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Veterinary Parasitology 175 (2011) 66–72

Contents lists available at ScienceDirect

Veterinary Parasitology

journa l homepage: www.e lsev ier .com/ locate /vetpar

nticoccidial and antiinflammatory activity of garlic in murine Eimeriaapillata infections

.A. Dkhil a,b,∗, A.S. Abdel-Bakia,c, F. Wunderlicha,d, H. Siesa,e, S. Al-Quraishya

Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi ArabiaDepartment of Zoology and Entomology, Faculty of Science, Helwan University, EgyptDepartment of Zoology, Faculty of Science, Beni-Suef University, EgyptDepartment of Molecular Parasitology, Heinrich Heine University, 40225 Duesseldorf, GermanyDepartment of Biochemistry and Molecular Biology, Heinrich Heine University, 40225 Duesseldorf, Germany

r t i c l e i n f o

rticle history:eceived 6 March 2010eceived in revised form 26 August 2010ccepted 9 September 2010

eywords:arlicoccidiosisnticoccidiumnti-inflammatory activity

a b s t r a c t

Coccidiosis with the protozoan parasite Eimeria as the infectious agent causes enormouseconomic losses, particularly in poultry farms. Here, we investigated the effects of garlicon the outcome of coccidiosis caused by Eimeria papillata in male Balb/c mice. The datashowed that mice infected with E. papillata revealed an output of 3260 ± 680 oocysts pergram faeces on day 4 p.i.. This output is significantly decreased to 1820 ± 415 oocysts ingarlic-treated mice. Infection also induced inflammation and injury of the liver. This wasevidenced (i) as increases in inflammatory cellular infiltrations, dilated sinusoids, and vac-uolated hepatocytes, (ii) as increased mRNA levels of inducible nitric oxide synthase (iNOS)and of the cytokines interferon gamma (IFN-�), and interleukin-6 (IL-6), (iii) as increasedplasma levels of alanine and aspartate aminotransferases, alkaline phosphatase, �-glutamyltransferase and total bilirubin, (iv) as increased production of nitric oxide derived products

(nitrite/nitrate) and malondialdehyde, and (v) as lowered glutathione levels and decreasedactivities of catalase and superoxide dismutase, respectively. All these infection-inducedparameters were significantly less altered during garlic treatment. In particular, garliccounteracted the E. papillata-induced loss of glutathione and the activities of catalase and

tase. Ouinjury o

superoxide dismuinflammation and

. Introduction

Coccidiosis is a widespread disease worldwide affectingany vertebrates. This disease is caused by the unicellu-

ar eukaryote Eimeria, which exhibits approximately 800ifferent species (Mehlhorn, 2001). Eimeria tenella is the

ost dangerous species causing dramatic economic losses

n poultry farms.Eimeria are common parasites in the digestive tract of

he hosts causing diarrhea and fluid loss. Infections begin

∗ Corresponding author at: Zoology Department, College of Science,ing Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia. Tel.:966 14675754; fax: +966 14678514.

E-mail address: mohameddkhil@yahoo.com (M.A. Dkhil).

304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2010.09.009

r data indicated that garlic treatment significantly attenuatedf the liver induced by E. papillata infections.

© 2010 Elsevier B.V. All rights reserved.

with oral uptake of Eimerian oocysts which release sporo-zoites in the intestine. These in turn invade enterocytesin which they multiply, and finally, oocysts are releasedagain with the faeces. Renaux et al. (2001) have also iden-tified an extra-intestinal route of migration of Eimerianparasites mainly in lymphoid organs. The coccidial par-asites are characterized by fast reproduction leading toinfection especially of young animals (Ryley and Robinson,1976; Licois et al., 1992; Gres et al., 2003; Pakandl, 2005).Infections with coccidia resulted in pathological changes

which can be detected in blood, urine and stool (Licois etal., 1978a,b; Jithendran and Bhat, 1996; Kulisic et al., 1998;Tambur et al., 1998a,b,c).

