Effects of praziquantel and sanguinarine on expression of immune genes and susceptibility to Aeromonas hydrophila in goldfish (Carassius auratus) infected with Dactylogyrus intermedius

Fish & Shellfish Immunology 35 (2013) 1301e1308

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Effects of praziquantel and sanguinarine on expression of immune genes and susceptibility to Aeromonas hydrophila in goldfish (Carassius auratus) infected with Dactylogyrus intermedius Chao Zhang, Fei Ling, Cheng Chi, Gao-Xue Wang* Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 May 2013 Received in revised form 3 August 2013 Accepted 4 August 2013 Available online 15 August 2013

Praziquantel and sanguinarine have been demonstrated positive therapeutic effects on monogenean Dactylogyrus intermedius; however, few studies have considered the post impacts of these antiparasitic chemicals on host after repelling the parasites. The changes of expression of selected immune genes (CCL-1, CXCL-8, IL-1b-1, IL-1b-2, TNFa-1, TNFa-2 and TGF-b) in gill, kidney and spleen and bacterial loads of Aeromonas hydrophila in gill, kidney, spleen and liver following bath administration of these antiparasitic chemicals were evaluated. The results showed that praziquantel and sanguinarine up-regulated to varying degrees of CXCL-8, IL-1b-1, IL-1b-2, TNFa-1 and TNFa-2 in gill, kidney and spleen. They both decreased the CCL-1 expression in gill while increased it in kidney and spleen. However, in all the tested tissues, the expression of TGF-b decreased in praziquantel treated goldfish whereas that increased in sanguinarine treated goldfish. The A. hydrophila challenge test showed that the praziquantel treatment enhanced the susceptibility to A. hydrophila while sanguinarine treatment decreased the susceptibility, as compared with the non-treated group. Overall, the results indicate that bath administration of praziquantel and sanguinarine modulates the immune related genes in goldfish and these may, to some extent, affect their ability to resist bacterial pathogens. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Fish Treatment Monogenean Gene expression Susceptibility

1. Introduction Today, aquaculture is the fastest growing food-producing sector in the world and contributes significantly to the world economy. The main drawbacks for aquaculture’s intensification and commercialization are associated with disease problems, including bacterial, viral, and parasitic infections [1,2]. The ectoparasitic monogenean such as Dactylogyrus intermedius, are usually attached to the gills of cyprinid fishes and caused symptoms of inflamed gills, excessive mucus production and accelerated respiration [3]. Moreover, mixed infections with other parasites and secondary bacterial infections are common, resulting in severe damages to the host such as loss of appetite, sluggish growth rate and high mortalities [4,5]. Application of drugs to control Dactylogyrus spp. is common and some traditional parasiticide drugs such as praziquantel [6], mebendazole [7] and toltrazuril [8] have been used for decades. * Corresponding author. Tel.: þ86 029 87092102 (office), þ86 02987091516 (home); fax: þ86 02987092164 (office). E-mail addresses: [email protected], [email protected] (G.-X. Wang). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.08.001

However, several drawbacks of chemical drugs (e.g. drug residues and drug resistance) drive to find a new effective therapy for the control of Dactylogyrus infestation. Recently, some plant secondary metabolites were demonstrated to have efficient anthelmintic activity on D. intermedius and therefore regarded as new plantderived antiparasitic drugs [9,10]. In recent works in our laboratory, some active natural compounds with property against D. intermedius were found and among them, sanguinarine stands out as the most effective one [9]. Although some drugs have their positive therapeutic effects, several hazards and side-effects on the host may also be induced. These include decreased growth, reproductive deficits, nephrotoxicity, immunosuppression and increased incidence of resistant bacterial strains [11]. For instance, oxytetracycline and sulfamerazine both suppressed the immune functions in rainbow trout [11,12]. Similarly, cyclosporin A induced immunosuppression when acted on parasitic infections [13]. On the contrast, levamisole stimulated both the specific and non-specific defense mechanisms in rainbow trout [14]. However, until now there have been only limited studies to evaluate the immunomodulatory effects of drugs on the fish immune related genes expression. Previous study reported antimicrobials oytetracycline could modulate the

