with T. sergenti infection and during the onset of anaemia, levels of MDA increased remarkably in proportion to the decrease of PCV and increase of parasitemia (Shiono et al. 2001, 2003). Similar observations have been reported in calves infected with T. sergenti (Haider 1992). The erythrocyte membrane is rich in polyunsaturated fatty acids, a primary target for reactions involving free radicals and is very susceptible to lipid peroxidation (May et al. 1998; Devasena et al. 2001). During the serious stage of anaemia, this oxidative index reached its maximum value. These observations further support the hypothesis that oxidative damage to the erythrocytes plays a crucial role in the pathogenesis of anaemia in bovine theileriosis.

The levels of antioxidants in erythrocytes are decreased during the progression of anaemia in cattle infected with T. sergenti (Shiono et al. 2003) and T. annulata (Abd Ellah et al. 2006). G6PD is an indicator of a metabolic disturbance of erythrocytes. This enzyme has a key role in the pentose phosphate pathway, which has critical significance in the survival of erythrocytes (Beutler 1984). G6PD enzyme is the principal source of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), which helps in maintaining glutathione at reduced state, thus protecting erythrocytes from oxidative stress. G6PD serves as an antioxidant enzyme, and the low activity of G6PD has been associated with increased haemolysis in cattle affected by theileriosis (Singari et al. 1991) and increased oxidative stress in endothelial cells (Leopold et al. 2003). Grewal et al. (2005) reported a significant increase in the activities of G6PD and GSH-PX in cattle naturally infected with T. annulata. They considered that this increase was a safe guard mechanism for protecting the erythrocytes from oxidative stress, in response to increased lipid peroxidation in erythrocytes. The variation of the G6PD activities might be related to the severity of the anaemia. Low G6PD activity can be followed by reduced activities of SOD and GSH-PX, because of the dependence of the activity of these enzymes on NADPH+H levels in the cell. Christophersen (1966), Flohe (1971) and Hafeman et al. (1974) also proposed that GSH-PX played a crucial role in preventing membranes from peroxide damage induced by lipid peroxides. Reduced glutathione is required for the disposal of hydrogen peroxide (H₂O₂) from erythrocytes by a reaction catalysed by GSH-PX. This reaction is important because the accumulation of H₂O₂ might decrease the lifespan of erythrocytes by increasing the rate of oxidation of haemoglobin to methemoglobin (Winterbourn 1985). It has been reported that CAT is of equal importance to GSH-PX in the defence of human erythrocytes against H₂O₂-generating reactions (Harvey 1989). It appears that, during theileriosis, SOD (similarly to glutathione (GSH)) plays an important role in the protection of erythrocytes against oxidative stress. Similar findings were reported in other parasitic infections. It has been reported that plasmodium-infected erythrocytes showed a decreased capacity of their antioxidant enzymes, including SOD (Friedman 1979; Wozencraft 1986; Erel et al. 1997), CAT, GSH-PX (Greve et al. 1999), G6PD (Roth et al. 1988), methemoglobin reductase (Stocker et al. 1985) and Vitamin E (Griffiths et al. 2001).

Nitric oxide (NO^· ) has been identified as at least one babesiacidal molecule produced by activated mononuclear phagocytes (MP) (Adler et al. 1995; Goff et al. 1996, 1998). When erythrocytes infected with Babesia bovis grown in culture are exposed to NO ^, either in a cell- free system (Johnson et al. 1996) or in the presence of activated MP (Goff et al. 1998), death of the parasites (crisis forms) occurs rapidly within the erythrocytes. In addition, B. bovis merozoites cultured with bovine MP can induce NO^· production with a babesiacidal effect (Stich et al. 1998).

Studies of Mabbott and Sternberg (1995) demonstrate a direct correlation of NO^· production with the development of anaemia in Trypanosoma brucei-infected mice, and the treatment with NO blockers led to a significant reduction of this anaemia. Pérez-Fuentes et al. (2007) found significant correlation between serum NO^· concentration and disease severity in chronic Chagas disease caused by Trypanosoma cruzi in humans.

