ORIGINAL PAPER

Th1 and Th2 cytokine gene expression in primary infectionand vaccination against Fasciola gigantica in buffaloesby real-time PCR

Niranjan Kumar & O. K. Raina & Gaurav Nagar &

Ved Prakash & Siju Susan Jacob

Received: 17 May 2013 /Accepted: 9 July 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Th1 and Th2 cytokine gene expression in buffalocalves during primary infection with Fasciola gigantica aswell as in response to immunization with the parasite recom-binant fatty acid binding protein (rFABP) and recombinantglutathione S-transferase (rGST) proteins was measured at14th week of infection by real-time PCR with the double-stranded DNA-binding dye SYBR Green. Experimental an-imals were randomly distributed into FABP, GST, cocktail,challenge and healthy groups. Animals in groups FABP andGSTwere immunized with 400 μg rFABP and rGST, respec-tively, and cocktail group with a mixture of 400 μg each ofrFABP and rGST in the neck and thigh muscles. All animalsreceived three immunizations at 3-week interval. Calveswere challenged per os with 400 viable metacercariae alongwith the unimmunized challenge control group 1 month afterthe last immunization. Expression of various cytokines inresponse to the immunization and parasite primary infectionwas measured by real-time PCR. Expression of IL-2 (4.5-fold) and IFN-γ (3.2-fold), followed by IL-6 (1.7-fold) andIL-4 (1.6-fold), with downregulation of TNF-α and IL-10was observed in response to F. gigantica infection in theseanimals. However, there was a sharp increase in the expres-sion of the IL-4 (211.93 and 111.81-fold) and IL-6 mRNA(219.22 and 48.29-fold) to GST and FABP immunizations,respectively. A downregulation of the IL-1α, a Th1 cytokinein response to FABP and GST immunization in these calves,

was also observed. Overall, a mixed type of Th1 and Th2cytokine environment was evoked to chronic F. giganticainfection and immunization with the above two recombinantproteins in buffaloes.

Introduction

Tropical fasciolosis is a wide spread economically importanthelminthosis caused by Fasciola gigantica and is consideredone of the limiting factors for domestic animal production. Itcauses significant economic losses estimated at US$ 3.2billion per annum to the animal husbandry sector worldwide(Spithill et al. 1999a) and is considered as a reemergingzoonosis in several countries (Mas-Coma et al. 2005;El-Rahimy et al. 2012). Anthelmintic therapy, the sole methodto control fasciolosis, has given rise to drug-resistant parasites(Martínez-Valladares et al. 2010; Devine et al. 2011). Vacci-nation is an urgent need to achieve environmentally friendlyand sustainable level of control (McManus and Dalton 2006;Foster and Elsheikha 2012). Various immunoprophylacticsagainst Fasciola hepatica/F. gigantica have shown potentialwith variable results (Spithill et al. 1999b; Nambi et al. 2005;Zhang et al. 2005; Kumar et al. 2012). Generally, helminthinfections are manifested by the suppression of the Th1 func-tion and induction of T cells, expressing cytokines character-istic of Th2 subset (Clery et al. 1996; Brady et al. 1999;Molina 2005). But various studies indicated that in helminthdiseases, an early phase of infection showed a mixed type ofTh1/Th2 response, and as the disease progresses, the cellularimmune response is polarized towards Th2 cytokines with thesuppression of Th1 cytokines (Hoyle and Taylor 2003;Waldvogel et al. 2004). The host genetic differences in thecellular immunity at intraspecies or interspecies level causingup or downregulation in cytokine expression are thought toinfluence the courses of several helminth infections (Molinaand Skerratt 2005; Raadsma et al. 2007). In the present study,

O. K. Raina :G. Nagar : S. S. JacobDivision of Parasitology, IVRI, Bareilly, India

V. PrakashBikaner, Rajasthan, India

Present Address:N. Kumar (*)Department of Parasitology, Vanbandhu Veterinary College, NAU,Navsari 396450, Indiae-mail: niruvet@gmail.com

Parasitol ResDOI 10.1007/s00436-013-3540-y

an attempt was made to determine the gene expression ofinterferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin(IL)-2, IL-1α, IL-4, IL-6, and IL-10 as the indicators of Th1 orTh2 type of immune response evoked in buffalo calves to aprimary infection with F. gigantica as well as in response toimmunization using recombinant fatty acid binding protein(rFABP) and glutathione S-transferase (rGST) in real-timePCR.

