The transmission dynamics of
pathogens by Ixodes hexagonus ticks
using an in vitro feeding system
Student:
Irene de Heer
Student number:
3155137
Duration:
21 October 2013- 24 January 2014
Location:
Utrecht Centre for Tick-borne Diseases
Utrecht University, the Netherlands
Supervisor:
Prof. Dr. F. Jongejan
Index
Abstract
3
Introduction
4-5
Materials and methods
6-10
Results
11-19
Discussion
20-21
Conclusion
22
References
23-34
Appendix A
25-37
Appendix B
38
Illustration: Two Dermacentor Reticulatus ticks attached to a
membrane.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
2
Abstract
The principal objective of this study was to determine the transmission dynamics of
pathogens by Ixodes hexagonus using an in vitro feeding system. Dermacentor reticulatus
were used in the assay as a control group, in order to evaluate the environmental
conditions in the in vitro feeding system.
Unfortunately the attachment rates of I. hexagonus were very low. In an attempt to
increase attachment rates different attachment stimuli were used. None of these increased
the attachment rate significantly and no conclusions can be drawn on the preferences of I.
hexagonus in an in vitro feeding system.
Thirty I. hexagonus female ticks were used, 4 of them attached to the membrane and
were tested for pathogens using PCR and RLB. No pathogens were detected, consequently
no conclusions could be drawn on transmission.
Further research is needed to optimize an in vitro feeding assay for I.hexagonus and to
determine the transmission dynamics of pathogens by Ixodes hexagonus.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
3
Introduction
Ticks are vectors of pathogens of veterinary and human medical importance. The tickbusters survey, which started in May 2005 had identified I. hexagonus as a vector for
Anaplasma phagocytophilum, Babesia divergens, Theileria annae and Rickettsia helvetica.
(1)
1.1 Objective of this research
The principal objective of this study is to determine the transmission dynamics of several
pathogens, including the ones mentioned above, by I. hexagonus ticks. For this purpose,
an in vitro feeding system will be used. Therefore this study can also examine and better
understand the preferences of I. hexagonus in an in vitro feeding system.
1.2 Ixodes Hexagonus
Taxonomy
Kingdom: Animalia
Phylum: Arthropoda
Figure 1: Fed Ixodes
Hexagonus female (10)
Class: Arachnida
Subclass: Acari
Order: Ixodida
Family: Ixodidae
Genus: Ixodes
Species: Ixodes hexagonus
I. hexagonus is a member of the family Ixodidae. Ixodidae are also referred to as hard
ticks because they are characterized by the presence of a tough, sclerotized plate on the
dorsal body surface, the scutum. Ixodidae comprises 13 genera and approximately 650
species. This is in contrast to the family Argasidae, or soft ticks, which comprise 5 genera
and approximately 170 species. This is illustrated in the diagram below (9)
Figure 2: Diagram illustrating systematic and evolutionary relationships
in ticks (9)
Life cycle
I. hexagonus lives primarily in nests and burrows. Like all ticks, it has 4 stages: an
embryonated egg stage, a larval stage, a nymphal stage and an adult stage. I. hexagonus
becomes sexually mature soon after adult emergence. In contrast to ticks of other genera,
Ixodidae mating doesn’t strictly occur on the host, but to initiate the gonotrophic cycle a
blood meal is needed, therefore they will find a host afterward. During feeding female
adults of ixodid ticks swell greatly and can excess 100 times their unfed body weight. In
Ixodidae, the female will die shortly after depositing a large batch of eggs. The eggs will
hatch into larvae, which begin active questing for a host. Ixodes is categorized as a ‘threehost’ species, because from larvae to adult the Ixodes will use three hosts. Larvae will
attach to the first host and feed for 3-7 days. Once they are fully engorged, the larvae will
drop off the host and molt to a nymph. The nymph will feed on a new host for 3-8 days,
drop off, molt to an adult and seek a new host. Under favorable conditions in the natural
environment the entire life cycle can be completed in less than 1 year. Under laboratory
conditions a life cycle can be completed in 3 or 4 months. (9, 16)
1.3 In vitro feeding
To study transmission of pathogens by ticks, normally experimental animals are needed.