Free radicals and endoparasites

Endoparasitic infection is among the major causes of oxidative stress. Lipid peroxidation was observed, and the activities and concentrations of antioxidants systems were decreased in the lungs of cattle infected with Dictyocaulus viviparous (Değer S. et al. 2008). Endoparasites such as Fasciola sp., Trichostrongylus sp. and Eimeria sp., coccidiosis and Taenia saginata larvae in muscle tissue of cattle (Dedra et al. 2004) had been reported to be associated with lipid peroxidation (Nabih and Toos 2002).

Fascioliasis

In fascioliasis, it was noticed that the phagocytic response of the rat liver to the parasite invasion and growth leads to free radical-mediated oxidative stress, which is the causative agent in the initiation and development of lipid peroxidation and increased MDA concentrations in the liver (Maffei Facino et al. 1989, 1993). In addition, infection with Fasciola hepatica reduces the antioxidant abilities in humans (Rehim et al. 2003; Kaya et al. 2007) and rats (Kolodziejczyk et al. 2005, 2006). Lipid peroxidation increased and activities or/and concentrations of antioxidant compounds were significantly changed in the liver of sheep with distomatosis (Değer Y. et al. 2008).

A decrease in plasma ascorbic acid concentrations due to impaired synthesis was found in experimental F. hepatica infections in sheep and rats (Gameel 1982; Kolodziejczyk et al. 2005, 2006), and an improved level of lipid peroxidation was detected in F. hepatica patients supplemented with ascorbate (Rehim et al. 2003). Peroxidative damage of membrane lipids was found to occur over the entire course of the murine liver fluke infection concomitant to a decrease in GSH levels (Maffei Facino et al. 1989; Kolodziejczyk et al. 2005, 2006), and GSH administration to experimentally infected rats greatly reduces the damage to membrane lipids of the liver tissue (Maffei Facino et al. 1993).

Gastrointestinal nematode

Ruminant gastrointestinal nematode infections are common and widespread, and their immunopathology closely resembles that of human gastrointestinal infection. However, research on intestinal parasites linking to oxidative stress in ruminants is still scarce. The responsiveness to nematode infection varies considerably between ruminant species, with goats being markedly more susceptible and less capable of developing immune resistance than sheep (Huntley et al. 1995; Hoste et al. 2008). Marked species differences in antioxidant status have been observed between sheep and goats, and it seems that these differences were influenced by nematode infection (Lightbody et al. 2001). Moreover, an intriguing relationship emerged between antioxidant status and differences in the relative susceptibility of sheep and goats to nematode infection. Compared with sheep, goats have higher plasma concentrations of albumin, SH groups, vitamins E and A, and total antioxidants capacity (TAC) (Lightbody et al. 2001). Thus it seems that the effect of nematode infection on TAC reflects increases in oxidative stress related to intestinal nematode infection. As goats have evolved as browsers, they are less likely than sheep (grazers) to be exposed to parasitic larvae (Torres-Acosta and Hoste 2008). Therefore goats might have been under less evolutionary pressure to develop natural resistance to gastrointestinal parasite infections. Therefore the higher antioxidant status in goats (Lightbody et al. 2001) and camels with intestinal nematode infestation (Abd Ellah et al. 2008) might allow them to counteract the potentially greater oxidative challenge in response to gastrointestinal parasite infection.

Coccidiosis

The pathological alterations in coccidiosis intensify free radical processes by stimulating catalytic activities of enzymes involved in the anti-oxidative protection, GSH-PX and SOD. However, during the disease, lipolysis from the lipid depots is increased due to smaller food intake and exhaustion of the organism by diarrhoea, which leads to intensification of free radical processes and formation of larger quantities of lipid peroxides in blood. Newly formed lipid peroxides and their degradation products are transported by blood stream to inactive organs and tissues having a toxic effect on them and generating cellular membrane damages. The organism then activates its anti-oxidative protection system. With the increased risk of peroxidation of lipids in blood, there is an increase in the enzymatic activity of GSH-PX. When Eimeria enter the digestive system, different developmental stages secrete specific metabolites that may be absorbed and induce changes in the enzymatic activity of the antioxidants in a variety of tissues.

