Laboratory rearing of Theileria annulata-free Hyalommaanatolicum anatolicum ticks
S. Ghosh Æ P. Azhahianambi
Received: 12 March 2007 / Accepted: 13 August 2007 / Published online: 13 September 2007� Springer Science+Business Media B.V. 2007
Abstract Hyalomma anatolicum anatolicum is a three-host tick which transmits Thei-leria annulata infection in Indian cattle. Laboratory rearing of ixodid ticks is an essential
requirement of any laboratory engaged with research on ticks and tick borne diseases. The
Entomology laboratory of Indian Veterinary Research Institute is fully equipped with all
the facilities and skilled manpower to maintain a homogenous H. a. anatolicum population
throughout the year. The continuous supply of eggs, larvae and adults of H. a. anatolicumis maintained to meet out the demand of different experiments viz., preparation of tick
antigens for immunization of animals, experimental challenge, isolation of genomic DNA
and RNA. Maintenance of a H. a. anatolicum colony free of T. annulata infection is
imperative for the experimental challenge infestation on cross-bred (Bos indicus ·B. taurus) calves, in order to prevent the transmission of T. annulata infection to the
experimental animals. A system has been developed in the laboratory in which the larvae
of H. a. anatolicum were fed on New Zealand white rabbits and the dropped fed nymphs
molted to adults are fed on cross-bred calves free of T. annulata infection. This synthetic
cycle prevents the transstadial transmission of T. annulata as the rabbits are unsusceptible
to T. annulata infection and only the adults were fed on cross-bred animals. Moreover,
absence of transovarial transmission of T. annulata prevents the chance of carry over
infection to experimental animals in the next cycle.
Keywords Hyalomma anatolicum anatolicum � Rearing of Theileria annulata free tick �rabbit-cattle cycle
Declaration: The experiments have been conducted in accordance with the approved guidelines ofCommittee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA). Besides,the institute animal ethics committee continuously monitored the animal experimentation.
S. Ghosh (&) � P. AzhahianambiEntomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, UP243122, Indiae-mail: [email protected]
123
Exp Appl Acarol (2007) 43:137–146DOI 10.1007/s10493-007-9100-3
Introduction
As per the last compilation reports on tick species of India, approximately 106 tick species
belonging to two families of Ixodidae and Argasidae are reported to infest domestic, wild
and game animals (Geevarghese et al. 1997). Amongst the reported tick species,
Hyalomma anatolicum anatolicum has been considered as one of the most widely dis-
tributed tick species infesting cattle, buffalo, sheep and goat and transmitting Theileriaannulata, T. buffeli, T. lestocardi (T. hirci) (Ghosh et al. 2006). The predicted distribution
of bovine tropical theileriosis in India and cattle and buffalo population at risk is well
documented (Minjauw and Mc Leod 2003). As per their estimation the cost of control of
theileriosis including the cost of control of ticks in India is about 441.5 million US$ per
annum. Beside the vectorial potential of the tick species, the significant direct effect of
ticks on livestock production (Biswas 2003) necessitated to develop methods to control the
tick species in an environmental friendly manner.
Under the national priority programme, the Division of Parasitology of the Indian
Veterinary Research Institute is continuously working on development of immunopro-
phylactic measures against the targeted tick species and the parasite, Theileria annualata(Singh and Ghosh 2003; Das et al. 2005; Ghosh et al. 2005). Continuous supply of
T. annulata-free stages of H. a. anatolicum is one of the mandatory requirements for the
different types of experiments conducted for the priority programme. We have previously
tried a cattle system for the generation of a tick colony but this strategy was not feasible.
Subsequently, a different strategy was designed and was found effective. The entire
strategy along with the life cycle data generated during the last 10 years is discussed.
Materials and methods
Animal
Healthy New Zealand white rabbits of 1 year old and 1.5–2 kg in weight were used for
feeding of ticks. The rabbits were maintained in clean disinfected rabbit cages of ca. 5 ·3.8 · 3.6 cm in size. Food and water were given ad libitum as per the ration developed by
the Laboratory Animal Research section of the institute. For raising good number of tick
stages for different type of experiments and to avoid stress on animals, 6–8 rabbits were
maintained simultaneously. Normally, two rabbits were utilized for each feeding cycle.