There are numerous reports indicating the efficacy ofgarlic in the prevention and treatment of a variety of

ry Paras

M.A. Dkhil et al. / Veterina

diseases and for validating its traditional uses. For instance,garlic has been described to exhibit antimicrobial activity(Chowdhury et al., 1991; Yoshida et al., 1998; Fleischaueret al., 2000), antitumor activity (Karasaki et al., 2001;Sundaram and Milner, 1996), as well as antithrombotic,antiarthritic, hypolipidemic, and hypoglycemic activities(Duraka et al., 2002; Kumar et al., 2003). Moreover, garlichas been reported to be effective against diverse para-sites such as Amoeba (Peyghan et al., 2008), Leishmania(Ghazanfari et al., 2006), Trypanosoma (Nok et al., 1996) andCryptosporidium (Wahba, 2003). The present study is aimedat investigating any possible effects of garlic on infectionswith Eimeria papillata in mice, particularly on the outcomeof disease and on the inflammatory response of the liver ofmale mice.

2. Materials and methods

2.1. Animals

Male Balb/c mice aged 8–9 weeks were obtained fromthe animal facilities of King Saud University, Riyadh, SaudiArabia. The mice were bred under specified pathogen-free conditions and fed a standard diet and water adlibitum. The experiments were approved by the stateauthorities and followed Saudi Arabian rules on animalprotection.

2.2. Infection with E. papillata

E. papillata used in this study is a pathogenic species ofthe distal small intestine (Danforth et al., 1992). Oocystswere collected from faeces of mice naturally infectedwith E. papillata and then surface sterilized with sodiumhypochlorite and washed at least four times in sterile salineprior to oral inoculation as described by Schito et al. (1996).These oocysts were used to inoculate mice by oral gavagingeach mouse with 103 sporulated oocysts of E. papillata sus-pended in 100 �l sterile saline. Once every 24 h, fresh faecalpellets were collected and weighed for each mouse andthe bedding was changed to eliminate reinfection. Oocystoutput was measured as previously described (Schito etal. 1996). Faecal pellets were suspended in 2.5% (wt/vol)potassium dichromate and diluted in saturated sodiumchloride for oocyst flotation. Oocysts were counted in aMcMaster chamber and expressed as number of oocystsper gram of wet faeces.

2.3. Garlic treatment

Garlic extract was prepared by homogenizing cloves ofgarlic in sterile saline at a final concentration of 20 mg/mlusing a tightly fitting Potter-Elveijhem teflon homoge-nizer as described previously (Balasenthil et al., 1999). Thehomogenate was centrifuged at 3000 × g for 10 min and thesupernatant was frozen at −20 ◦C until use. Three groups of

mice, with 6 animals per group, were investigated. The firstgroup was inoculated only with sterile saline and servedas the control group. The second and third groups wereinfected with 103 sporulated oocysts of E. papillata. Onlymice of the third group were treated by oral gavage with

itology 175 (2011) 66–72 67

100 �l garlic extract (20 mg/ml) daily for 4 days during theinfection.

2.4. Preparation of plasma and livers

Blood was taken from retro-orbital plexus of all 18 mice,and plasma was prepared and stored in liquid nitrogenuntil use. Then, mice were killed by cervical dislocation.Livers were aseptically removed, cut up in sterile saline,and stored in liquid nitrogen until use—if not otherwisestated.

2.5. Histology

Liver pieces, prepared from the 3 groups of mice onday 4 p.i., were fixed in 10% neutral buffered formalin atroom temperature for 6 h before embedding in paraffin andsectioning. The sections were stained with haematoxylinand eosin according to the method described by Drury andWallington (1980).

