The beneficial effect of dietary zinc supplementation on anaemia andimmunosuppression in Trypanosoma brucei infected ratsJ.I. Eze a,*, L.C. Ayogu a, F.O. Abonyi b, U.U. Eze a
a Department of Veterinary Medicine, University of Nigeria, Nsukka, Nigeriab Department of Animal Health and Production, University of Nigeria, Nsukka, Nigeria
Zinc is an essential trace element crucial for normal development and function of cells mediating non-specific immunity and protects bio-molecules from oxidative damage. This study was designed to assessthe effects of dietary zinc supplementation on anaemia and immunity of trypanosome-infected rats. Thirtyrats, divided into five groups (A–E) of 6 each, were used for the study. Parameters used to assess the effectof the supplementation are antibody response to Sheep RBC using direct haemagglutination test,parasitaemia using the rapid matching method, WBC count using the improved Neubauer haemocytometermethod, haemoglobin concentration using the cynomethaemoglobin technique while PCV was deter-mined using the microhaematocrit method. The pre-infection supplementation did not prolong the pre-patent period significantly (p > 0.05). However, it significantly (p < 0.05) increased the packed cell volume(PCV), haemoglobin (Hb) concentration, leucocyte count, and antibody titre by day 7 on the supplemen-tation (OTS). Following infection on day 7 OTS, the PCV and Hb decreased but remained significantly(p < 0.05) higher than the infected not supplemented (INS) group, while on day 14 OTS, they main-tained a significantly (p < 0.05) higher antibody titre as compared to other groups. On day 21 OTS, the
weight of 8 ppm and not infected not supplemented (NINS) groups was significantly (p < 0.05) higherbut the relative organ weight of their liver and spleen was significantly (p < 0.05) lower than 2 ppm, 4 ppmand INS groups. On day 21 OTS, the parasitaemia levels of INS group was significantly (p < 0.05) higherthan the supplemented groups. From the results, dietary zinc supplementation can be useful in themanagement of anaemia and immunosupression caused by trypanosomes in rats.
Animal and human trypanosomosis remains responsible for sub-stantial global morbidity and mortality in tropical and subtropicalregions (Espuelas et al., 2012). Animal trypanosomosis causes directlosses to livestock production, with more than 20 million Ameri-can dollars spent per annum on trypanocidal drugs and indirectlosses related to the opportunity cost of land and other resourcescurrently not used for livestock production due to the presence oftsetse fly (FAO, 2005). Also, approximately 70 million people dis-tributed over a surface of 1.55 million km2 are estimated to be atdifferent levels of risk of contracting Human African trypanosomosis(Simarro et al., 2012). Major modifications of immune systemand erythrocytic indices have been observed, thus makingimmunosupression and anaemia the major features in Africantrypanosomosis.
In the absence of safe and efficient vaccines, chemotherapy, to-gether with vector control, remains the most important measureto control the disease. Nevertheless, the current chemotherapeu-tic treatments are clearly inadequate because of their toxic effects,generation of resistance as well as route and schedule of adminis-tration not well adapted to the field condition (Espuelas et al., 2012).Also, trypanosomes, especially the Trypanosoma brucei sub-groups,are known for their antigenic variation and subsequent escape fromimmune clearance. The qualitative and quantitative aspects of thehost immune response play an important role in the disease processand seem to be essential for the control of the early parasite rep-lication, which is associated with host resistance (Trishmann, 1986).
In view of the above, many researchers have used immuno-stimulants or immunomodulatory agent such as vitamins andmicronutriets in the management of African trypanosomosis (Ezeand Ochike, 2007; Eze et al., 2011; Toma et al., 2008). The immunesystem is a highly proliferative, complex and integrated networkof cells and organs, and therefore can be strongly influenced by thesemicronutrients and vitamins (Wellinghausen and Rink, 1998).
Zinc biology is a rapidly developing field, and recent researchreveals zinc’s strategic role in most organ systems. Zinc, an essen-tial trace element, is required by all organisms and modulates theimmune response, influencing cellular growth and affecting the de-velopment and integrity of immune system (Dardene, 2002). Also,zinc functions as an antioxidant and is involved in many critical bio-chemical reactions. Zinc deficiency results in dysfunction of plasmamembrane proteins, which present with some pathological fea-tures (Sharifian et al., 2012; Wellinghausen and Rink, 1998). It hasbeen found that zinc deficiency is associated with alteration of theimmune response, anaemia, increased erythrocyte fragility, perni-cious anaemia, as well as some other bodily functional abnormalities.Generally, zinc deficiency has been shown to impair host defencesfrom a variety of bacterial, parasitic, fungal and viral diseases(Pekarek et al., 1977; Shankar and Prasad, 1998; Van Eeckhout et al.,1976; Wellinghausen, 2001).