With the development of in vitro feeding a lot of these experimental animals are replaced.
This makes it a more favorable method from an ethic point of view, also the high costs of
maintaining suitable hosts for ticks are circumvented and the controlled conditions make
transmission studies easier.
The first in vitro feeding assay was started in 1965 with Boophilus microplus. Embryonated
hen eggs were used here to feed the larvae of B. micoplus. (22) In this current study a
silicon membrane was used. The first silicon membrane used in a feeding assay was the
silicon membrane of Habedank and Hiepe.(23) Kröber and Guerin further improved the
silicon membrane, making it softer and thinner (60-150µm). (21)
The in vitro feeding system of Kröber and Guerin was successfully introduced at the
Utrecht Centre for Tick-Borne Diseases in 2009 and further optimized. (3)
In ordered to successfully feed the ticks, attachment stimuli are needed. In this study
several are tested.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
5
Materials and methods
In vitro feeding
Ticks
I. hexagonus ticks were sent to the lab from different places in the Netherlands. A total of
30 female adults were used. In each feeding unit 5 adult females were placed.
D. reticulatus were used as a control group. These ticks where derived from the colony of
the UCTD. In each feeding unit 5 adult females and 5 adult males were placed.
Blood
Every week cow blood was collected from the department of farm animals at the Faculty of
Veterinary Medicine. During the experiment four cows were used, every week a different
one to minimize the discomfort. The blood was taken from the jugular vein; the skin of the
cow was disinfected with cotton drenched in alcohol. The blood was collected by use of a
needle, a catheter and a sterile glass bottle. To prevent contamination, the handlers wore
gloves. When the bottle was filled up to 300ml, the blood was stirred with a sterile 10mL
pipette for 15 minutes to take out the coagulation factors. With stirring a blood clot formed
around the glass pipette, and could be removed from the bottle.
In the laboratory glucose at 2 g/L was added to stabilize the erythrocytes. Then, the blood
was poured into sterile 50mL falcon tubes. To ensure this was done as sterile as possible,
the opening of the glass bottle and of the falcon tubes were sterilized by swiftly taking
them through a Bunsen burner flame. The falcon tubes were then stored at 4˚C.
Membranes
Membranes where attached to the bottom of the feeding units to mimic the skin of a host.
In order to prepare these membranes a layer of plastic film (17 µm) was pulled tightly
over a glass plate (40 x 30 cm) and fixed with tape. Then tape was used to correct
wrinkles in the film by pulling the film up en fixing it to the sides.
Eight lens cleaning papers were placed at equal distance on the film and fixed with tape.
A mix of 15g E4 silicone glue, 4,5g silicone oil, 2,9g hexane and a drop of white color glue
was spread out evenly over the lens cleaning papers by moving a scraper up and down
along the papers.
After drying for at least 24 hours at room temperature, the thickness of the membrane
was measured at six spots per lens cleaning paper by using a micro calipers. Only
membranes with a thickness between 70 and 110µm were used, to ensure the hypostome
of I.hexagonus could penetrate the membrane.
These where then secured on a six-well plate lid with tape, in order to create a smooth
surface were the feeding units, up to 4 per plate, could be glued on.
Attachment stimuli
For optimal attachment of the ticks to the membrane, the membrane must be provided
with the smell of a host. Bovine hair had previously been used successfully by Kröber and
Guerin (11). For this purpose membranes were rubbed on the back of a cow, as described
by Lenssen (3). This was done in the morning before assembling the units, when a new
group of ticks where put into the experiment in order to keep the smell as strong as
possible. During the experiment different attachment stimuli were used. These are
described and motivated in the results.
Feeding units
The feeding units where handmade, after the example of Kröber and Guerin (11). The
units consist of tube with a height of 45mm, a diameter of 26mm and a wall thickness of
2mm. 4mm from one side of the tube ring is constructed around it.
In preparation the units are glued on the six-well plate lid the membrane is stretches over,
and left to dry for at least 3 hours.