Caprine eimeriosis was reported to be associated with decrease in erythrocyte antioxidant enzyme activities including GSH-PX, SOD and CAT of Eimeria-infected kids (Cam et al. 2008) and GSH level (Gibson et al. 1980), which could be attributed to the release of excessive free radicals during the infection or the decrease in production of these enzymes as a result of liver damage. These radicals may result in the formation of protein peroxide and inactivation of detoxifying enzymes, such as GSH-PX via splitting of peptide chain (Georgieva et al. 2006).

Parasites, such as Trichostronglylidae sp., Eimeria sp. and Babesia sp. in kids induced lipid peroxidation due to increased ROS generation to an extent that overcomes the cellular antioxidants resulted in oxidative stress (Mates 2001; Dede et al. 2002). In addition, there is increased production of aldehydic compounds such as MDA, which is considered one of the bio-products in lipid peroxidation and a marker of oxidative stress (Lee et al. 2004).

Free radicals and external parasites

In skin diseases, the body possesses an array of a potent antioxidant protection such as SOD, CAT, glutathione (GSH), GSH-PX and the antioxidant vitamins A, E and C (Bickers and Athar 2006). Synergistic and co-operative interactions of these antioxidants rely on the sequential degradation of peroxides and free radicals (Portugal et al. 2007). Dimri et al. (2008) found increased SOD and decreased reduced-GSH and CAT in dogs with demodicosis. Decreased SOD and CAT were observed in tissues of sheep with psoroptic mange (Dimri et al. 2010). Furthermore, Dimri et al. (2008) reported decrease in antioxidant enzyme activities and trace mineral concentrations in buffaloes suffering from sarcoptic mange, which suggested that sarcoptic mange in buffaloes is associated with compromise in antioxidant defence, and oxidative stress may play an important role in pathogenesis.

Tategaki et al. (2003) found increased NO^· production in splenic macrophages of mice exposed to mite antigen in a dose-dependent manner. Increased NO^· production was reported also in humans with cutaneous leishmaniasis and other inflammatory skin diseases (Serarslan et al. 2005; Bickers and Athar 2006). Despite that responsiveness is related to the hosts' ability to develop resistance against parasitic infection (Brunet 2001), NO^· may react with O₂ ^·−leading to production of peroxynitrite anion (ONOO⁻), or by the Fenton reaction to produce hydroxyl radical (^·OH) (Radi et al. 2001; Allen et al. 2009). Both ONOO⁻ and ^·OH are the most potent nitrative and oxidative agents which can change lipids and protein structure (Halliwell and Chirico 1993). In scabby camels, the increase blood NO^· was coupled with stimulation of MDA (Saleh et al. 2011). The authors hypothesised that ONOO⁻ and ^·OH formation is increased and can exert their oxidising and cytotoxic effects in scabby camels. Portugal et al. (2007) assumed also that increased ONOO⁻ formation is common in various types of skin diseases.

Conclusion

The infection of the animal body with internal or external parasites is associated with excessive release of free radicals, which may be attributed to decrease the nutrients availed by the body to synthesise antioxidants and may be due to destruction of cells produced by the activity of the parasites. Free radicals are involved in the pathogenesis of most of the parasitic infestations, like T. annulata and T. sergenti, Babesiosis, Trypanosomosis, D. viviparous, Fasciola sp., Trichostrongylus sp., Eimeria sp., coccidiosis and Taenia saginata larvae, that had been reported to be associated with lipid peroxidation. Other parasitic infection in different animal species needs further research, especially the relation between the degree of infestations and the effect on antioxidants defence mechanisms.