The role of zinc in immune modulation and its influence in thecourse and outcome of infections is being increasingly recognisedin recent years (Bhaskaram, 2002). Zinc is known to play a centralrole in the immune system, and zinc-deficient persons experienceincreased susceptibility to a variety of pathogens (Shankar andPrasad, 1998) such as Trypanosoma cruzi (Fraker et al., 1982) andTrypanosoma musculi (Lee et al., 1983). Severe zinc deficiency is
characterised by severely depressed immune function, frequent in-fections, bullous pustular dermatitis, diarrhoea, alopecia, and mentaldisturbances (Shankar and Prasad, 1998).
Mwangi et al. (1995) reported a decrease in serum zinc level inrabbits infected with Trypanosome b brucei. Also, Olurode et al. (2009)reported an enhanced chemotherapeutic efficacy of diaminazineaceturate in T. brucei infected rats fed zinc supplemented diets. Zincdeficiency can be reversed with zinc supplementation and nutri-tional doses of zinc supplements prevent alteration of the immunefunction and improve resistance to infections. The effects of zincsupplementation have been studied in Chagas disease caused by Try-panosoma cruzi (Gonçalves-Neto et al., 2011) and Tyrpanosoma evansi(Brazão et al., 2009; Dalla Rosa et al., 2012).
Zinc levels may be an influential factor determining suscepti-bility or resistance of West African cattle to trypanosomiasis(Traoré-Leroux et al., 1985). In an investigation, decrease in zinc levelscoincided with the onset of T. brucei gambiense in peripheral bloodof rabbit (Mwangi et al., 1995). Zinc-deficient animals showed threetimes the number of trypanosomes as that of the complete and pair-fed mice (Lee et al., 1983).
This study was therefore undertaken to investigate the effect ofzinc supplementation in T. brucei brucei infected rats and the pos-sible protective effects on anaemia and immune response.
2. Materials and methods
2.1. Experimental animals
Thirty (30) growing rats were used for this study. The rats wereobtained from the Department of Veterinary Medicine laboratoryanimal unit and kept in rat cages in a fly proof departmental ex-perimental room. The rats were fed and given water ad libitum. Theywere allowed 7 days for acclimatisation.
2.2. Trypanosome isolate
The Trypanosoma brucei brucei used for the experiment was iso-lated from a naturally infected dog presented at the VeterinaryTeaching Hospital, University of Nigeria, Nsukka. The isolate wasproperly characterised and maintained as UNVTH 007 through serialpassage in rats.
2.3. Zinc oxide
Zinc as zinc oxide was used for the study and was produced byZinc National Monterrey, NL, Mexico.
2.4. Sheep red blood cells (SRBC)
Fresh sheep blood was obtained from sheep in the animal houseof the Department of Veterinary Parasitology and Entomology, Uni-versity of Nigeria, through their jugular vein. Before use, the red bloodcells were washed three times with 1 part of blood to 9 parts ofphosphate-buffered saline (PBS), pH 7.2, by centrifugation at3000 rpm for 10 min on each occasion. After the final wash, theSRBCs were suspended in PBS as a 2% suspension (based on packedcell volume) for the serological tests and as a 10% suspension for
88 J.I. Eze et al./Experimental Parasitology 154 (2015) 87–92
immunization of the rats. A 1 ml amount of the 2% suspension con-tained approximately 5 × 108 red blood cells.
2.5. Experimental design
The rats were randomly assigned into five groups (A–E), witheach group having six (6) rats each. Groups A, B and C rats werefed with feed supplemented with 2, 4 and 8 parts per million (ppm)of zinc. The supplemented groups and group D rats were infectedwith 5.34 × 105 trypanosomes per mouse on day 7 on the supple-mentation. Group E served as uninfected control.