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expression of immune related genes such as C3 and IL-1b in gilthead seabream [15]. Antibiotics oxolinic acid and florfenicol both altered the transcriptional profiles of inflammation genes (e.g. IL1b, IL-8), which were dependent on the type of antibiotics that was administered and the time of sampling [16]. Furthermore, identification of gene transcripts that are regulated by some drugs might serve as useful biomarkers in the fish to monitor the effects of drugs during feeding [16]. The host resistance against bacterial or viral infection is one of the methods for detection of immunotoxic chemicals or drugs and in this regard, the commonly used tests are based on the measurement of the inhibitory influence on the survival rate or mortality after infection with virulent strains of bacteria or viruses [17e 19]. Some commonly used aquaculture drugs or aquatic contaminants have been investigated on the susceptibility or resistance to bacterial using mortality as the indicator [15,16,20e22]. In recent decades, polymerase chain reaction (PCR) has become an important method for the rapid, sensitive and specific detection of bacterial and viral agents. Previous studies have shown that real-time PCR could be used for a definite quantitative determination of some strains of bacteria, such as Flavobacterium columnare [23,24], F. psychrophilum [25], Edwardsiella ictaluri [26], Aeromonas salmonicida [27] and A. hydrophila [28]. Taking into account all these previous considerations, the primary aim of this study was to examine the expression of immune related genes (CCL-1, CXCL-8, TNFa-1, TNFa-2, IL-1b-1, IL-1b-2, and TGF-b) in gill, kidney and spleen following praziquantel and sanguinarine exposure. The second objective was to determine whether praziquantel and sanguinarine would alter the susceptibility to A. hydrophila of the host by comparing the bacterial loads in gill, kidney, spleen and liver. To our knowledge, this is the first study to examine the immune related gene expression associated with antiparasitic drugs use in aquaculture. 2. Materials and methods 2.1. Chemicals Praziquantel was obtained from Hanzhong Tianyuan Company (Hanzhong, China) and sanguinarine was purchased from the Xi’an Hongsheng Company (Xi’an, China). Both chemicals were analytical grade. 2.2. Fish, parasites and bacteria One-year-old healthy goldfish (11.83  2.09 g and 8.10  0.21 cm), obtained from Shaanxi Fisheries Research Institute (Shaanxi, China), were maintained under laboratory conditions (temperature 25.0  2  C, pH 6.9  0.4, dissolved oxygen 6.0e 7.8 mg L1) for 7 days prior to infection. Ten goldfish were checked randomly to verify pathogen free status of parasites and bacteria prior to the trial. After acclimatization, all fish were cohabitated with those infected with D. intermedius in a ratio of 5:1. Three weeks later, ten fish were randomly killed and checked for the intensity of parasites under a light microscope (Olympus BX41, Tokyo, Japan) at 10  4 magnification [29]. Fish were chosen for the test when the infestation rate reached 100% and the moderate infestation intensity (40e60 parasites per fish) was obtained. A strain of A. hydrophila (2WCL-103) obtained from Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China) was used for the study. This bacterial has previously been used in other freshwater studies for this purpose [28]. Prior to challenge trial, this strain of A. hydrophila was used for experimental infection of goldfish in order to test the bacterium was pathogenic to goldfish. It was cultured in tryptic soy broth (TSB) at 28  C in a shaker and used