Then the units are carefully cut loose from the sixwell-plate lid and the thin layer of foil is
removed. To test for leakage the units are placed in a sixwell-plate unit filled with
demineralized water. When after 20 minutes no leaking occurs the units are ready to be
used.
5 I. hexagonus females were places per unit. In the control unit 10 D. reticulatus ticks
were placed, 5 male and 5 female. For I. hexagonus no males were used, because these
were not available.
To prevent the ticks from escaping the unit is closed off with a ‘bonbon’. This bonbon is
made by wrapping a piece of 6x6 cm lace curtain around a perforated stopper. The bonbon
was places approximately 0, 5 cm from the membrane, to prevent the tick from wandering
and encourage feeding.
Figure 3: The completely assembled feeding units used.
Water bath
In order to mimic natural feeding conditions a water bath was used. The water bath was
filled with demineralized water and kept at a constant temperature of 37˚C. Glass blocks
were places in the water bath and an aquarium of 40x25x25 cm was placed on top. The
aquarium was filled with a potassium sulphate solution of 120 grams per liter to reach just
below the water level of the water bath. The feeding units where placed inside and the
aquarium was covered with triangular formed lid made of metal. Humidity and
temperature where constantly monitored and checked twice a day, in order to be kept at
approximately 90% and 27 ˚C.
Figure 4: The water bath used.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
7
Feeding procedure
The four outer wells of a sterile six-well plate are each filled with 3, 1 milliliter blood.
Before taking the blood from the falcon tube it is fortexed 10-15 seconds. Then the six-well
plate is put in the aquarium for 15 minutes to warm the blood, mimicking the in vivo
situation. When the ticks are placed into the units and secured with the bonbons they are
transferred to the six-well plate filled with blood. This has to be done carefully to prevent
air bubbles.
The blood is replaced twice a day and every 24 hours the ticks are checked for attachment
and mortality. When the blood is replaced the bottom of the unit is washed with a PBS
solution.
After blood replacement the feeding units with the ticks are put back into the aquarium.
When there was attachment of I. hexagonus ticks, a sample was taken from that well. 0, 5
ml of blood was pipetted into an Eppendorf tube and stored at -20˚C.
Protocols of the in vitro feeding procedures were made during the project and they are
included in appendix A.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
8
PCR and Reverse Line Blot hybridization
0, 5 ml blood of the wells above which I. hexagonus ticks attached was sampled twice a
day and stored at -20˚C. Ticks which attached to the membrane of the in vitro feeding
system were stored in 70% ethanol at 4˚C. In order to determine transmission of
pathogens, first the ticks were examined making use of PCR and RLB-techniques. When a
pathogen is present in the tick, the blood samples are tested as well using PCR and RLBtechniques in order to determent transmission.
In order to test the ticks for pathogens, DNA-extraction will be performed. The protocol
used for this is added in appendix A. In Summary DNA is extracted by disrupting the tick
down mechanically and chemically and separating the DNA from the rest of the material.
The DNA will then be amplified by PCR.
PCR
PCR was invented in the 1980s and stands for polymerase chain reaction.
By heating the sample up to 95˚C the double stranded DNA is split in two strands. This is
called denaturation (step 2 in the figure below). Then the temperature is set to 50-70˚C, so
the primers that are added can attach, after which the temperature is set to 68-72 ˚C and
the polymerase enzyme and the nucleotides are added. This process is repeated, so de DNA
template is exponentially amplified. Billions of copies are typical.
The primers are chemically synthesized, therefore PCR can only be used to clone DNA whose
beginning and end sequences are known. (17, 18)
Since I. hexagonus has been identified as a vector for Anaplasma phagocytophilum, Babesia
divergens, Theiieria annae and Rickettsia helvetica. (1) In this research the primers for
Ehrlichia spp., Anaplasma spp., Theileria spp. and Babesia spp. were used.
By means of gel electrophoresis the PCR results were visualised.
Figure 3: Illustration of the PCR methode. (17)
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
9
Reverse Line Blot hybridization
When the amplification of DNA by PCR has been proven successful by gel electrophoresis,
Reverse Line Blot hybridization (RLB) can be performed.
