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Veterinary Parasitology 180 (2011) 332– 335

Contents lists available at ScienceDirect

Veterinary Parasitology

jou rn al h om epa ge: www.elsev ier .com/ locate /vetpar

yalomma anatolicum anatolicum and Hyalomma dromedarii (Acari:xodidae) imbibe bovine blood in vitro by utilizing an artificial feedingystem

. Tajeri ∗, G.R. Razmiepartment of Pathobiology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

r t i c l e i n f o

rticle history:eceived 19 October 2010eceived in revised form 28 February 2011ccepted 8 March 2011

a b s t r a c t

Hard ticks have great importance because of blood feeding and transmitting dangeroushuman and animal diseases. Each year, they cause a lot of economical damage to the live-stock industry. Control of ticks and tick-borne diseases is a major priority in most parts of theworld and many studies have been done in this field. We know that studying haemopar-asites and assessing the effect of different compounds on ticks requires a lot of money,

eywords:n vitro feedingicksyalomma anatolicum anatolicumyalomma dromedariirtificial membrane

support and sometimes it is time consuming. Considering all of these problems, today,in some research laboratories throughout the world, artificial in vitro feeding of ticks hasbecome common. Development and application of such methods provide a cheap and acces-sible background for investigating haemoparasitic diseases under controlled conditions. Forthe first time we report successful in vitro feeding of two important ixodid ticks of the genus

Hyalomma.

. Introduction

Ticks transmit a wider range of animal and humanathogens than any other arthropod vectors and theireeding behavior damages the host directly (Jongejannd Uilenberg, 2004). Hyalomma anatolicum anatolicums widely distributed over most geographical regions inran (Nabian et al., 2009), where it is considered the

ain vector of Theileria annulata, the causative agentf tropical theileriosis. Moreover, it transmits otherathogens such as Theileria lestoquardi (Kirvar et al., 1998)nd Crimean-Congo haemorraghic fever virus whereasyalomma dromedarii is a vector of minor importance with

onfined distribution over desert areas and mainly infestsamels. The role of this species in disease transmissions not well understood but it is capable of transmittingheileria annulata under laboratory conditions (Walker

∗ Corresponding author. Tel.: +98 511 8763851; fax: +98 511 8763852.E-mail address: shahin tajery@yahoo.com (Shahin Tajeri).

304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2011.03.014

© 2011 Elsevier B.V. All rights reserved.

et al., 2003). Because of their inevitable role in the live-stock industry, developing novel methods for studyingdifferent aspects of these arthropods, specifically in vitrofeeding devices, have substantial importance. An in vitrofeeding system provides a cheap, precise and ethicallyacceptable assay for systemic and topical acaricides. Inaddition, it permits a variety of investigations, such as val-idation of vector competence for pathogens, unravelingvector–pathogen interactions, identifying novel vaccinecandidates and analysis of the complex sialome of ticks.Generally, ticks can feed in vitro through two methods,capillary tube feeding and artificial membranes (Waladdeet al., 1996). Off-host feeding of different ixodid speciesover a variety of membranes has been studied; theseinclude Rhipicephalus (Boophilus) microplus (Kemp et al.,1975; Waladde et al., 1979), Dermacentor andersoni (Paine

et al., 1983), D. nuttali (Habedank et al., 1994), Amblyommahebraeum (Kuhnert et al., 1995) A. variegatum (Voigt et al.,1993; Young et al., 1996; Barre et al., 1998), A. cajennense(de Moura et al., 1997), Hyalomma anatolicum excavatum(Habedank et al., 1994) Ixodes holocyclus (Stone et al., 1983),

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S. Tajeri, G.R. Razmi / Veterina

I. ricinus (Bonnet et al., 2007) and Rhipicephalus appendic-ulatus (Waladde et al., 1991, 1993; Musyoki et al., 2004).The second method, capillary feeding, has been utilized forRhipicephalus (Boophilus) microplus (de la Vega et al., 2000),Ixodes scapularis (Korshus et al., 2004; Soares et al., 2005;Broadwater et al., 2009), I. ricinus, I. hexagonus (Bouwknegtet al., 2010), Rhipicephalus appendiculatus (Purnell andJoyner, 1967), R. bursa (Bouwknegt et al., 2010), Derma-centor reticulatus (Bouwknegt et al., 2010) and D. variabilis(Kocan et al., 2005). An absolutely defined method wasrecently designed by Kröber and Guerin (2007a,b) for Ixodesricinus. The present study was carried out to investigate theapplication of this in vitro system for Hyalomma anatolicumanatolicum and Hyalomma dromedarii.