Table 1 Sequences of primer pairs used in real-time PCR. Primer name

Sequence (50 e30 )

b-actin forward b-actin reverse

GAT GAT GAA ATT GCC GCA CTG ACC GAC CAT GAC GCC CTG ATG T AAG GTC ACC GAA CCC ATC AG TCG TCA CAT GAT GGC CTT CA CTG AGA GTC GAC GCA TTG GAA TGG TGT CTT TAC AGT GTG AGT TTG G GCG CTG CTC AAC TTC ATC TTG GTG ACA CAT TAA GCG GCT TCA C GAT GCG CTG CTC AGC TTC T AGT GGG TGC TAC ATT AAC CAT ACG CAT TCC TAC GGA TGG CAT TTA CTT CCT CAG GAA TGT CAG TCT TGC AT TCA TTC CTT ACG ACG GCA TTT CAG TCA CGT CAG CCT TGC AG GTA CAC TAC GGC GGA GGA TTG CGC TTC GAT TCG CTT TCT CT CGC CAG CTG GTC AAG ACT GT CCA GTT GGT GGC TGT GTC GT

CCL-1 forward CCL-1 reverse CXCL-8 forward CXCL-8 reverse IL-1b-1 forward IL-1b-1 reverse IL-1b-2 forward IL-1b-2 reverse TNFa-1 forward TNFa-1 reverse TNFa-2 forward TNFa-2 reverse TGF-b forward TGF-b reverse Aero forward Aero reverse

to challenge fish. The concentration (colony forming units per milliliter CFU ml1) of A. hydrophila was determined through serial 1:10 dilutions using standard plate-counts. 2.3. Experimental design and sampling procedure Goldfish experimentally infected with D. intermedius were divided into three groups and received the following treatments: (1) treated by praziquantel at its therapeutic concentration (13.5 mg L1); (2) treated by sanguinarine at its therapeutic concentration (0.7 mg L1) [29]; (3) used as control group without any treatments. The doses of the chemicals used are the lowest concentrations to completely kill D. intermedius in 48 h. After 48-h exposure, the fish from each group were moved to a 20-L aquarium filled with 15-L aerated water. Water was exchanged daily and the quality was monitored three times per day. For immune genes expression, three individuals were sampled at 2 h, 1 day, 3 days and 6 days post treatment (p.t.). Fish were anesthetized with overdose of MS-222 (Geruien, China) and sampled using aseptic technique. Immediately after the fish were dissected using aseptic technique, one piece of gill filament and a small piece of kidney and spleen (w20 mg) were immersed in TRIzol reagent and stored in 80  C until RNA extraction. The rest of gill filaments were biopsied to determine the number of parasites. For the bacterial challenge, three fish in each group were taken at 1 day and 6 days p.t. Firstly, the goldfish were immersed in water with A. hydrophila at a concentration of 3  108 CFU ml1 for 2 h. Fish not exposed to A. hydrophila were kept in water with the same amount of TSB for the same time and used as a blank control. Then the fish were returned to tanks with aerated water for 2 h. The gill, kidney, spleen and liver from each fish were sampled using aseptic technique, weighed and stored at 80  C for genomic DNA (gDNA) isolation. The mortality of goldfish was recorded and dead fish were checked for D. intermedius and A. hydrophila infection once daily for one week. 2.4. RNA extraction, reverse transcription, and real-time PCR Total RNA was extracted using TRIzol reagent (Invitrogen, Spain) and treated with DNAse I (Fermentas, USA) to minimize the contamination of genomic DNA. RNA quality was verified by electrophoresis on ethidium bromide staining 1.0% agarose gels and by A260 nm/A280 nm ratio. Total RNA was reverse transcribed into cDNA using the reverse transcriptase kit (TaKaRa, Japan) following the

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and there were 3.2 and 2.4 parasites on a fish exposed to praziquantel and sanguinarine on 6 days p.t., separately (Table 2).