DNA strands from the PCR are labeled with biotin and put onto a membrane containing
covalently-bound oligonucleotide probes. These probes consist of a reverse nucleotide
sequence from the pathogen tested. The different probes used were: Ehrlichia/Anaplasma
catch-all, Anaplasma centrale, Anaplasma marginale, 3 types of Anaplasma
phagocytophilum, Anaplasma bovis, Anaplasma platys, Ehrlichia canis, Ehrlichia chaffeensis,
Ehrlichia ruminantium, Ehrlichia sp Omatjenne, Theileria/Babesia catch-all, 2 types of
Babesia catch-all, Babesia felis, Babesia divergens, Babesia microti, Babesia bigemina,
Babesia bovis, Babesia rossi, 2 types of Babesia canis, Babesia vogeli, Babesia major, 2
types of Babesia caballi, Babesia venatorum sp EU1, Theileria equi, Theileria equi-like,
Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, Borrelia valaisiana,
Rickettsia catch-all, Rickettsia conorii, Rickettsia helvetica, Rickettsia massiliae and
Rickettsia raoultii.
When the strands of DNA and the probes match they will bind. When the strands and probes
don’t match, the DNA strands will be washed out with buffer solution.
Then streptavidin-peroxidase and a chemo luminescent reagent are added to the membrane.
Streptavidin-peroxidase will bind to the biotin label and the chemo luminescent reagent will
cause a reaction with the peroxidase. This reaction produces light. The membrane is then
placed in a lightproof cassette, together with a light sensitive film. When the film is developed
and printed, the results can be interpreted. (19)
Figure 4: Illustration of the RLB methode. (20)
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
1
0
Results
Every week a new feeding was started. Ticks were sorted by size and weight and similar
sizes were put together. Per feeding unit either 5 female I. hexagonus ticks were placed,
or 5 female D. reticulatus together with 5 male D. reticulatus. Male Ixodes hexagonus were
not used. D. reticulatus was used as a control group to evaluate the feeding system.
The results are shown in the following tables:
(U stands for feeding Unit.)
Feeding 1
In this feeding 4 units were made, the first two held both 5 female and 5 male D.
reticulatus ticks. The second two held both 5 female I. hexagonus ticks. Before
assembling, membranes were rubbed on the back of cows as an attachment stimulus.
Two adult D. reticulatus ticks attached to the membrane, one of them detached between 43
and 67 hours after they were first put into the feeding unit. Figure 5 shows a picture of the
remaining attached tick. Mortality went up to 60% in both D. reticulatus units. I. hexagonus
neither attached nor died.
U1:
U2:
U3:
U4:
Dermacentor Reticulatus (5♂ + 5♀)
Dermacentor Reticulatus (5♂ + 5♀)
Ixodes Hexagonus (5♀)
Ixodes Hexagonus (5♀)
U1:
Time
(hours)
19:15
Attachment
(%)
10
Death
(%)
10
43:05
10
50
67:05
0
91:05
U2:
Time
(hours)
19:15
Attachment
(%)
10
Death
(%)
30
43:05
10
60
60
67:05
10
60
0
60
91:05
10
60
Attachment
(%)
0
Death
(%)
0
Attachment
(%)
0
Death
(%)
0
43:05
0
0
43:05
0
0
67:05
0
0
67:05
0
0
91:05
0
0
91:05
0
0
U3:
Time
(hours)
19:15
U4:
Time
(hours)
19:15
Figure 5: Dermacentor Reticulatus attached to the membrane
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
1
1
Feeding 2
In this feeding 4 units were made, the first held both 5 female and 5 male D. reticulatus
ticks. The second three each held 5 female I. hexagonus tick. Before assembling,
membranes were rubbed on the back of cows as an attachment stimulus.
This time more I. hexagonus ticks were used to better evaluate if there is a difference in
their size/weight and the attachment rate. Expectation was that smaller ticks would attach
more easily, because they would need to feed more before being able to complete their life
cycle.