2.6. Quantitative real-time PCR

Total RNA of liver pieces was isolated using Trizol(Invitrogen). Contaminating genomic DNA was digestedwith DNase using the DNA-freeTM kit (Applied Biosys-tem, Darmstadt, Germany). Then, the QuantiTect® ReverseTranscription kit (Qiagen, Hilden, Germany) was used tosynthesize cDNA. Real time PCR was performed in a Taq-Man7500 (Applied Biosystems) using the QuantiTectTM

SYBR® Green PCR kit (Qiagen) and the gene-specificQuantiTectTM primer assay (Qiagen) according to themanufacturer’s instructions. The primers for induciblenitric oxide synthase (iNOS), interferon gamma (IFN-�),interleukin-6 (IL-6) and 18S rRNA were purchased fromQiagen (Hilden, Germany). Following an initial incubationat 50 ◦C for 2 min, Taq polymerase was activated at 95 ◦Cfor 10 min, 45 cycles followed at 95 ◦C for 15 s, at 60 ◦Cfor 35 s, and for 30 s at 72 ◦C. PCR product was measuredas SYBR green fluorescence at the end of the extensionphase. All PCR reactions yielded only a single product ofthe expected size as revealed by melting point analysisand gel electrophoresis. Relative quantitative evaluationof amplification data was done using Taqman 7500 sys-tem software v.1.2.3f2 (AppliedBiosystems) and the 2−��c

T method (Livak and Schmittgen, 2001). Expression of thegenes was compared to 18S rRNA (Krücken et al., 2009).

2.7. Liver enzymes in plasma

2.7.1. AminotransferasesAlanine aminotransferase (ALT) and aspartate amino-

transferase (AST) activities in plasma were determinedby the method of Reitman and Frankel (1957). Substrates(0.5 ml) (�-ketoglutarate (2 mM) and either �-alanine or �-l-aspartate (0.2 mM)) were incubated for 5 min at 37 ◦C in a

water bath. Serum (0.1 ml) was then added and the volumewas adjusted to 1.0 ml with 0.1 M phosphate buffer (pH7.4). The reaction mixture was incubated for 30 and 60 minfor ALT and AST, respectively. Then, 0.5 ml of 1 mM dinitro-phenylhydrazine was added to the reaction mixture and

6 ry Parasitology 175 (2011) 66–72

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eft for another 30 min at room temperature. Finally, theolor was developed by addition of 5.0 ml of NaOH (0.4 N)nd absorbance read at 505 nm in a Jasco-Japan-V530 spec-rophotometer.

.7.2. �-Glutamyl transferase�-Glutamyl transferase (�GT) was estimated in plasma

omogenate according to the method described by Szasz1969). The substrate l-�-glutamyl-4-nitroanilide, in theresence of glycylglycine, is converted by �GT in the sam-le to 4-nitroaniline which was measured at 405 nm in a

asco-Japan-V530 spectrophotometer.

.7.3. Alkaline phosphataseAlkaline phosphatase (ALP) activity in plasma was

ssayed according to the method of Ellis et al. (1971).lkaline phosphatase in the sample hydrolysed phenylhosphate to form phenol and phosphate at pH 10.0. The

iberated phenol is measured colorimetrically in the pres-nce of 4-aminophenazone and potassium ferricyanide.

.8. Bilirubin

Total bilirubin (TB) in plasma was assayed according tohe method of Walters and Gerade (1970). The reactionetween bilirubin and the diazonium salt of sulfanilic acidroduced azobilirubin which show a maximum absorbancet 535 nm in an acid medium. In the presence of dimethyl-ulfoxide (DMSO) the total bilirubin participates in theeaction and, in the absence of (DMSO), only conjugatedilirubin reacts.

.9. Lipid peroxidation

Lipid peroxidation in plasma and liver homogenateere determined according to the method of Ohkawa et

l. (1979) by using 1 ml of trichloroacetic acid 10% and 1 mlf thiobarbituric acid 0.67%, followed by heating in a boilingater bath for 30 min. Thiobarbituric acid reactive sub-

tances were determined by the absorbance at 535 nm andxpressed as malondialdehyde (MDA) equivalents formed.

.10. Nitrite

The assay of nitrite in serum and liver homogenate wereone according to the method of Berkels et al. (2004). Incid medium and in the presence of nitrite the formeditrous acid diazotises sulphanilamide, which is coupledith N-(1-naphthyl) ethylenediamine. The resulting azoye has a bright reddish-purple color which was measuredt 540 nm.