Table 2 Numbers of Dactylogyrus intermedius of infected fishes. Detection time

Abundance  SD (minemax) Praziquantel

Sanguinarine

Control

Prior to infection Hour 2 Day 1 Day 3 Day 6

46.4  7.2 (34e57) 0 0 0 3.2  0.7 (1e5)

46.4  7.2 (34e57) 0 0 0 2.4  0.5 (0e4)

46.4  7.2 (34e57) 38.7 43.2 40.1 47.9

   

8.9 5.6 4.7 6.2

3.2. Expression of immune-related genes

(25e44) (29e53) (33e49) (38e54)

manufacturer’s instructions. Real-time qPCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) and SYBR Premix Ex Taq II kit (TaKaRa). Primers for goldfish CCL-1, CXCL-8 (¼IL-8), TNFa-1, TNFa-2, IL-1b-1, IL-1b-2, and TGF-b were obtained in previous studies and listed in Table 1 [30,31]. The cycle parameters consisted of one cycle of 10 min at 95  C and then 40 cycles with 15 s at 95  C, 20 s at 58  C and 10 s at 72  C. Each individual sample was run in triplicate wells. 2.5. Genomic DNA isolation and bacterial quantify Total gDNA of fish tissues and the bacterium A. hydrophila were extracted using DNeasy Tissue kit (Qiagen, USA) following the manufacturer’s instructions. For fish tissues, per milligram tissue of gDNA was eluted with 10 ml sterile water. DNA yield and purity were determined spectrophotometrically using Nanodrop ND1000. The gDNA of A. hydrophila was made 10-fold serial dilutions from 10 ng mL1 to 10 fg mL1 with sterile water or tissue extracts to make standard curve (threshold cycle (Ct) values vs. DNA concentration of A. hydrophila). The bacterial quantity in each tissue was determined by qPCR with a pair of A. hydrophila-specific primers (Aero, Table 1) [28]. The qPCR parameters were as follows: 95 for 10 min followed by 40 cycles of 95  C for 15 s and 60  C for 1 min. The concentration of gDNA of different tissues was measured via the standard curve. Total amount of A. hydrophila in each mg tissues was further calculated through bacterial DNA concentration multiply eluted volume/tissue weight, taking into account the dilution factor. The concentration of A. hydrophila in fish tissues was expressed as genome equivalents per mg of tissue (GEs/mg) based the 4.7 mbp genome size of A. hydrophila and a conversion factor of 1 pg ¼ 978 mbp [28,32,33]. The quantification limit has been determined as 10.4 genomes, making 50 fg of A. hydrophila DNA [28]. 2.6. Statistical analysis All data were expressed as mean  SD and were analyzed by one-way ANOVA after normalization. The RT-qPCR data were OOC analyzed by the 2 T method [34]. The concentrations of A. hydrophila DNA were compared with Duncan multiple range tests. Analysis of the data was done using SPSS17.0 statistical soft. A probability level of P < 0.05 was considered significant. 3. Results 3.1. Effects of praziquantel and sanguinarine on D. intermedius Prior to the test, the detection of ten random fish showed all fish were infected with D. intermedius and the mean abundance was 46.4 parasites (34e57 parasites) per fish (Table 2). Both praziquantel and sanguinarine showed positive therapeutic effect on D. intermedius. No parasites were observed at 2 h and 3 days p.t.,