The parameters used to assess the effect of the supplementa-tion were as follows: change in body weight, level of parasitaemia,antibody titre, haemoglobin concentration, packed cell volume, totalwhite blood cell count and relative organ weight. The parameterswere taken on day 0 and every other 7th day. The rats were hu-manely sacrificed on day 21 on the supplementation after samplecollection, and their liver, spleen and heart carefully dissected outfor determination of relative organ weight (ROW). Animal studieswere in compliance to the ethical procedure of the Animal Use andCare Committee, Faculty of Veterinary Medicine, University ofNigeria, Nsukka, which corresponds with NIH guidelines (NIH, 1996).
2.6. Detection of parasites
Following infection of rats with T. brucei, the parasitaemia weremonitored in the rat blood using the wet mount and haematocritcentrifuge methods on a daily basis until all rats were positive.
2.7. Blood sample collection
Blood sample was collected from the retro-bulbar plexus of themedian canthus of the eyes of the rats using microhaematocrit cap-illary tube. The blood was collected into two different sample bottles,one containing anticoagulant (EDTA) and the other without anti-coagulant for haematology and serology respectively.
2.8. Determination of antibody titre
Sheep RBCs (0.1 ml of 10% sheep RBC) were used to immunisethe rats by intraperitoneal injection and challenged by similar IPinjection of the same amount in day 5 post immunization (PI). Onthe 7th day post challenge or day 0 (day supplementation started)and subsequent 7 days, the antibody response was determined usinghaemagglutination test as described by Nelson and Mildenhall(1967). Booster doses of the sheep RBC were given every other 14thday following challenge.
2.9. Determination of PCV, haemoglobin concentration, totalleucocyte count and parasitaemia
The packed cell volume and Hb concentration were deter-mined by the microhaematocrit methods (Jain, 1986) andcyanomethaemoglobin method (Jain, 1986) using SP6-500UV spec-trophotometer (PYE UNICAM, England) respectively. The totalleucocyte count were carried out manually using the improvedNeubauer haemocytometer method, as described by Jain (1986).Parasitaemia was estimated using the matching method of Herbertand Lumsden (1976).
2.10. Determination of body weight and relative organ weight
The weight of individual rat was determined using electronicweighing balance. Whereas the relative organ weights (ROW) of dif-ferent organs were calculated using the formula:
Relative organ weightAbsolute organ weightWhole animal we
=iight
2.11. Statistical analysis
The data collected were subjected to analysis of variance(ANOVA). Variant means were then separated using Duncan’s mul-tiple range tests. Differences between means were consideredsignificant at p < 0.05.
3. Results
The supplementation did not prolong the pre-patent period sig-nificantly (p > 0.05), with the pre-patent period being 5.32 ± 0.36,5.64 ± 0.61, 6.02 ± 0.83 and 4.88 ± 0.44 days for 2 ppm, 4 ppm,8 ppm, infected not supplemented (INS) and not infected notsupplemented (NINS) groups respectively. The pre-infection supple-mentation made no significant changes on the packed cell volume(PCV) in all the groups (Fig. 1). However, on day 14 on the supple-mentation (OTS), 4 ppm, 8 ppm and NINS groups were significantly(p < 0.05) higher than 2 ppm and INS groups. Also, on day 21 OTS,NINS group was highly significantly (p < 0.01) higher than 2 ppm,4 ppm and INS groups, but significantly (p < 0.05) higher than 8 ppmgroup. Also from Fig. 2, the pre-infection supplementation with zincincreased the haemoglobin concentration of 8 ppm group signifi-cantly (p < 0.05) on day 7 OTS when compared with other groups.Following infection on day 7 OTS, the Hb concentration on days 14and 21 OTS decreased in all the infected groups, with INS group beingsignificantly (p < 0.05) lower than other groups. From Fig. 3, the pre-infection supplementation significantly (p < 0.05) increased the WBCcount of the supplemented groups when compared with theunsupplemented groups on day 7 on the supplementation (OTS).On day 14 OTS, INS and NINS groups were significantly (p < 0.05)lower than 2, 4 and 8 ppm groups. However, on day 21 OTS, NINSgroup was significantly (p < 0.05) higher than other groups. Thesupplementation led to increase in antibody titre of the supple-mented groups (Fig. 4). The antibody titre increased significantly(p < 0.05) in the supplemented groups when compared tounsupplemented groups on day 7. Following infection, the supple-mented groups maintained significantly (p < 0.05) higher antibodytitre when compared with other groups on days14 OTS. However,on day 21 the antibody response declined in all supplemented groupsbut not to the level of pre-supplementation values, with 8 ppm groupbeing significantly (p < 0.05) higher than other groups. Figure 5 shows
Fig. 1. Bar chart of mean packed cell volume (%) of Trypanosoma brucei infectedfed diet supplemented with different levels of zinc.