After each feeding cycle, the animals were kept free for at last 20 days–1 month.
For feeding on large animals, cross-bred male calves of 6–10 months old and more than
60 kg in weight, were maintained from the initial stage of birth in the tick and fly proof
shed of the division of Parasitology. Each animal was kept in well-ventilated shed with
slatted floor. The size of the individual shed was ca. 2.4 · 2.3 m (as per the recommen-
dation of CPCSEA). The animals were examined regularly to maintain the disease free
condition. To keep the animals stress free, 5–6 animals were maintained simultaneously
and 2–3 animals were used for each feeding cycle. After each feeding cycle, the animals
were kept free for 15–20 days. To protect the animals from natural tick infestation, the
animals were fed with dry fodder and water ad libitum. During winter (the outside tem-
perature of the animal shed is going below 4�C) the temperature of animal rooms were
maintained using room heater. The T. annulata/Babesia bigemina free status of the calves
were ascertained by periodical examination of Giemsa stained blood smears.
138 Exp Appl Acarol (2007) 43:137–146
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Ear bag for rabbit and cattle
Ear bags for rabbit (7 · 10 cm) and for cattle (13 · 17 cm) were fabricated using mouslin
cloth. Stitching of bags was done meticulously so that the tick stages cannot come out from
the bag. The typical design of the tick-feeding bag is given in Fig. 1a, b.
Head collar
For feeding on rabbits, head collars of the diameter of ca. 22 cm were used. The centre hole
(for putting the collar on neck) was of ca. 6.5 cm in diameter for keeping the tick feeding
bags undamaged on ear and also allow rabbit to take food and water easily.
Collection of ticks from the field (Izatnagar)
The engorged H. a. anatolicum ticks were collected from the resting places of cattle in and
around the institute, Izatnagar, UP, India. Collections were made from the cracks and
Fig. 1 (a) Showing tick feeding bag for rabbit system; (b) showing tick feeding bag for cattle system
Exp Appl Acarol (2007) 43:137–146 139
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crevices with the help of forceps. Collection of engorged H. a. anatolicum from the animal
shed is advantageous over the flag dragging method to collect the questing larvae as it is
easier to identify the ticks at the adult stage and it does not require any lint flag.
The collected ticks were identified as H. a. anatolicum by morphological key given by
Sen and Fletcher (1962).
Rearing of collected ticks
After proper identification, the fully engorged adults collected from the field were rinsed in
distilled water and placed on filter paper kept inside the tick rearing glass tubes (5 cm in
height and 2.5 cm in diameter) covered with mouslin cloth with the help of rubber band.
Each vial bore a label bearing the name of the species and the date of recovery. The glass
tubes were kept in desiccators where 85% relative humidity (RH) was maintained. The
desiccators were kept at 28�C or at 18–20�C for oviposition.
Egg hatching
After completion of the oviposition, the dead female H. a. anatolicum were removed from
the glass tubes in order to avoid the fungal growth on the dead ticks and subsequent
contamination of the eggs. The freshly laid eggs were kept at two different temperatures of
18–20�C and 28�C with 85% RH.
Feeding of H. a. anatolicum larvae on rabbits
Larvae were fed on New Zealand white rabbits. Before releasing ticks on animals, the ear
pinnas were shaved without damaging the skin. For each feeding cycle, eggs laid by one
tick were divided into two parts and were kept in separate tubes. The larvae hatched in each
tube were used for each rabbit. Before releasing on rabbits the larvae were transferred from
the glass tube to ear bags. The bags along with larvae were inserted into the ear pinna and
the threads were gently tightened around the base of the ear pinna. Care should be exer-
cised in such a way not to tight the rope around the base of the pinna as it might lead to the
formation of edema around the pinna. The optimum tying of the bags with ear pinna
required some expertise and experience in handling of animals for tick feeding. After
releasing the tick larvae, the animals were checked daily twice, once in the morning and
again in the evening to avoid the loss of tick stages. It was observed that when H. a.anatolicum larvae feeds on rabbits it has a two-host life cycle. After feeding, the engorged
larvae are remaining on rabbit to moult into unfed nymphs, which then attach and engorge.