In unit 4, which held the biggest and heaviest ticks 1 attached after 43 hours. Figure 6
shows this tick. None of the I. hexagonus ticks died, in contrast to the 40% mortality rate
with the D. reticulatus ticks.
U1:
U2:
U3:
U4:
Dermacentor reticulatus (5♂ + 5♀)
Ixodes hexagonus (5♀) – mean weight: 0,406mg
Ixodes hexagonus (5♀) - mean weight: 0,226mg
Ixodes hexagonus (5♀) - mean weight: 0,608mg
U1:
Time
(hours)
19:16
Attachment
(%)
20
Death
(%)
0
43:53
30
0
67:23
10
90:48
U2:
Time
(hours)
19:16
Attachment
(%)
0
Death
(%)
0
43:53
0
0
0
67:23
0
0
0
40
90:48
0
0
Attachment
(%)
0
Death
(%)
0
Attachment
(%)
0
Death
(%)
0
43:53
0
0
43:53
20
0
67:23
0
0
67:23
20
0
90:48
0
0
90:48
20
0
U3:
Time
(hours)
19:16
U4:
Time
(hours)
19:16
When the female tick attaches, she often secretes a cement-like cone to securely attach to
the skin. This phenomenon is shown in the pictures below. The tick has been cut loose
from the cement to show it more clearly.
Figure 6: Ixodes Hexagonus and its cone.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
1
2
Feeding 3
In this feeding 4 units were made, the first held both 5 female and 5 male D. reticulatus
ticks. The second three each held 5 female I. hexagonus ticks. Before assembling,
membranes were rubbed on the back of cows as an attachment stimulus.
No attachment was seen this round. Mortality of D. reticulatus was much less then in the
previous feeding, whereas mortality of I. hexagonus increased.
U1:
U2:
U3:
U4:
Dermacentor reticulatus (5♂ + 5♀)
Ixodes Hexagonus (5♀) - mean weight: 1,036mg
Ixodes Hexagonus (5♀) - mean weight: 0,729mg
Ixodes Hexagonus (5♀) - mean weight: 0,545mg
U1:
Time
(hours)
19:20
Attachment
(%)
0
Death
(%)
10
42:43
0
10
67:43
0
91:48
U2:
Time
(hours)
19:20
Attachment
(%)
0
Death
(%)
0
42:43
0
0
10
67:43
0
0
0
10
91:48
0
0
Attachment
(%)
0
Death
(%)
0
Attachment
(%)
0
Death
(%)
0
42:43
0
0
42:43
0
0
67:43
0
0
67:43
0
0
91:48
0
0
91:48
0
20
U3:
Time
(hours)
19:20
U4:
Time
(hours)
19:20
Feeding 4
In this feeding 4 units were made, the first two held both 5 female and 5 male D.
reticulatus ticks. The second two held both 5 female I. hexagonus ticks.
In an attempt to improve attachment rates, two different attachment stimuli were
compared. The membranes used for two units of this feeding were rubbed over cows, once
a day for four days the week before this feeding was started, instead of only once. The
membranes used for the other two units were sprayed with cow perfume. This perfume
was assembled at the Utrecht Centre for Tick-Borne Diseases on 11-04-2012. The perfume
was made by extracting cow hair in 96% ethanol. It was sprayed onto the membranes
until droplets were visible. Then the membranes were air dried and sprayed again. When
the membranes were completely dry the units were assembled.
Attachment rates of D. reticulatus are noticeably higher than previous weeks, with the
membranes that were more frequently rubbed on cows as well as with the perfume.