Materials and methods

Experimental animals

Buffalo calves (n=34), aged 8–10 months, were purchasedthrough a local animal contractor from F. gigantica non-endemic areas of North India. The experimental animalswere dosed with tetramisole hydrochloride (10 mg/kg bodyweight) to clear the gastrointestinal parasitic infections. Bothserological and fecal screening of these calves was carriedout to rule out previous exposure to F. gigantica. The calveswere maintained on concrete floor in the divisional experi-mental shed, adopting standard animal husbandry practices.Experimental procedures were carried out as per the guide-lines of the institute animal ethics committee.

The experimental animals were randomly assigned to fivegroups of seven calves each but with a cocktail group of sixcalves only. Animals in groups FABP and GST were immu-nized each with 400 μg rFABP and rGST, respectively, andcocktail group received a mixture of 400 μg each of FABPand GST. The recombinant proteins were expressed forimmunization study as described earlier (Kumar et al.2012). Other two groups were maintained as infected chal-lenge and uninfected control animals, respectively. Both theantigens were delivered to the host with Montanide 70 M-VG (SEPPIC, France) at 70:30 adjuvant–antigen ratios in theneck and thigh muscles with three immunizations given at 3-week interval. Animals were challenged per os, each with adose of 400 viable metacercariae 30 days after last immuni-zation. Unimmunized challenge control calves were infectedeach with a dose of 400 metacercariae. Metacercariae wereharvested on polyethylene sheets from F. gigantica-infectedLymnaea auricularia, and each infection dose was preparedin a wheat flour bolus. Metacercariae viability was con-firmed by in vitro excystation before experimental infectionof the animals.

Total RNA extraction and cDNA synthesis

Blood samples, collected at 14th week of challenge infectionfrom the jugular vein of the experimental calves, wereprocessed for peripheral blood mononuclear cells (PBMCs)isolation by histopaque gradient (Histopaque 1077, Sigma,

USA). Isolated PBMCs were washed in RPMI-1640 (Sigma,USA), centrifuged, and used for RNA isolation. The PBMCswere solubilized in 1 ml Trizol reagent (Invitrogen, USA)and RNA isolated following standard protocols. The totalRNA was used for cDNA synthesis by reverse transcriptionof the gene signal with oligo-dT primer (18 mer) and M-MLV reverse transcriptase enzyme, following reversetranscriptase-PCR protocol. The cDNAwas used in the studyof cytokine gene expression by real-time PCR.

Real-time PCR for gene expression

Four calves from each group were randomly selected forstudies on cytokine expression. cDNA synthesized fromthe total RNA from these animals was used as a templatefor the amplification of different cytokine genes viz, IFN-γ,TNF-α, IL-1α, IL-2, IL-4, IL-6, and IL-10 and a housekeeping gene (β-actin). The primer sequences used in thePCR for the amplification of these genes were designed asper Konnai et al. (2003). The following combinations ofprimers were used for the amplification of β-actin and dif-ferent cytokine genes: β-actin (227 bp)—forward 5′-CGCACCACTGGCATTGTCAT-3′ and reverse 5′-TCCAAGGCGACGTAGCAGAG-3′, IFN-γ (218 bp)—forward 5′-ATAACCAGGTCATTCAAAGG-3′ and reverse 5′-ATTCTGACTTCTCTTCCGCT-3′, TNF-α (350 bp)—forward5′-CAAGTAACAAGCCGGTAGCC-3′ and reverse 5′-TGGAAGACCCCTCCCTGGTA-3′, IL-2 (217 bp)—forward 5′-TTTTACGTGCCCAAGGTTAA-3′ and reverse 5′-GTTTACTGTTGCATCATCA-3′, IL-1α (173 bp)—forward 5′-GATGCCTGAGACACC CAA-3′ and reverse 5′-GAAAGTCAGTGATCGAGGG-3′, IL-4 (181 bp)—forward 5′-CAAAGAACACAACTGAGAAG-3′ and reverse 5′-AGGTCTTCAGCGTACTTGT-3′, IL-6 (236 bp)—forward 5′-TCCAGAACGAGTATGAGG-3′ and reverse 5′-CATCCGAATAGCTCTCAG-3′, and IL-10 (186 bp)—forward 5′-TGCTGGATGACTTTAAGGG-3′ and reverse 5′-AGGGCAGAAAGCGATGACA-3′. The PCR reactions were set in25 μl volume containing QuantiTect SYBR Green PCR mas-ter mix with SYBR Green 1 dye, ROX passive reference dye,Hot Start Taq DNA polymerase and dNTPs (Qiagen, GmbH,Germany), 0.25μMconcentration of each gene-specific prim-er, and 1 μl of cDNA template in optical 96-well reactionplates at an initial incubation at 95 °C for 15 min, followed by40 cycles of amplification with denaturation at 95 °C for 15 s,primer annealing at 60 °C for 30 s, and primer extension at72 °C for 30 s. The reactions were performed in duplicate,with duplicate negative controls to determine the specificity ofthe PCR reaction, usingMx3000 P system (Stratagene, USA).A dissociation curve of each sample was analyzed in compar-ison to the negative controls. Mean cycle threshold (Ct) valuesof the duplicate test and negative control samples were used forstatistical analysis using Student’s t test. The delta Ct values