In the praziquantel-treated group, there was a significant upregulation of CCL-1 in the kidney and spleen after treatment, while a significant down-regulation was found in gill (Fig. 1A). Except for a slight down-regulation in gill after 2 h p.t., the CXCL-8 level was significantly up-regulated in the three tissues and it remains high throughout the observation period (Fig. 1B). Both IL-1b1 and IL-1b-2 were up-regulated in goldfish gill, kidney and spleen, while a higher expression was observed of IL-1b-2 (Fig. 1C). Similarly, up-regulation was also observed in TNFa-1 and TNFa-2 in all the tested tissues, while a higher expression was observed of TNFa2 (Fig. 1D). On the contrast, a significant down-regulation of TGF-b gene expression was found in the three tissues throughout the observation period, with the exception of the gene expression in gill at 2 h p.t. and in kidney at 6 d p.t. (Fig. 1E). In the sanguinarine treatment group, the expression of CCL-1 significantly increased in kidney and spleen, while it significantly decreased in gill (Fig. 2A) during the observation period. Upregulation was also observed in CXCL-8 in all the tested tissues from 2 h to 6 days p.t. (Fig. 2B). Similarly, higher expression of IL1b-1 and IL-1b-2 in gill, kidney and spleen were induced significantly after exposure to sanguinarine. However, the magnitude of IL-1b-2 increment was much higher compared to that observed for IL-1b-1 (Fig. 2C). A significant increase in expression of TNFa-2 was observed in gill, kidney and spleen p.t. with sanguinarine, while the magnitude of TNFa-1 expression was lower but still significant (Fig. 2D). The expression of TGF-b was significantly up-regulated in gill at 6 days p.t. and in kidney at 3 and 6 days p.t., while its expression was higher but not significant in spleen (Fig. 2E). 3.3. Infection amount of A. hydrophila in fish tissues There was no A. hydrophila detected in fish tissues before immersion in A. hydrophila and no mortality were observed at the treated group, or in the control non-inoculated group (date not shown). In the group treated with praziquantel, the infection amount of A. hydrophila in all fish tissues was higher than that in control group at 1 day and 6 days post exposure (dpe) to A. hydrophila (Table 3). On day 1, the amounts of A. hydrophila in gill, spleen and liver were 2.84, 3.06 and 1.44 fold than that in control group (P < 0.05). The bacterial amount in kidney was also higher than non-treated goldfish, although not significant. Though all tested tissues demonstrated higher bacterial loads at 6 dpe to A. hydrophila, only the kidney and spleen showed statistically significant difference (1.21 and 1.15 fold). On the contrast, all tissues from goldfish in the group treated with sanguinarine showed lower bacterial loads than that in control group (Table 3). The bacterial loads ranged from 419.8 to 4885.4 GEs/mg at 1 dpe to A. hydrophila in different organs from fish exposed to sanguinarine (Table 3). The bacterial loads decreased significantly (P < 0.05) in all the tissues of sanguinarine treated fish compared to non-treated fish after exposure to A. hydrophila. All treated fish also showed lower bacterial loads in the tested tissues at 6 dpe to A. hydrophila than non-treated fish, though not significant in liver. 4. Discussion Although praziquantel and sanguinarine have been demonstrated positive therapeutic effect on D. intermedius by previous studies [6,9] and this report (Table 2), the post effects of

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5

A CCL-1

Kidney

Gill

4

Spleen

* *

3

*

*

*

*

*

*

2 1

* *

*

0

6

B

CXCL-8

5 *

4 3

*

*

* 2

*

*

*

*

Expression relative to β-actin

1 0 3.5 3

C

1L-1β -1 1L-1β -2

2.5

*

2

*

*

*

*

*

*

*

*

1.5 1 0.5 0

4

D

TNFα-1 TNFα-2

3

*

*

* *

*

2

*

*

*

*

1

0

2

E

TGF-β

1.5

1 *

* *

*

0.5 *

*

*

* *

*

6d

3d

1d

2h

6d C on tro l

3d

1d

2h

6d C on tro l

3d

1d

2h

C on tro l

0

Time post treatment with praziquantel Fig. 1. Quantitative expression analysis of immune genes in the gill, kidney and spleen of praziquantel treated goldfish. A: CCL-1; B: CXCL-8; C: IL-1b-1 and IL-1b-2; D: TNFa-1 and TNFa-2; E: TGF-b. The expression data were normalized against those of the 0 h time point for each gene. The results are the mean  SEM from three individual fish. * represents significantly different (p < 0.05) from controls.