The engorged nymphs were collected. In contrast, when the larvae were fed on cattle or
gerbils it has a three-host life cycle and engorged larvae detach naturally and moult to
unfed nymphs whilst off the host.
Rearing of nymphs
For moulting to next stage of development, freshly collected engorged nymphs were
thoroughly cleaned before placing in tick rearing glass vials. The engorged nymphs were
140 Exp Appl Acarol (2007) 43:137–146
123
kept in-group of 50 in each vial. The tube containing engorged nymphs were kept at two
different temperatures and in 85% RH for moulting. The moulting pattern was recorded
daily.
Feeding of H. a. anatolicum adults on cattle
The freshly hatched adults were kept unfed for 7 days before release on disease free male
cross-bred calves. Before releasing the unfed adults, the ear pinnas were shaved smoothly
for ease of attachment of ticks. For each infestation, 50–70 unfed adults (males and females
together) were taken in each ear bag and the bag was tied with the base of ear pinna.
Extreme care was taken to avoid the formation of odema due to improper tying of bag with
ear pinna. Usually the adults were released in the morning hours of the day and were
closely observed for 24 h. During the initial stage of tick attachment (up to 12 h of release)
the animals suffered from irritation and try to remove the tick-feeding bag. The ear bags
were checked daily, collected the fed adults, cleaned, weighed, labelled and kept singly in
the glass tube and were kept in the above mentioned temperatures and 85% RH for
oviposition.
Control of temperature and humidity
The tick colonies were maintained in dessicators which were kept at 18–20�C and at 28�C
in incubator. Except when the light was switched on for observation, the interior of the
incubator was constantly kept dark. The humidity was maintained by keeping a 10%
solution of KOH at the base of the desiccators (Solomon 1951). The condition provided a
RH between 85% at 28�C temperature.
Collection of data
The tick stages are maintained for more than 10 years in the Entomology Laboratory,
Division of Parasitology, Indian Veterinary Research Institute employing the above-
mentioned strategy to generate a homogenous colony. The data collected during this long
period were analyzed and the mean data were presented for each and every parameter. All
the tick feeding data were generated under the strict supervision of trained and experienced
scientific and technical staff.
Results
Identification of tick species
The collected ticks were identified as H. a. anatolicum. The females were identified by the
possession of hexagonal basis capitulum; capitulum short and palps broad; presence of
knob like genital operculum which is circular in outline; scutum is usually longer than wide
with a narrowly rounded posterior margin, with punctuation in central field and in scapular
areas; the cervical fields are shallower with parallel sides.
Exp Appl Acarol (2007) 43:137–146 141
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The males were identified by the following descriptions: small elongated body; scutum
punctuated with distinct lateral grooves; eyes spherical and orbital; basis capitulum sub
triangular or broadly hexagonal dorsally; elongate adanal shields; subanal shields situated
on the axis of adanal shields.
Oviposition of engorged H. a. anatolicum
The pre-oviposition period of the laboratory bred adult ticks varied with the temperature. If
the ticks were kept at 18–20�C and 85% RH, the pre-oviposition period ranged between 25
and 62 days (Mean = 38.4 days). However, at a constant temperature of 28�C and RH
85%, the pre-oviposition period ranged between 10 and 12.2 days (Mean = 6.4 days).
The oviposition period was also varied with the temperature. At a temperature of 18–
20�C and RH of 85% the oviposition period ranged from 25 to 38 days (Mean = 30.4
days). While at 28�C and 85% RH the oviposition period ranged between 18 and 22.4 days
(Mean = 21.8 days). The rate of oviposition attained the peak between 6 and 11 days when
the ticks were maintained at 18–20�C, whereas, the pattern of oviposition was completely
different when the ticks were maintained at 28�C. Within four days, the egg laying reached
at peak and continued for another 24–48 h. This could be attributed to the higher rate of
digestion and absorption of blood at higher temperature. There was a linear relationship
between the egg masses and the weight of the engorged adults. After oviposition, the
females are dying within 1–7 days (Mean = 4.2 days) at 18–20�C, whereas it was 1–
6 days (Mean = 3.4 days) at 28�C.