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Fig. 1 Real-time PCR showingcytokine expression in healthyand F. gigantica-infected calvesat 14th week of infection

Table 1 Th 1 cytokine expression in healthy and F. gigantica-infected groups (14th week postinfection)

Name ofcytokines

Samples Animalnumber

Ct referencegene

Ct targetgene

Dct targetgene

Average Dcttarget gene

Calibrator Ddct targetgene

Foldchange

IFN-γ Healthy 1 16.53 26.5 9.97 10.30 10.30 0 12 16.3 28.5 12.2

3 17.1 26 8.9

4 17.34 27.45 10.11

Infected 1 18.5 27.45 8.95 8.58 10.30 −1.72 3.292 17.75 26.2 8.45

3 15.75 25.1 9.35

4 18.65 26.2 7.55

TNF-α Healthy 1 16.5 23.88 7.38 6.24 6.24 0 12 17.1 24.73 7.63

3 16.3 21 4.7

4 17.21 22.45 5.24

Infected 1 20.1 30 9.9 8.78 6.24 2.54 0.172 17.7 25.1 7.4

3 15.7 23.4 7.7

4 18.5 28.6 10.1

IL-2 Healthy 1 16.5 31.5 15 15.30 15.30 0 12 17.1 31.3 14.2

3 16.3 32.8 16.5

4 17.21 32.7 15.49

Infected 1 20.1 31.1 11 13.13 15.30 −2.17 4.512 17.7 31.45 13.75

3 15.7 30.5 14.8

4 18.5 31.45 12.95

In Tables 1, 2, 3, 4 and 5

Delta cycle threshold (Dct) target gene = ct target gene − ct reference gene (β-actin gene)

Delta dct (Ddct) target gene = average Dct target gene – calibrator

Fold change = 2-Ddct target gene

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Table 2 Th 2 cytokine expression in healthy and F. gigantica infected groups (14th week postinfection)

Name ofcytokines

Samples Animalnumber

Ct referencegene

Ct targetgene

Dct targetgene

Average Dcttarget gene

Calibrator Ddct targetgene

Foldchange

IL-4 Healthy 1 16.53 27.8 11.27 11.17 11.17 0 12 16.3 27.56 11.26

3 17.1 28.1 11

4 17.34 28.5 11.16

Infected 1 18.5 27.7 9.2 10.44 11.17 −0.74 1.662 17.75 28.5 10.75

3 15.75 27.9 12.15

4 18.65 28.3 9.65

IL-6 Healthy 1 16.5 31.45 14.95 14.18 14.18 0 12 17.1 31.64 14.54

3 16.3 29.75 13.45

4 17.21 31 13.79

Infected 1 20.1 31.9 11.8 13.41 14.18 −0.77 1.712 17.7 32.12 14.42

3 15.7 30.12 14.42

4 18.5 31.5 13

IL-10 Healthy 1 16.53 25.75 9.22 8.61 8.61 0 12 16.3 24.7 8.4

3 17.1 25.36 8.26

4 17.34 25.9 8.56

Infected 1 18.5 29.8 11.3 11.06 8.61 2.45 0.182 17.75 29.5 11.75

3 15.75 26.5 10.75

4 18.65 29.1 10.45

Fig. 2 Real-time PCR showingcytokine expression inimmunized and unimmunizedcalves at 14th week postchallenge

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were calculated after subtracting the mean Ct values ofβ-actingene from the mean Ct values of different cytokines.