C. Zhang et al. / Fish & Shellfish Immunology 35 (2013) 1301e1308

9 8 7

A CCL-1

Gill

1305

Spleen

Kidney * *

6 5

*

4 3

*

*

* *

2 1

*

*

*

0 4

B CXCL-8 3

* *

2

*

*

Expression relative to β-actin

1

0 8 7

C

1L-1β-1

*

*

1L-1β-2

6

*

5 4

*

* *

3 *

2

*

*

*

*

*

*

*

1 0

6 5

D

TNFα-1

*

TNFα-2

4

* *

*

3

*

*

*

*

*

* *

2

*

*

*

*

*

*

*

*

*

*

*

*

1 0 9 8

E TGF-β

7

*

6 5 4 3

*

*

2 1

6d

3d

1d

2h

6d C on tro l

3d

1d

2h

6d C on tro l

3d

1d

2h

C on tro l

0

Time post treatment with sanguinarine Fig. 2. Quantitative expression analysis of immune genes in the gill, kidney and spleen of sanguinarine treated goldfish. A: CCL-1; B: CXCL-8; C: IL-1b-1 and IL-1b-2; D: TNFa-1 and TNFa-2; E: TGF-b. The expression data were normalized against those of the 0 h time point for each gene. The results are the mean  SEM from three individual fish. * represents significantly different (p < 0.05) from control.

praziquantel and sanguinarine on fish are usually neglected after repelling D. intermedius. Therefore the present study was conducted to investigate the expression of immune related genes and susceptibility to A. hydrophila in goldfish following treatment with

praziquantel and sanguinarine. The results showed that praziquantel and sanguinarine can modulate the expression of a series of immune genes of CCL-1, CXCL-8, IL-1b-1, IL-1b-2, TNFa-1, TNFa-2, and TGF-b in gill, kidney and spleen.

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Table 3 Quantification of genome equivalents of Aeromonas hydrophila per mg of tissues (GEs/mg) between treated and un-treated goldfish at day 1 and day 6 after drugs exposure. Within a tissue, means followed by different letters are statistically different (P < 0.05). Sampling time

Treatment

Genome equivalents of A. hydrophila per mg of tissues Gill

Day 1

Day 6

Praziquantel Sanguinarine Control Praziquantel Sanguinarine Control

30445.2 419.8 10720.9 2945.4 1856.8 2635.1

Kidney      

a

1357.2 121.8c 1141.8b 332.7a 128.2b 221.3a

It’s well known that IL-1b plays an important role in the immune response and acts as one of the most important and fastest inflammatory mediator [31,35]. The present work demonstrated that constitute expression of IL-1b was observed in gill, kidney and spleen of fish after exposure to praziquantel or sanguinarine. This result is consistent with Cantas et al.’ research which showed that flumequine caused a significant increase in the expression of IL-1b in zebrafish [36]. Besides, other studies also demonstrated an increase of IL-1b in fish induced by external chemicals, such as aquatic drugs or contaminants [35,37,38]. As an important inflammatory mediator, the increase of IL-1b suggested that praziquantel and sanguinarine could induce a local inflammatory reaction after treatment. Similar to IL-1b, another interleukin-type cytokine IL-8, namely CXCL-8, also showed overall upward trend in the experiment, although not significant in gill of sanguinarine treated goldfish. This result was in accordance with previous study that both antibiotics oxolinic acid and florfenicol increased the IL-8 in Atlantic cod [16], and flumequine also induced the overexpression of IL-8 in zebrafish [36]. Fast et al. had reported that IL-1b could augment IL-8 release [39], so it’s not surprising that IL-8 exhibited higher expression. Another up-regulation cytokine induced by the two drugs was TNF-a, which is also an important component during the initial of inflammatory response. Our results showed that both TNFa-1 and TNFa-2 were up-regulated by the two drugs in the three tissues during the observation period. What’s more, TNFa-2 expression levels were higher than those of TNFa-1, which was also observed in goldfish by Grayfer et al. [40]. Similar result was reported by Cuesta that lindane induced the increment of TNF-a expression in gilthead seabream head-kidney [38]. On the contrast, a down-regulation of TNF-a expression was observed in the zebrafish’s intestine by tetracycline treatment [41]. The different effects of immunomodulating might depend on the drug types, administration modes and fish species [16]. The increase of IL-1b, CXCL-8 and TNF-a in the gill, kidney and spleen may be the result of host stress response to praziquantel or sanguinarine. Whatever, the results showed the influence mode of praziquantel and sanguinarine on these genes was similar and it’s not tissue-specific. However, the CCL-1 gene exhibited obvious variation in different tissues. In the both treatment, CCL-1 significantly decreased in gill, while it significantly increased in kidney and spleen. Although the specific mechanisms has yet to be determined, we inferred that D. intermedius mainly infected on the gills of fish was one of the most probable reason. CCL-1 was also observed increment in goldfish when infected with Trypanosom acarassii [30,42]. Until now little is currently known about the specific function of CCL-1 in fish under external chemical conditions, the present study suggested that CCL-1 could be influenced by anthelmintic drugs, such as praziquantel and sanguinarine, and therefore may act as a potential therapeutic target. Another remarkable finding of the present study was the opposite impact of the two drugs on the expression of TGF-b. Down-regulation of TGF-b expression in tissues tested was