2. Materials and methods

2.1. Feeding units

We constructed the feeding units as described by Kröberand Guerin (2007a), and set them up in polystyrene six welltissue culture plates (Jet Biofil®, Guangzhou, China).

2.2. Artificial membrane

Anti fungal silicone sealant (American dolphin®, AlMuqarram Industry, Sharjah, U.A.E) was mixed withtoluene solvent (108325, Merck®, Germany) with a ratio of4:1 by using a glass rod inside a beaker. This mixture wasthen applied to Tiffen® lens cleaning paper (Hauppauge,NY, USA) with a silicone rubber. The sealant was allowedto dry and polymerize on the paper for about 12 h; follow-ing this period, the membrane was cut into pieces in theform of squares (3 cm × 3 cm) and then a layer of plasticmosquito netting cut in the shape of a circle (24 mm diam-eter) was glued (silicone sealant) to the center of each ofthe 3 cm × 3 cm membranes which were then attached toone side of the feeding tubes with silicone glue. After about3 h the excess membrane around the edges of the feedingunits was cut off with fine scissors or a razor blade.

Permeability of the membrane was assessed withethanol and distilled water. Leaking pores were correctedwith silicone glue.

2.3. Attachment stimuli for ticks

Granules of tick feces, a plastic cross, freshly shavedbovine hair and its extract were the attachment stimuli(Kröber and Guerin, 2007a). 70% ethanol was used insteadof dichloromethane to prepare bovine hair extract. Theextract was applied to the internal surfaces of each feedingchamber with a swab.

2.4. Ticks

Colonies of Hyalomma anatolicum anatolicum and

Hyalomma dromedarii were kept at the Laboratory AnimalSection, Veterinary Clinic of Ferdowsi University. FemaleNew Zealand White rabbits were used to culture these ticks.In this study, second generation unfed adults of the two tickspecies, about 7–8 months old and kept in an incubator

sitology 180 (2011) 332– 335 333

(27 ◦C, 85–90% relative humidity and complete darkness,except when the door of the incubator was opened forexamination) were used. Before introducing the ticks intothe feeding units they were exposed to sunlight for about2 h.

2.5. Blood preparation

Blood from Holstein cattle was collected weekly fromthe central industrial slaughterhouse of Mashhad and wasprepared according to Kröber and Guerin (2007a) exceptthat here the blood was not supplemented with ATP.

2.6. The experiment

At the start of the experiment 3 ml of refrigerated bloodwas poured into each well. After that, the tissue cultureplate was floated on the surface of warm water (39 ◦C)inside a water bath. We transferred active ticks to the pre-pared feeding units (all attachment stimuli applied) andclosed the upper end with rubber caps to prevent escape ofticks. Feeding units were placed in wells after about 20 min,providing enough time for the blood to warm. The lid of thewater bath was closed at night and was semi-closed duringdaytime.

Blood was replenished every 12 h (at 8 a.m. and 8 p.m.).To keep the feeding units and plates devoid of bacteria orfungi, these were treated with physiological saline, antibi-otics and Nystatin (Kröber and Guerin, 2007a).

The ticks were examined two or three times a day andthe observations were recorded. Dead ticks were removedwith forceps to prevent fungal growth. Replete femaleticks were collected, stabilized in petri dishes with zincoxide adhesive plaster (MehecoNova®, Changzhou HualianHealth Dressing Co., China) and kept in the incubator(27 ◦C, 85–90% relative humidity and complete darkness)for oviposition.

