Alnassan et al 2015 Allicin Eimeria tenella

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SHORT COMMUNICATION. In vitro efficacy of allicin on chicken Eimeria tenella sporozoites. Alaa Aldin Alnassan1. & Ahmed Thabet1. & Arwid Daugschies1.
 

        

   

             

          

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Author's personal copy Parasitol Res DOI 10.1007/s00436-015-4637-2

SHORT COMMUNICATION

In vitro efficacy of allicin on chicken Eimeria tenella sporozoites Alaa Aldin Alnassan 1 & Ahmed Thabet 1 & Arwid Daugschies 1 & Berit Bangoura 1

Received: 22 June 2015 / Accepted: 16 July 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Chicken coccidiosis is a major parasitic disease caused by Eimeria spp. It is controlled and treated using chemical anticoccidial agents. Development of partial or complete resistance toward these anticoccidials is considered a major problem in poultry industry. Allicin is an organosulfur compound produced as a result of the reaction between alliin and alliinase after hacking of garlic. In this study, tenfold dilution from 180 mg/ml to 1.8 ng/ml of allicin in distilled water was tested against E. tenella in vitro. The percent of inhibition in allicin was from 99.9 to 71.53 % using 180 mg/ml and 180 ng/ml, respectively. The percent of inhibition was 56.24 % using 1.8 ng/ml. We used allicin as a treatment from plants against chicken coccidiosis; however, in vivo study should be performed to confirm these results. Keywords Allicin . Chicken coccidiosis . Eimeria tenella . Anticoccidial

Introduction Chicken coccidiosis is a protozoal disease and leads to high annual economical losses in poultry industry due to high mortality, reduction of body weight and treatment costs. Eimeria tenella is one of the most important Eimeria species, causing caecal haemorrhages and high mortality (McDougald and FitzCoy 2013). Chicken coccidiosis is controlled using chemical

* Berit Bangoura [email protected] 1

Institute of Parasitology, Centre of Infectious Diseases, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 35, 04103 Leipzig, Germany

anticoccidials in feed or drinking water worldwide (McDougald 1982; Blake and Tomley 2014) or application of a live vaccine. The main problem regarding control schemes based on drug administration is the development of resistance following repeated use of the compounds. In the last years, several plant extracts were tested for potential use against chicken coccidiosis. Garlic is one of the most important plants to prevent other poultry diseases in field (Peinado et al. 2012; Jimoh et al. 2013). Allicin is not found directly in garlic but produced as an active defence metabolite from garlic by the reaction between alliin and alliinase after hacking of garlic (Borlinghaus et al. 2014). It has been shown that allicin has antibacterial and antifungal properties of potential medical importance (Borlinghaus et al. 2014). Garlic extract as well as isolated secondary metabolites like propyl thiosulfinate oxide and propyl thiosulfinate have been shown to exhibit partial anticoccidial activity in vivo and in vitro (Kim et al. 2013; Khalil et al. 2015). In this study, efficacy of targeted allicin treatment on the development of chicken E. tenella sporozoites in cell culture was studied.

Material and methods E. tenella (Houghton strain) was kindly provided by Dr. D. P. Blake, Royal Veterinary College, University of London, UK. E. tenella oocysts were passaged in chickens as described before (Shirley 1977) and stored at 8 °C in 2 % potassium dichromate until use at the Institute of Parasitology, University Leipzig, Germany. Oocysts were washed in phosphatebuffered saline (PBS; pH=7.2) and sterilised using 12 % sodium hypochlorite for 15 min to prevent any bacterial contamination. Oocyst walls were broken using 0.5-mm glass beads (BioSpec Products, Inc., Bartlesville, OK, USA), and free sporocysts were then incubated in 0.25 % trypsin and 4 %