Results

Expression of various cytokine genes viz IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 in buffalo calves in response toprimary infection with F. gigantica was studied at 14th weekof infection. Expression of IL-2 (4.5-fold) and IFN-γ (3.2-fold), followed by IL-6 (1.7-fold) and IL-4 (1.6-fold), was

detected in response to F. gigantica infection in these exper-imental animals (Fig. 1; Tables 1 and 2). This indicated amixed type of Th1 and Th2 cytokine environment evoked toF. gigantica infection in buffaloes. However, the expressionof two cytokines viz TNF-α and IL-10 was observed to bedownregulated in the host in response to this helminth infec-tion (Fig. 1; Tables 1 and 2).

Also, cytokine responses of IL-4, IL-6, and IL-1α (Fig. 2;Tables 3, 4, and 5) were studied in response to the immuni-zation of the calves with FABP and GST recombinant anti-gens. In contrast to the level of expression of IL-4 and IL-6 in

Table 3 IL-4 cytokine expression of unimmunized and immunized groups at 14th week post-challenge

Samples Animalnumber

Ct referencegene

Ct targetgene

Dct targetgene

Average Dct targetgene

Calibrator Ddct targetgene

Foldchange

Healthy 1 16.53 27.80 11.27 11.17 11.17 0 12 16.30 27.56 11.26

3 17.10 28.10 11.00

4 17.34 28.50 11.16

Challenge–control

1 18.50 27.70 9.20 10.44 11.17 −0.74 1.662 17.75 28.50 10.75

3 15.75 27.90 12.15

4 18.65 28.30 9.65

FABPimmunized

1 22.44 25.75 3.31 4.37 11.17 −6.81 111.822 28.20 34.44 6.25

3 23.19 24.61 1.42

4 22.79 27.80 5.01

GST immunized 1 25.27 28.17 2.91 3.45 11.17 −7.73 211.942 24.39 26.64 2.25

3 28.22 32.85 4.63

4 25.80 29.80 4.00

Table 4 IL-6 cytokine expression in unimmunized and immunized groups at 14th week post-challenge

Samples Animalnumber

Ct referencegene

Ct targetgene

Dct targetgene

Average Dcttarget gene

Calibrator Ddct targetgene

Foldchange

Healthy 1 16.50 31.45 14.95 14.18 14.18 0 12 17.10 31.64 14.54

3 16.30 29.75 13.45

4 17.21 31.00 13.79

Challenge–control 1 20.10 31.90 11.80 13.41 14.18 −0.77 1.712 17.70 32.12 14.42

3 15.70 30.12 14.42

4 18.50 31.50 13.00

FABP immunized 1 17.59 23.98 6.40 6.41 14.18 −7.78 219.222 25.41 29.43 4.02

3 16.10 24.91 8.81

4 18.05 24.45 6.40

GST immunized 1 17.43 26.86 9.43 8.59 14.18 −5.59 48.292 17.51 26.33 8.82

3 20.06 27.38 7.32

4 17.55 26.35 8.80

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primary infection of the host, there was a sharp increase in theexpression of the IL-4 mRNA (211.93 and 111.81-fold) inresponse to GST and FABP immunizations, respectively.Likewise, IL-6 expression showed 219.22 and 48.29-foldincrease to FABP and GST immunization, respectively. How-ever, a downregulation of the IL-1α cytokine in response toFABP and GST immunization in these calves was observed(Fig. 2; Tables 3, 4, and 5). These changes in IL-4 and IL-6cytokines were statistically significant (p<0.05) between theunimmunized and immunized groups of calves.

Discussion

An understanding of how helminths are able to generateimmune environments which favor pathogen persistence,and chronic infection is important for developing successfulstrategies for control of helminthic diseases. Recent studieshave indicated a strong Th2 and T regulatory immune re-sponses in F. hepatica while simultaneously suppressingTh1-driven immune responses (Vukman et al. 2013). Studiesin cattle and buffaloes have demonstrated that T cell responseis polarized towards the Th2 bias in F. hepatica and F.gigantica infection (Mulcahy and Dalton 2001; Waldvogelet al. 2004; Ingale et al. 2008, 2010). A detectable level ofIL-4 cytokine was measured in goat in response to glutathioneS-transferase immunization against F. hepatica followed bythe challenge with metacercariae (Buffoni et al. 2010). How-ever, some contrasting observations have been reported whichindicate that a Th0 immune response occurs during the earlyphase of infection, with concurrent expression of IFN-γ, IL-2,and IL-4 (Mulcahy and Dalton 2001; Hoyle and Taylor 2003).