Hatching of eggs
The incubation period of the freshly laid eggs varied between 50 and 60 days (Mean =
57.9 days) when maintained at 18–20�C, while at 28�C, the incubation period was 20–
28 days (Mean = 21.7 days). Hatched larvae were ready to feed on animals at approxi-
mately 6–7 days after hatching.
Feeding of H. a. anatolicum larvae on rabbits
The larvae attached to the body of the rabbits usually within 18–36 h of release. A
significantly high percentage of fed larvae did not drop off after engorgement and molted
to nymphs and reattached on the same host, thus maintaining two-host pattern of feeding.
In all the experiments, not more than 60–65% larvae succeeded in feeding on rabbit host.
Among the successful larvae, more than 97% were recovered as fed nymphs and 2.5–2.8%
dropped as fed larvae. The fed nymphs started dropping after 13th–16th day (Mean = 14th
day) and continued for 5–6 days. The general pattern of dropping of fed nymphs was
nocturnal.
Rearing of nymphs for moulting
The engorged nymphs stopped movement after 6–7 days of keeping in the rearing tubes
and moulted to next stage after 13–15 days (Mean = 14 days) of incubation. The complete
142 Exp Appl Acarol (2007) 43:137–146
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molting to adult stage takes another 4–7 days of initiation of moulting. Immediately after
completion of moulting, the unfed adult started upward movement and attached with the
mouslin cloth fixed on the tubes.
Feeding of H. a. anatolicum adult on cattle
The 7 days old unfed adult ticks usually take 24–48 h for attachment. Immediately after
attachment the adults starts feeding and takes about 8–15 days (Mean = 10.5 days) for
complete engorgement. The feeding process was slow during pre-repletion phase which
usually lasted two days before dropping. In the repletion phase the feeding was rapid and
most of the blood was ingested during this phase. The largest number of engorged females
normally dropped on 6th and 7th day. The weight of fed adults ranged between 305.4 to
336.7 mg (Mean = 312.6 mg) and on average 165.3 to 202.4 mg egg masses (Mean =
191.7 mg) were laid by individual adult.
Longevity of the H. a. anatolicum life stages
For getting continuous supply of the different tick stages of H. a. anatolicum for several
experiments, specific strategies were used. For example, freshly hatched larvae, nymphs
and adults as well as engorged adult female ticks were kept at 4�C with 80% RH for 3–
4 months. Long-term preservation at 4�C leads to 20 to 30 % mortality of the tick stages.
Besides, a significant percentage of tick stages could not attach with the natural host after
release. When not released on the host, the longevity of each stage at 28�C with 80% RH
was: larvae, 20–25 days; nymphs, 15–35 days and adults, 30–40 days. Usually the lon-
gevity of unfed adults was significantly higher than the fed ones. This may be attributed to
a general sluggish physiological process which enables them to survive longer period
without feeding. For getting good results, the tick stages should be released within 6–
7 days after moulting to the subsequent stage.
High fecundity of H. a. anatolicum
It was observed that the egg masses laid by individual tick (Mean = 191.7 mg ± 11.4,
n = 1,414) was four times higher than the other most prevalent tick species, Boophilusmicroplus (Mean = 49.38 ± 5.6, n = 356) infesting livestock.
Discussion
In India, H. a. anatolicum, a three host tick which transmits Bovine Tropical Theileriosis in
cross-bred cattle. For any experiments to evaluate immunogenic potential of tick molecules
in cattle system and to study the T. annulata transmission limiting potential of the iden-
tified molecules necessitate the requirement of continuous supply of good quantity and
quality of active H. a. anatolicum population free of T. annulata infection. Though it
would be easier and cost effective to maintain the T. annulata infection free H. a. anat-olicum population on rabbit-rabbit cycle, the quality and quantity of H. a. anatolicumpopulation obtained from the rabbit-rabbit cycle were not encouraging in nature. In our
Exp Appl Acarol (2007) 43:137–146 143
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experiments, besides fewer yields, the individual size of the adult H. a. anatolicum was
phenomenally reduced with extremely male biased sex ratio while fed on rabbit alone. On
the other hand, feeding of H. a. anatolicum on rabbit-cattle cycle (Fig. 2) yielded more
number of good quality T. annulata infection free H. a. anatolicum adults. The strategy
was designed to feed the larvae that hatched out from the egg masses of field collected H.a. anatolicum on New Zealand white rabbits and the resultant fed nymphs (a two host
cycle) dropped from the rabbits and molted to adults and the adults were fed on cross bred
calves. The larvae resulting from the egg masses obtained from the engorged H. a.anatolicum were used in the subsequent cycles to feed on rabbits. Laboratory rearing of T.annulata infection free H. a. anatolicum on rabbit-cattle cycle strategy was driven by three
principles viz., absence of transovarial transmission of T. annulata in H. a. anatolicum,
unsusceptible nature of rabbits to T. annulata infection and blockage of transstadial
transmission of T. annulata by feeding only the adults on cattle. This strategy alleviate the
problem of maintaining H. a. anatolicum on rabbit-rabbit cycle as feeding of H. a. anat-olicum larvae on rabbit and adults on cattle appears to simulate the natural life cycle of the
multi-host tick, H. a. anatolicum.