Perfume looks like a good alternative for rubbing cows. With the I. hexagonus ticks
attachment rates unfortunately didn’t increase.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
1
3
U1:
U2:
U3:
U4:
Dermacentor reticulatus (5♂ + 5♀) (rubbing)
Dermacentor reticulatus (5♂ + 5♀) (cow perfume)
Ixodes hexagonus (5♀) (rubbing)
Ixodes hexagonus (5♀) (cow perfume)
U1:
Time
(hours)
18:45
Attachment
(%)
10
Death
(%)
0
43:00
20
0
65:27
10
91:45
U2:
Time
(hours)
18:45
Attachment
(%)
10
Death
(%)
0
43:00
20
0
10
65:27
50
10
0
10
91:45
0
10
Attachment
(%)
0
Death
(%)
0
Attachment
(%)
0
Death
(%)
0
43:00
0
0
43:00
0
0
65:27
0
0
65:27
0
0
91:45
0
0
91:45
0
20
U3:
Time
(hours)
18:45
U4:
Time
(hours)
18:45
Feeding 5
In this feeding 4 units were made, the first two held both 5 female and 5 male D.
reticulatus ticks. The second two held both 5 female I. hexagonus ticks.
In order to investigate if the lack of attachment by I. hexagonus is caused by a dislike of
cow smell, the membranes used this week were either sprayed with dog perfume or
covered with small pieces of cow hair. The dog perfume was made at the Utrecht Centre
for Tick-Borne Diseases. The perfume was made by extracting cow hair in 96% ethanol.
Attachment rates did not increase for any of the groups. Mortality in unit containing D.
reticulatus and dog perfume went up to 50%. Mortality in the units containing I.
hexagonus also increased, making it 20 % in the unit with dog perfume and 40% in the
unit with cow hair.
U1:
U2:
U3:
U4:
Dermacentor Reticulatus (5♂ + 5♀) (colony) (cow hair)
Dermacentor Reticulatus (5♂ + 5♀) (colony) (dog perfume)
Ixodes Hexagonus (5♀) (cow hair)
Ixodes Hexagonus (5♀) (dog perfume)
U1:
Time
(hours)
17:54
Attachment
(%)
10
Death
(%)
0
43:07
10
0
67:07
10
75:07
10
U2:
Time
(hours)
17:54
Attachment
(%)
0
Death
(%)
10
43:07
0
40
0
67:07
0
40
10
75:07
0
50
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
1
4
U3:
Time
(hours)
17:54
Attachment
(%)
0
Death
(%)
0
43:07
0
0
67:07
0
75:07
0
U4:
Time
(hours)
17:54
Attachment
(%)
0
Death
(%)
0
43:07
0
0
0
67:07
0
0
40
75:07
0
20
Feeding 6
Possibly the lack of attachment has to do with climate factors. This week the temperature
of the water bath was turned up to 39˚C. Hypothetically this should give better attachment
rates.
In an ecological study conducted by Bunnel et al. (15) it was reported that I. hexagonus
ticks were attracted to the fecal odor of sick hedgehogs compared with healthy ones. By
using gas chromatography differences in odor profile were analyzed. Sick animals tended
to have raised levels of the volatile aromatic heterocyclic compound indole in their feces.
When given the choice between indole and a solvent control, ticks were attracted to indole.
However, fecal matter from healthy hosts, with the addition of indole, was not attractive
for ticks. This suggests that ticks are capable of detecting their host immune status, and
prefer sick animals. Fever being one of the major signs of illness, higher temperatures
might be attractive for ticks. And therefore the raised temperature of the water bath might
get better attachment rates.
4 units were made, the first two held both 5 female and 5 male D. reticulatus ticks. The
second two held both 5 female I. hexagonus tick.
There seems to be no noticeable difference in attachment rates or mortality for D.
reticulatus, though a difference is seen between unit 1 and 2. For I. hexagonus there is a
20% attachment rate in unit 3, and a 20% mortality rate in both unit 3 and 4.