The results of the present preliminary study on cytokineexpression indicated that the T cell response of the buffalocalves with a chronic infection of F. gigantica (14th week)was of mixed type, with a co-dominance of Th1 and Th2cytokines. In an early experimental infection in cattle andbuffaloes with F. gigantica, an expression of higher level ofIL-6 but absence of IFN-γ was observed by Molina (2005).Waldvogel et al. (2004) observed the production of Th2cytokine IL-4 but absence of IFN-γ in F. hepatica-infectedcattle during the early phase of infection, but during its chronicphase, non-polarized Th0 response was observed, wherein IL-2, IL-4, and IFN-γ were produced. In a study on F. gigantica-infected bovine calves, no IFN-γ production was observed at10th and 30th days postinfection, but at 75th day postinfection,some of the infected calves showed expression of IFN-γ,suggesting a shift to Th0 immune response during the chronicphase of infection (Ingale et al. 2008, 2010). The three cyto-kines, IL-10, IFN-γ, and TNF-α work in concert during theacute and chronic stages of human fasciolosis. In the acutestage, a significant increase was observed in IL-10, IFN-γ, andTNF-α, while in the chronic phase, a significant increase ofIL-10 and TNF-α with lower level of IFN-γ was observed.The mixed type of Th1/Th2 response helped in overcomingthe harmful effect of the parasites in the early phase of infec-tion and with time and after maturation of the escaping worms,Th2 action predominates (Osman and Abo-El-Nazar 1999).Increased serum levels of IL-4, IL-6, IL-8, or IL-10 cytokineshave been observed in cattle, buffaloes, sheep, and humaninfected with F. gigantica or F. hepatica (Khalil et al. 1999;Molina 2005; Zhang et al. 2006). Modulation of macrophageand dendritic cell function and interaction with toll-like recep-tors are thought to be some of the fundamental events

Table 5 IL-1 α cytokine expression in unimmunized and immunized groups at 14th week post-challenge

Samples Animalnumber

Ct referencegene

Ct targetgene

Dct targetgene

Average Dcttarget gene

Calibrator Ddct targetgene

Foldchange

Healthy 1 16.80 29.00 12.20 8.96 8.96 0 12 16.09 24.27 8.18

3 17.00 25.00 8.00

4 16.80 24.27 7.47

Challenge–control 1 20.10 31.90 11.80 13.41 8.96 4.45 0.052 17.70 32.12 14.42

3 15.70 30.12 14.42

4 18.50 31.50 13.00

FABP immunized 1 17.59 36.45 18.86 17.37 8.96 8.41 0.002 25.41 37.45 12.05

3 16.10 36.17 20.08

4 18.05 36.55 18.50

GST immunized 1 17.43 36.31 18.88 16.84 8.96 7.88 0.002 17.51 33.96 16.45

3 20.06 34.31 14.25

4 17.55 35.35 17.80

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involved in establishing the immune regulation. The cytokineenvironment in which they do so will influence the T cellsubsets that later develop. Several Fasciola antigens includingperoxiredoxins, cathepsin-L, have been identified to causesuppression of Th1 immune response by inducing a Th2response in the infected host (Donnelly et al. 2005, 2008;Golden et al. 2010).

Immunization of the buffalo calves with FABP and GSTantigens in the present study caused significantly (p<0.05)higher expression of the Th2 cytokines (IL-4 and IL-6), withdownregulation of the Th1 cytokine, IL1-α. Deceased expres-sion of IL1-α in response to immunization with these twoantigens could not be explained. This study measured only afew cytokines; though the expression of other cytokines needsto be studied for drawing a conclusion on the cytokine re-sponse to the F. gigantica antigens. Some contrasting obser-vation was observed by Dowling et al. (2010) while examin-ing the interactions between recombinant proteins cathepsin Land sigma class glutathione transferase of F. hepaticawith thedendritic cells, both the antigens failed to enhance dendriticcell phagocytosis or induce Th2 immune responses in vivo.Flynn and Mulcahy (2008) suggested roles of IL-10 andtransformation growth factor-beta in controlling IL-4 andIFN-γ production during experimental F. hepatica infectionin cattle leading to the suppression of IFN-γ production,which may mediate the parasite survival.

To conclude, comprehensive studies are required on therole of various factors including parasite-derived ones andcytokines that influence the host–parasite interactions fordeveloping a successful vaccine against this pathogen.

Acknowledgments The authors are thankful to the Department ofBiotechnology, Government of India, NewDelhi for providing grants tothis research project. We are thankful to the Director, Indian VeterinaryResearch Institute, Izatnagar for providing necessary facilities for theresearch work. The first author is also thankful to IVRI for providingfellowship during PhD.

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