89J.I. Eze et al./Experimental Parasitology 154 (2015) 87–92
the relative organ weights of the liver and spleen of the supple-mented and control groups. The relative organ weight of the liverof 2 ppm, 4 ppm and INS groups were significantly higher than 8 ppmand NINS groups. On the other hand, the relative organ weight ofthe spleen showed that NINS group was significantly lower thangroups A, B and D, but not with group C. The supplementation didnot cause any significant change between the infected groups onday 14 OTS (Fig. 6). However, on day 21 OTS the parasitaemia levelsof INS group had significantly (p < 0.05) higher parasitaemia thanother infected groups. From Fig. 7, the supplementation with zincbefore infection with Trypanosoma brucei brucei did not signifi-cantly (p > 0.05) affect the weight of the different groups. However,following infection, the weight of 8 ppm and NINS groups was sig-nificantly (p < 0.05) higher than other groups on day 21 OTS.
4. Discussion
The pre-patent period was not significantly increased follow-ing pre-infection supplementation with zinc. This differs with thereport that zinc supplementation prolonged the pre-patent periodof supplemented groups when compared with the control in
Trypanosoma evansi infected rats (Dalla Rosa et al., 2012). The vari-ation could be attributed to the difference in species, route ofadministration or dosage and duration of supplementation. Packedcell volume and haemoglobin concentration were used to assess theanaemia in this study. Increase in PCV values and haemoglobin con-centrations were recorded before infection. Zinc plays an importantrole in haemoglobin synthesis by activating delt-aminolaevutinicacid (ALA) dehydrogenase, an enzyme essential for the formationof porphobilinogen from two ALA molecules (Akhtar et al., 2003;Jaffe and Lawrence, 2013). Also, the increase in PCV values can beattributed to the anti-oxidative effect of zinc. Zinc concentrationsin cell membranes appear to be important in preserving their in-tegrity (Bray and Bettger, 1990) and, together with glutathioneperoxidase and superoxide dismutase, plays a crucial role in the
Fig. 2. Bar chart of mean haemoglobin concentration (g/dl) of Trypanosoma bruceiinfected fed diet supplemented with different levels of zinc.
Fig. 3. Bar chart of mean leucocyte count (103 cells per ml) of Trypanosoma bruceiinfected fed diet supplemented with different levels of zinc.
Fig. 4. Bar chart of mean antibody titre (mlU/mL) of Trypanosoma brucei infectedfed diet supplemented with different levels of zinc.
Fig. 5. Bar chart of mean relative organ weight (10−3 g) of Trypanosoma brucei in-fected fed diet supplemented with different levels of zinc.
90 J.I. Eze et al./Experimental Parasitology 154 (2015) 87–92
antioxidant system and thus protects biomolecules from oxidativedamage (Mustafa et al., 2010; Prasad, 2014).
The PCV and haemoglobin values of the supplemented groupsremained significantly higher than the infected unsupplementedgroup but did not differ with the uninfected control following in-fection. This result is consistent with the works of Silva et al. (2006)and Polat (2011). This could be attributed to the beneficial effectof zinc as an antioxidant and immunostimulant. Oxidative stressof the red blood cells (RBC) and their subsequent life span reduc-tion are suggested to play an important role in the development ofanaemia in African trypanosomsosis (Eze et al., 2008; Igbokwe andMohammed, 1992; Taiwo et al., 2003). Zinc is a component of dozensof vital enzymes within the body and in these enzymes the zinc mol-ecule acts directly as an anti-oxidant, protecting the biochemicalstructure of the enzyme from free radical attack. Secondly, zinc actsto stabilise proteins which may otherwise react with highly unsta-ble minerals, particularly iron and copper, to form free radicals. Zincdeficiency has been found to be associated with anaemia, in-creased erythrocyte fragility, as well as some other bodily functionalabnormalities (Akhtar et al., 2003; Jaffe and Lawrence, 2013). There-fore, it is expected that zinc supplementation would reduce oxidative
stress, prolong RBC survival, and thus maintain high PCV and hae-moglobin levels.