23802.2 4885.4 20694.3 2728.9 570.4 1559.4

Spleen      

a

2841.2 672.8b 1892.7a 177.3a 138.7c 231.8b

12935.7 1416.2 4223.2 8849.5 718.5 4111.5

Liver      

a

1217.3 190.2c 230.5b 1021.7a 241.8c 734.9b

5530.4 2708.1 3832.4 4373.4 2852.2 3560.9

     

230.5a 190.2c 217.3b 552.8a 309.1b 429.3ab

observed among fish exposed to praziquantel, while overall upregulation trend in the three tissues was observed in sanguinarine treatment. As a well-known immunoregulatory cytokine, TGF-b plays an important role in maintaining the immunological balance and re-establishing immune homeostasis by possibly downregulating expression of other cytokines and cytokine-induced effect [43,44]. The up-regulation of TGF-b by sanguinarine was consistent with earlier studies which showed TGF-b increased when juvenile chinook salmon exposed to pesticide and it seem to be connected to inflammatory processes [45,46]. Others hypothesized that the up-regulation of TGF-b was induced by inflammatory mediator increment such as IL-1b or might be aimed to restrict the inflammatory process [47]. Although the reason for the downregulation of TGF-b induced by praziquantel was not known, we speculate that the toxic and accumulation effects of praziquantel in vivo which previous had reported [48] may be responsible for such findings. Nevertheless, further studies are needed in the future to shed new light on the current outcomes and to clarify the potential mechanisms. The pharmacokinetics of a drug, i.e., the absorption, distribution, penetration and elimination may partly be responsible for the drug effect on host immune responses [49]. The absorption site for most drugs were the gastrointestinal tract or skin (based on the different modes of administration), and through which, drugs can permeate into the body, distribute throughout the body and accumulate in different tissue. Drugs that are accumulated or concentrated in immune organs could stimulates immune cells and thus influence immune responses [50]. Previous study had demonstrated praziquantel was readily absorbed from the gastrointestinal tract of the fish by oral administration and retained in serum, muscles, liver, bile fluid and kidney [51,52]. Our follow-up study found sanguinarine also shows different distribution and clear up mode in different tissues, such as the gill, kidney, spleen and liver (data not publish). Hence, we speculate that the tissue-specific genes expression discussed above may be related to the different accumulation content and elimination types in different organs of the two drugs. The host resistance challenge assay is regarded as the most definitive test of immune system function with the highest level of biological relevance since it measures an integrated immune response at the whole organism level [19]. Therefore the bacterial challenge test after withdrawal of the two drugs was conducted. This trial demonstrated that praziquantel treated fish had a higher load of A. hydrophila in organs than un-treated fish. The present study and previous study [16] support the notion that some chemical drugs can alter bacterial pathogen proliferation in host when given at their therapeutic concentrations. As is discussed above, the variation of immune functions by praziquantel might be responsible for the changes of infection probability of opportunistic pathogens. The results are in agreement with previous findings. Shelley et al. found [20] atrazine and nonylphenol could cause increased disease susceptibility in juvenile rainbow trout for their