.11. Glutathione

Glutathione (GSH) was determined chemically in liveromogenate using Ellman’s reagent (Ellman, 1959). The

ethod is based on the reduction of Ellman’s reagent

5,5′dithiobis(2-nitrobenzoic acid) with GSH to produce aellow compound. The chromogen is directly proportionalo GSH concentration, and its absorbance was measured at05 nm.

Fig. 1. Mice oocyst output on day 4 p.i. with 103 sporulated E. papillataoocysts. Infected mice without garlic treatment (−garlic). Mice infectedand treated with garlic (+garlic). All values are means ± SD. *p < 0.001 for−garlic vs. +garlic.

2.12. Activities of catalase and superoxide dismutase

Catalase activity in plasma was assayed by the methodof Aebi (1984). Catalase reacts with a known quantity ofH2O2. The reaction is stopped after exactly one minutewith a catalase inhibitor. In the presence of horseradishperoxidase, remaining H2O2 reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone toform a chromophore with a color intensity inversely pro-portional to the amount of catalase in the original sample,and measured at 240 nm). Superoxide dismutase activ-ity in plasma was assayed by the method of Nishikimi etal. (1972). This assay relies on the ability of the enzymeto inhibit the phenazine methosulphate-mediated reduc-tion of nitroblue tetrazolium dye, which was measured at560 nm.

2.13. Statistical analysis

Values in tables are presented as means ± standarddeviation (n = 6). Two-way ANOVA was carried out, and thestatistical comparisons among the groups were performedwith Duncan’s test using a statistical package program(SPSS version 17.0). Data obtained with qRT-PCR were com-pared with student’s t-test.

3. Results

Infection of mice with E. papillata resulted in an outputof 3260 ± 680 E. papillata oocysts per gram faeces on day 4p.i., as shown in Fig. 1. Treatment of mice with garlic extractsignificantly decreased the oocysts expulsion by about 44%,i.e. only 1820 ± 415 oocysts were released per gram feceson day 4 p.i.

E. papillata infections caused a moderate inflamma-

tory response of the liver as indicated by inflammatorycellular infiltrations, cytoplasmic vacuolation and degen-eration of hepatocytes. In addition, the hepatic sinusoidswere dilated and apparently contained more Kupffer cellsas compared to livers of the non-infected control of mice

M.A. Dkhil et al. / Veterinary Parasitology 175 (2011) 66–72 69

Fig. 2. Protective role of garlic against infection with E. papillata in hep-atic tissue of mice. (A) Non-infected mouse liver. (B) Infected mouse liveron day 4 p.i. showing inflammatory cellular infiltration and dilated sinu-soids. Some hepatocytes appeared vacuolated. (C) Liver treated with garlicextract with lowered inflammation and damage. Sections stained withhematoxylin and eosin. Bar = 50 �m.

Fig. 3. Real-time RT-PCR analysis of iNOS, IFN-� and IL-6 in the liver.Expression was analyzed in hepatic mRNA from infected and garlic treated

group of mice on day 4 p.i. Signals for genes of interest were normalizedto 18S rRNA signals, and relative expression is given as fold increase com-pared to control mice on day 0 p.i. All values are means ± SD. *p < 0.001for −garlic vs. +garlic groups.

(Fig. 2A and B). Treatment of mice with garlic extractlargely prevented the E. papillata-induced histopatholog-ical changes in the liver as indicated by a diminution ininflammatory cellular infiltration and hepatocyte damage(Fig. 2C). The E. papillata-induced inflammatory responseof the liver was also indicated by the highly significantincrease in the mRNA of iNOS and of the cytokines IFN-� and IL-6 as determined by quantitative RT-PCR. Again,

garlic treatment was associated with a significant lower-ing in the E. papillata-induced increases in iNOS, IFN-� andIL-6 (Fig. 3). Moreover, infections with E. papillata causedliver injury, evidenced as elevated plasma levels of hepaticenzyme such as ALT, AST, ALP and �GT by approximately

70 M.A. Dkhil et al. / Veterinary Parasitology 175 (2011) 66–72

Table 1Effect of garlic extract on plasma ALT, AST, ALP, �GT and total bilirubin in male Balb/c mice infected with E. papillata.