3. Results

Both tick species attached readily to the non-livingmembrane (Fig. 1); females fed for about six days untilengorgement and took most of their meal during the final12 h of blood feeding. Engorged females oviposited after8–10 days and larvae hatched within one month, being ableto feed normally on rabbit ears. When a tick attached tothe membrane, the hypostome was visible on the otherside of the membrane macroscopically or under a stere-omicroscope. After a day or two, the tick started to secreteits cement around the hypostome, which afterwards grewbigger and reached its final volume. The cement cone had acreamy-white color and a small pore was always detectablein the center of it. Usually a sticky dark red fluid dis-tinct from tick feces flowed from anus of engorging ticksand coagulated after a while. In females this phenomenonsometimes prevented full engorgement and in the case of

male ticks it caused them to stick to the membrane, beingunable to detach themselves from the membrane, there-fore we decided to separate the material from the tick bodywith fine forceps. Male ticks attached, fed and mated withfemale ticks regularly and those which could not pierce

334 S. Tajeri, G.R. Razmi / Veterinary Parasitology 180 (2011) 332– 335

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ig. 1. Inside view of feeding chambers. Hyalomma dromedarii female tickeflected by differences in tick sizes (A). Another feeding unit showing sours of attachment (B).

he membrane succumbed to death after 4–5 days. Femaleicks did not attach synchronously (Fig. 1A), and this usuallyorced us to keep exchanging the blood for longer periodsabout 15–16 days) in order to have more ticks engorged.

hen an attached tick was detached manually with forcepshe blood did not leak into the feeding chamber, indicatinglasticity of the membrane.

. Discussion

Kröber and Guerin (2007a) method has been repeatedy Bouwknegt et al. (2010) and Campbell et al. (2010) withhe same tick species, Ixodes ricinus. There is no report onevelopment and application of this artificial feeding sys-em to other tick species. In our investigations we were ableo successfully feed two other tick species, suggesting highdaptability of this artificial system.

In similar studies, attachment rates of 75–100% forxodes ricinus females (Kröber and Guerin, 2007a) and2–78% for Amblyomma hebraeum (Kuhnert et al., 1995)ere obtained, which are higher than our findings (55%

nd 75% for H. dromedarii and H. anatolicum anatolicum,espectively). Recently, Campbell et al. (2010) reportedn attachment rate of 90% within the first 24 h for Ixodesicinus. In the case of H. dromedarii low percentages ofttachment and engorgement can be associated with theype of the blood we used. We know that this tick has

greater tendency to feed on camels. Another reasonor these poorer results might be due to not adding ATPo the blood and the way we prepared the bovine hairxtract. Besides, the silicone sealant we used had a higherhore A hardness (21◦ versus 16◦ in other experiments), anndicator of the degree of hardness of the sealant. Notwith-tanding these weak points in the present work, successn establishing artificial in vitro feeding of these important

ectors justifies our observations.

We are now trying to modify this artificial feedingssay (constructing thinner membranes and smaller feed-ng chambers) in such a way that other life cycle stagesf these tick species can be maintained artificially. After

ing on silicone membrane. Asynchronous attachment of ticks is obvious,ale and female Hyalomma anatolicum anatolicum ticks during their first

achieving this goal, we could complete their life cycles invitro, and eventually establish a system for infecting tickswith pathogens such as Theileria annulata for further workon vector–pathogen interactions.

Acknowledgments

Dr. Lesley Bell-Sakyi (The Roslin Wellcome Trust TickCell Biobank, The Roslin Institute and Royal (Dick) Schoolof Veterinary Studies, University of Edinburgh) is thankedfor critically reading the manuscript.

We are grateful to Dr. Thomas Kröber (Institute of Biol-ogy, University of Neuchâtel, Switzerland) for the kind giftof silicone rubber and a sample of the feeding unit. Dr.Alireza Haghparast is acknowledged for providing the lenscleaning papers after his trip to Japan. Our sincere thanksgo to Mr. G.A. Azari and Mr. H. Eshrati for their technicalsupport in maintaining tick colonies.

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