C. Zhang et al. / Fish & Shellfish Immunology 35 (2013) 1301e1308

immune-toxic effects. However, a totally opposite orientation result was observed in the sanguinarine treated fish with the decrement of bacterial infection. Hypothetically, the significant reduction of A. hydrophila could be very likely due to the antibacterial activity of sanguinarine [53,54]. Similar results were reported that antibiotics could result in a decreased proliferation of the bacterial pathogens in Atlantic cod, which lasted until 5 days postwithdrawal of the antimicrobial drugs [16]. This could be related to the depletion of the drugs residues in the fish during this time. Based on the above discussion about the opposite behavior of TGFb, we propose the hypotheses that the TGF-b might be a key point induced by different parasitic drugs and responsible for the differential bacterial infection. Additional studies are still needed in order to clearly establish the link between treatments of the anthelmintic drugs to bacterial infection in fish. In conclusion, the present study has shown that bath administration of praziquantel and sanguinarine at their therapeutic concentrations could result in differential effects on the expression levels of the immune-related genes and differences in the proliferation of the bacterial pathogens, when goldfish were infected D. intermedius. These variations might be associated to the type of anti-parasites drugs. To understand the mechanisms by which the two drugs modulate the expression of immune genes and bacterial susceptibility in goldfish, detailed studies are necessary particularly on the pharmacokinetics of the drugs and their effects on the physiological level. Functional studies of each gene in relation to administration of anti-parasites drugs are also the focus point in future studies. Acknowledgments This research was supported by Natural Science Foundation of China (no. 31072242). References [1] Meyer FP. Aquaculture disease and health management. J Anim Sci 1991;69: 4201e8. [2] Bondad-Reantaso MG, Subasinghe RP, Arthur JR, Ogawa K, Chinabut S, Adlard R, et al. Disease and health management in Asian aquaculture. Vet Parasitol 2005;132:249e72. [3] Reed P, Francis-Floyd R, Klinger R. FA 2 8/FA0 33: Monogenean parasites of fish. EDIS delectronic data information source dUF/IFAS extension. University of Florida; 17 May 2009. http://edis.ifas.ufl.edu/FA033. [4] Woo P, Bruno D. Diseases and disorders of finfish in cage culture. Malaysia: CABI; 2009. [5] Wang GX, Han J, Feng TT, Li FY, Zhu B. Bioassay-guided isolation and identification of active compounds from Fructus Arctii against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus). Parasitol Res 2009;106: 247e55. [6] Harnett W. The anthelmintic action of praziquantel. Parasitol Today 1988;4: 144e6. [7] Schmahl G, Mehlhorn H, Haberkorn A. Sym. triazinone (toltrazuril) effective against fish-parasitizing Monogenea. Parasitol Res 1988;75:67e8. [8] Treves-Brown K. Availability of medicines for fish. J Fish Vet Soc 1999;4: 40e55. [9] Wang GX, Zhou Z, Jiang DX, Han J, Wang JF, Zhao LW, et al. In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa (Maxim) Fedde against Dactylogyrus intermedius in Carassius auratus. Vet Parasitol 2010;171: 305e13. [10] Wang Y, Wu ZF, Wang GX, Wang F, Liu YT, Li FY, et al. In vivo anthelmintic activity of bruceine A and bruceine D from Brucea javanica against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus). Vet Parasitol 2011;177:127e33. [11] Saglam N, Yonar ME. Effects of sulfamerazine on selected haematological and immunological parameters in rainbow trout (Onchorhynchus mykiss, Walbaum, 1792). Aquac Res 2009;40:395e404. [12] Siwicki AK, Anderson DP, Dixon OW. Comparisons of nonspecific and specific immunomodulation by oxolinic acid, oxytetracycline and levamisole in salmonids. Vet Immunol Immunopathol 1989;23:195e200. [13] Chappell LH, Wasting JM. Cyclosporin A antiparasite drug, modulator of the host-parasite relationship and immunosuppressant. Parasitology 1992;105: S25e40.

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