Group ALT (IU/L) AST (IU/L) ALP (IU/L) �GT (IU/L) Bilirubin (mg/dL)

Non-infected 47.9 ± 3.7 75.5 ± 6.0 39.7 ± 2.9 45.2 ± 3.1 3.1 ± 0.2Infected (−garlic) 74.6 ± 2.5a 116.4 ± 3.2a 67.9 ± 3.6a 69.6 ± 5.6a 5.0 ± 0.2a

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Infected (+garlic) 65.9 ± 2.0b 100.1 ± 4.0b

alues are means ± SD (n = 6).a Significant change at p < 0.05 with respect to non-infected mice.b Significant change at p < 0.05 with respect to infected mice.

0%, 40%, 70% and 60%, respectively (Table 1). Also, infec-ion significantly increased total bilirubin from 3.1 ± 0.2 to.0 ± 0.2 mg/dl (Table 1). Again, garlic treatment was asso-iated with a significant lowering in these liver parameterss can be seen from Table 1.

E. papillata infections also induced a highly significantncrease in hepatic nitrite/nitrate and MDA by approxi-

ately 80% and 90%, respectively (Table 2). In blood plasma,ncreases in nitrite/nitrate and MDA were less pronouncedTable 2). Again, garlic treatment significantly lowered the. papillata-induced increase in both Nitrite/Nitrate andDA, respectively (Table 2).Finally, we also determined some major components

nvolved in the downregulation of substances formeduring oxidative stress such as GSH, catalase and SOD,espectively (Table 3). Conspicuously, these substancesere significantly downregulated by E. papillata infections,

nd these effects were largely prevented by garlic treat-ent (Table 3).

. Discussion

Here, we show that garlic exhibits anticoccidial activ-ty, evidenced as a significant lowering in the output of E.apillata oocysts with the faeces of the infected mice. Thisiminished output reflects that garlic impairs the devel-pment of parasites in the host before the relatively inertocysts are formed and finally released. The fact that gar-ic possesses anticoccidial activity has been also reportedn rabbit coccidiosis (Toula and AL-Rawi, 2007). Such anmpairment and even inhibition of the intracellular devel-pment of parasites by garlic are also known to occurith most anticoccidial drugs. There exist numerous drugs

urrently in use to treat and even prophylactically pre-ent coccidiosis. These drugs can be categorized according

o their presumed actions into three classes (Greif et al.,001). The first class summarizes those drugs which inhibitarasite DNA-synthesis, as e.g. clopidol, decoquinate andiverse sulfonamide-derivatives. The second class com-rises those drugs which interfere with parasite protein

able 2ffect of garlic extract on the level of nitrite/nitrate and malondialdehyde in liver

Groups Nitrite/nitrate

�mol/g liver �mol/L

Non-infected 99.0 ± 7.0 45.2 ±Infected (−garlic) 166.0 ± 4.1a 49.0 ±Infected (+garlic) 135 ± 9.0a,b 42.6 ±alues are means ± SD (n = 6).a Significant change at p < 0.05 with respect to non-infected mice.b Significant change at p < 0.05 with respect to infected mice.

58.3 ± 2.7b 50.4 ± 3.2b 4.1 ± 0.2b

synthesis, as e.g. dindamycin, spiramycin, clarithomycinand paromomycin. The third class sums up those drugswhich ultimately cause destruction of parasite membraneintegrity, as e.g. the polyethers moneusin, narasin, lasalocidand salinomycin (Greif et al., 2001).