Author's personal copy Parasitol Res

sodium taurocholic acid by 41 °C for 90 min. The excysted sporozoites were purified using a DE-52 anion exchange method (Schmatz et al. 1984). Allicin was provided commercially (Allicin-AllimaxDeutschland GmbH, Syke, Germany) as powder capsules (180 mg sterilised allicin 100 % per capsule). Allicin powder was dissolved in distilled water (180 mg/ml) under bench to prevent any bacterial contaminations. Tenfold dilutions were carried out from original stock (180 mg/ml) until the end concentration (1.8 ng/ml). Madin-Darby bovine kidney (MDBK) cells (DSMZ, Braunschweig, Germany) were grown overnight in 24-well plates (TPP, Trasadingen, Switzerland) at an initial density of 2×105 cells per well in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4.5 g/l) and L-glutamine (Gibco, Invitrogen, Karlsruhe, Germany) and 5 % foetal bovine serum. One percent penicillin/streptomycin and 1 % amphotericin B (GE Healthcare, Germany) were added during infection of cells with sporozoites to prevent any bacterial and fungal contamination. 2×105 sporozoites were incubated in allicin solutions for 1 h and then washed two times in PBS. Four replicates were prepared for each dilution. The pellet of each replicate was dissolved in DMEM, and then the cells were

infected and incubated for 24 h at 41 °C. MDBK cells were washed with PBS after 24 h and incubated until 96 h after infection. Two hundred microlitre of trypsin was added to each well and incubated at 41 °C for 15 min. Detached cells were washed and transferred into microcentrifuge tubes. Trypsin was eliminated from these cells by centrifugation (1540g for 5 min), and the pellets were suspended in 200 μl PBS. DNA mini kit (Qiagen GmbH, Germany) was used for DNA extraction. The concentration of DNA was measured using NanoDrop 2000 (Thermo Scientific, Braunschweig, Germany) at 260 nm. All the samples were diluted to a concentration of 20 ng/μl. Stratagene Mx3000P (Stratagene, La Jolla, USA) was used for quantitative PCR assay (target fragment length 147 bp) as described by Thabet et al. (2015). Reaction solution consisted of 10 μl SYBR Green master mix (Thermo Scientific, Germany), 0.4 μl of a 25-μM stock of primers ET forward (tggaggggattagagga) and ET reverse (caagcagcatgtaacggaga) and 7.2 μl nuclease-free water (Kawahara et al. 2008; Thabet et al. 2015). The template volume was 2 μl (final volume 20 μl). Cycling reaction was carried out under the following conditions: 5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 20 s at 62 and 20 s at 72 °C. Inhibition rate was analysed and calculated as follows:

% of inhibition ¼ 100  ð1−ðnumber of E: tenella gene copies in treated sample=number of E: tenella gene copies in non‐treated controlÞÞ

Results and discussion In this study, a standard curve was generated using the ITS-1 gene of E. tenella from a pSCA-amp/kan plasmid. Six tenfold dilutions of plasmid were performed from 4.82×107 to 4.82× 102, and the efficiency of qPCR was 92.7 %. Table 1

The inhibition rate was from 99 % by 1.8 mg (p < 0.05) to 56.24 % by 1.8 ng/ml of allicin (p = 0.057; Table 1). The correlation between the applied allicin dose and the observed inhibition of parasite replication was calculated using Spearman rank correlation test (Table 1).

Inhibition of E. tenella replication in garlic-treated in vitro cultures Concentration

Gene copies (single replicates)

Average of treatment group Inhibition rate (%)

180 mg

18 mg

1.8 mg

180 μg

18 μg

1.8 μg

180 ng

18 ng

1.8 ng

Positive control

0.09 0.49 0.44

1.50

6.20

104.80

345.20

17.38

161.00

249.40

373.40

389.80

4.75 5.51

2.74 2.88

72.73 160.20

66.46 22.71

89.08 99.94

270.30 140.20

125.80 63.14

512.30 310.40

684.80 1129.00

0.01

5.66

2.93

172.90

30.04

136.50

337.70

789.70

201.60

990.20

0.26

4.36

3.69

114.16

116.10

85.73

227.30

307.01

349.43

798.45

99.97*

99.45*

61.55

56.24

99.54*

85.70*

85.46*

89.26*

71.53*

The correlation between the applied allicin concentration and the observed parasite replication inhibition was very strong (Spearman rho=0.909 with p