Presently there are no available invasive or non-invasive techniques to identify
T. annulata infection status of H. a. anatolicum without harming the life of the tick.
Though the T. annulata infection status of the field collected H. a. anatolicum is not
known, the New Zealand white rabbits which were fed with larvae of field origin never
showed any sign of T. annulata infection. The absence of transovarial transmission of
T. annulata by the H. a. anatolicum might be the reason behind it. Although a report
Fig. 2 Schematic diagram showing strategy to rear T. annulata infection free H. a. anatolicum tick inrabbit-cattle cycle
144 Exp Appl Acarol (2007) 43:137–146
123
citing the T. annulata infection in rabbit transmitted by Hyalomma rufipes (Dipeolu and
Ogunji 1977), generally the rabbits are considered as non-susceptible host for T. an-nulata infection (Bhattacharyulu et al. 1975). Though properly screened T. annulatainfection free cross bred calves were used in the experiment for feeding of adult H. a.anatolicum, the possibility of getting infection through the ticks present in green fodder
can not be ruled out. Once again the absence of transovarial transmission of T. annulataand the unsusceptible nature of rabbits facilitate the production of T. annulata infection
free H. a. anatolicum Izatnagar isolate. We prefer dry fodders to feed the calves kept for
the purpose of feeding adults of H. a. anatolicum to avoid natural field infestation.
The influence of temperature in the duration of pre-oviposition and oviposition periods
observed in the present investigation was also reported by many workers and subject has
been extensively reviewed (Snow 1969). It was also observed that there is a significant
difference in the life cycle parameters of the tick reared in the Entomology Laboratory and
parameters recorded by Geevarghese and Dhanda (1987). This may be due to continuous
inbreeding of the present tick species for the last more than 10 years in the laboratory while
the data generated by Geevarghese and Dhanda (1987) were from out bred population.
Hyalomma spp. ticks are well known for transmission of Congo-crimean hemorrhagic
fever (CCHF) virus to man (Camicas et al. 1994; Estrada-Pena and Jongejan 1999).
Though there is no report of tick borne human diseases reported in and around Izatnagar,
due care was taken while handling the tick stages to prevent any possibility of transmission
of tick borne diseases to technicians and students of the Entomology Laboratory. By
wearing latex gloves and handling the ticks with forceps, the accidental tick bites were
avoided while handling them.
High fecund nature of H. a. anatolicum over the B. microplus could be directly
attributed to the bigger size of the former. On viewing this data, from the evolutionary
angle suggests that the multi host ticks like H. a. anatolicum should produce more juve-
niles to persist in the environment as unlike one host ticks they have to search for new hosts
after each dropping and the survivability of the dropped tick stages depend on the envi-
ronmental condition and availability of suitable host.
This experiment and our experience conclude that the rearing of T. annulata infection
free H. a. anatolicum on rabbit-cattle cycle is a suitable strategy to raise large number of H.a. anatolicum with optimal size and activity which is essential for any experiments to get
repeatable results.
Acknowledgements Sincere thanks are due to the Director of the institute for providing laboratoryfacilities. Thanks are due to Department of Biotechnology, Government of India for providing necessaryfunding. The authors are grateful to Mr. Laxmi Lal and Mr. Naresh Kumar for technical support rendered bythem to maintain the tick colony continuously in the laboratory.
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