The liver is the first-pass organ which is directly con-nected with the intestine by the portal vein. E. papillataparasites infect the distal small intestine and normally donot invade the liver, though different lymphoid organssuch as spleen and lymph nodes have been described tobe infected by E. coecicola parasites (Renaux et al., 2001).Our data indicate that the inflammation of the intestineinduced by E. papillata is also associated with an inflamma-tory response of the liver without being directly affectedby the parasite. Moreover, our study shows that garlicdoes not only target Eimeria parasites in hosts, but alsoexhibits antiinflammatory activity thus protecting host tis-sues. Indeed, E. papillata infections cause an inflammatoryresponse in the liver of mice. This response manifests itselfas a perturbed liver structure and as a significant increasein iNOS and production of NO as evidenced by increasednitrite/nitrate levels as well as in the cytokines IFN� andIL-6. In addition, garlic decreases hepatic injury due to E.papillata infections which is indicated by the lowered activ-ities of ALT, AST, ALP and �GT. Previous studies have shownincreased activities of ALT, AST and ALP (San Martin-Nunezet al., 1988) and of �GT activity due to cholestasis in rabbitssuffering from hepatic coccidiosis (Joyner et al., 1983; Heinand Lammler, 1978). Garlic significantly diminishes liverinjury and inflammation and therefore can be consideredas a hepatoprotective agent.

Gedik et al. (2005) have reported that one of the majorprotective functions of garlic is to decrease the oxidativedamage in liver. Indeed, garlic prevents the infection-induced loss of GSH and decrease of activities of catalase

and SOD. These components are normally lowered dur-ing oxidative damage induced by infection as it has beenalso described by Georgieva et al. (2006) who showedan increase in MDA levels of E. tenella-induced coccidio-sis. In accordance, Kiruthiga et al. (2007) have found that

and plasma of infected balb/c mice with E. papillata.

Malondialdehyde

�mol/g liver �mol/L

1.0 0.47 ± 0.03 0.17 ± 0.020.7a 0.80 ± 0.03a 0.19 ± 0.07a

1.3b 0.60 ± 0.05b 0.16 ± 0.05b

M.A. Dkhil et al. / Veterinary Parasitology 175 (2011) 66–72 71

Table 3Effect of garlic extract on liver glutathione, catalase and superoxide dismutase in male Balb/c mice infected with E. papillata.

Group GSH (�mol/g liver) CAT (U/g liver) SOD (U/g liver)

Non-infected 0.92 ± 0.07 2.05 ± 0.07 5.65 ± 0.26Infected (−garlic) 0.80 ± 0.04 1.73 ± 0.02a 4.79 ± 0.15a

b b b

roxide d

Infected (+garlic) 1.1 ± 0.80

Values are means ± SD (n = 6). GSH, glutathione; CAT, catalase; SOD, supea Significant change at p < 0.05 with respect to non-infected mice.b Significant change at p < 0.05 with respect infected mice.

garlic significantly decreased lipid peroxidation andincreased GSH, catalase and SOD. The antioxidative prop-erty of garlic has been previously ascribed mainly to its fourmajor chemical components, i.e. allinin, allyl cysteine, allyldisulfide, and allicin (Chung, 2006).

Finally, it is noteworthy that Raso et al. (2001) haveviewed that the oxidative stress is mainly due to NO pro-duced by the stimulated iNOS. This finding also fits ourdata showing increased iNOS and nitrite/nitrate induced byinfection. The inhibitory effect of garlic is mainly attributedto the impaired action of iNOS (Gu et al., 2003; Chang andChen, 2005).

Collectively, our data indicate that garlic exhibits asignificant anticoccidial activity and, coincidently, an anti-inflammatory activity protecting host tissue from injuriesinduced by parasites.

Acknowledgments

The authors thank Denis Delic (Department of Molec-ular Parasitology, Heinrich Heine University, Duesseldorf,Germany) and Ahmed Esmat (Department of Zoology, Fac-ulty of science, Helwan University, Egypt) for technicalassistance. This work was supported by the distinguishedscientist fellowship program, King Saud University, SaudiArabia.

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