Increase in WBC count as reported in this work has been shownto indicate improved immune response (Ufele et al., 2007). The in-creases due to zinc supplementation have been previously reported(Baltaci et al., 2003; Sajadifar, 2012). Leucocyte counts during stressand infectious disease is a measure of immune response (Dufva andAllander, 1995; Hardie et al., 1991; Ufele et al., 2007) as they areimportant in protecting our body against infection. Improvementof WBC counts in zinc supplemented groups has been attributedto antioxidant role of zinc that is known to modulate immune func-tion by protecting cells from damaging effects of oxygen radicals(Bray and Bettger, 1990).
The mean antibody titres of the supplemented groups were sig-nificantly higher than other groups both pre- and post-infection.This is in agreement with other reports that zinc supplementationincrease antibody response to antigens (Brazão et al., 2008a). Bothprimary and secondary antibody responses have been reported todecrease in Zn deficient mice. Studies in young adult Zn-deficientmice have shown greatly depressed responses to both T-lymphocyte-dependent and T-lymphocyte-independent antigens. The decreasenoted in the mean leucocyte count and antibody titre on day 21 OTScould be attributed to the overwhelming influence of trypanosoposis.The relative organ weight of the spleen, liver and heart at the highestdose (8 ppm) did not differ from the uninfected control. This is anindication that zinc supplementation was able to prevent/reducesplenomegally, an important pathology in trypanosomosis (Taylorand Authie, 2004). The increase in spleen has been related toanaemia. Enlargement occurs in animals and humans as a result ofphysiological adaptation, metabolic abnormalities, toxic effects, in-flammatory processes or proliferative diseases (Williams andIatropoulos, 2002).
The supplementation led to decrease in parasitaemia of the supple-mented groups which agrees with earlier reports by Brazão et al.(2008b) and Gonçalves-Neto et al. (2011). Zinc has been reportedto influence host resistance mechanisms, thus altering the suscep-tibility to infectious diseases (Traoré-Leroux et al., 1985). Mwangiet al. (1995) reported a decrease in serum zinc level in rabbits in-fected with Trypanosome b brucei. Hence, decreasing zincconcentrations in vivo impair natural killer (NK) cell activity, phago-cytosis of macrophages and neutrophils, and certain functions likechemotaxis and generation of the oxidative burst (Keen and Gershwin,1990; Wellinghausen and Rink, 1998). For instance, in zinc defi-ciency a reduced capability of mononuclear phagocytes to killintracellular Trypanosoma cruzi is reported by Wirth et al. (1989)and Cook-Mills et al. (1990). It has also been reported that zinc supple-mentation led to an effective host immune response by up-modulatingthe host’s immune response, thus contributing to a reduction of bloodparasites and the harmful pathogenic effects of infections (Brazãoet al., 2009). Reports have shown that increase in parasitaemia intrypanosomosis corresponds with a rise in rectal temperature, rapidweight loss, packed cell volume decline and decrease in totalplasma protein in all the infected animals (Eze et al., 2013;Katugunka-Rwakishaya et al., 1995). In view of the above, suppres-sion of parasitaemia following zinc supplementation in trypanosomesinfected animal would ameliorate the pathology of the disease.
The supplementation with zinc before infection with Trypano-soma brucei brucei did not significantly (p > 0.05) affect the bodyweight of the different groups. This is in line with other results thatzinc supplementation did not alter the weight of the animals (Hahnand Baker, 1993; Malcolm-Callis et al., 2000). However, a study byMayland et al. (1980) on calves showed that zinc supplementa-tion can cause significant increases in weight gain.
In conclusion, zinc supplementation enhanced the immune re-sponse and reduced anaemia in Trypanosoma brucei brucei infectedrats.
Fig. 6. Bar chart of mean parasitaemia (106 parasites /ml of blood) of Trypanosomabrucei infected fed diet supplemented with different levels of zinc.
Fig. 7. Bar chart of mean body weight (g) of Trypanosoma brucei infected fed dietsupplemented with different levels of zinc.
91J.I. Eze et al./Experimental Parasitology 154 (2015) 87–92
Conflict of interest
There were no conflicts of interests related to this work.
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