U1:
U2:
U3:
U4:
Dermacentor reticulatus (5♂ + 5♀) (colony) (cow rubbing)
Dermacentor reticulatus (5♂ + 5♀) (colony) (cow rubbing)
IxodeshHexagonus (5♀) (cow rubbing)
Ixodes hexagonus (5♀) (cow rubbing)
U1:
Time
(hours)
16:35
Attachment
(%)
10
Death
(%)
0
40:31
0
10
63:45
10
88:40
U2:
Time
(hours)
16:35
Attachment
(%)
0
Death
(%)
0
40:31
0
10
10
63:45
0
10
20
30
88:40
0
10
Attachment
(%)
0
Death
(%)
0
Attachment
(%)
0
Death
(%)
0
40:31
0
0
40:31
0
0
63:45
0
0
63:45
0
0
72:22
20
0
72:22
0
0
88:40
20
20
88:40
0
20
U3:
Time
(hours)
16:35
U4:
Time
(hours)
16:35
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
1
5
Feeding 7
In order to obtain more results, in this feeding 8 units were made, of which 6 each held 5
female I. hexagonus ticks and two both held 5 female and 5 male D. reticulatus ticks.
The standard attachment stimulus of rubbing the membrane on cows was compared to a
membranes sprayed with cow perfume and covered in small hair. Cow perfume was
sprayed onto the membranes until droplets were visible. The membranes were thereafter
air dried and sprayed again. Small pieces of cow hair (2-4 mm) were put onto the
membrane, and left to dry. When the membranes were completely dry the units were
assembled. Figure 7 shows a picture of the membrane right before the units were
assembled. And Figure 8 shows a picture after assembling the units.
The attachment rate for I. hexagonus is noticeably higher in two of the units using cow
perfume combined with small hair than in the units using cow rubbing as an attachment
stimulus. In U7 a leak was detected, therefore all the ticks died before the first checkpoint.
U1:
U2:
U3:
U4:
U5:
U6:
U7:
U8:
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Dermacentor reticulatus (5♂ + 5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow rubbing)
Ixodes hexagonus (5♀) (cow rubbing)
Ixodes hexagonus (5♀) (cow rubbing)
Dermacentor reticulatus (5♂ + 5♀) (cow rubbing)
U1:
Time
(hours)
16:37
Attachment
(%)
0
Death
(%)
0
24:01
0
0
40:42
0
48:12
U2:
Time
(hours)
16:37
Attachment
(%)
20
Death
(%)
0
24:01
20
0
0
40:42
20
0
20
0
48:12
20
0
64:22
20
0
64:22
20
20
72:03
20
20
72:03
20
20
88:42
20
20
88:42
20
20
Attachment
(%)
0
Death
(%)
0
Attachment
(%)
0
Death
(%)
0
24:01
0
0
24:01
20
0
40:42
0
40
40:42
30
0
48:12
0
40
48:12
30
0
64:22
0
60
64:22
10
20
72:03
0
60
72:03
10
20
88:42
0
100
88:42
10
30
U3:
Time
(hours)
16:37
U4:
Time
(hours)
16:37
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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U5:
Time
(hours)
16:37
Attachment
(%)
0
Death
(%)
0
24:01
0
0
40:42
0
48:12
U6:
Time
(hours)
16:37
Attachment
(%)
0
Death
(%)
0
24:01
0
0
0
40:42
0
0
0
0
48:12
0
0
64:22
0
0
64:22
0
0
72:03
0
0
72:03
0
0
88:42
0
0
88:42
0
0
Attachment
(%)
10
Death
(%)
0
U7:
Time
(hours)
16:37
U8:
Time
(hours)
16:37
Attachment
(%)
0
Death
(%)
100
24:01
0
100
24:01
10
0
40:42
0
100
40:42
20
0
48:12
0
100
48:12
20
0
64:22
0
100
64:22
10
0
72:03
0
100
72:03
10
0
88:42
0
100
88:42
0
20
Figure 7: the membrane of feeding 7
Figure 8: The assembled feeding unit of feeding 7
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Feeding 8
In order to obtain more results, this week also 8 units were made, of which 6 each held 5
female I. hexagonus ticks and two both held 5 female and 5 male D. reticulatus ticks.
Because the combination of cow perfume and small hairs seemed to improve the
attachment rates last week, this combination was used again this week. Unfortunately it
did not have the same result. None of the I. hexagonus ticks attached, except for one in
unit 7, but that detached before the next checking round. In unit 4 a leak was detected,
therefore all the ticks died before the first checkpoint. In unit 3 there also was a leak
detected, three ticks died because of the leak and formed a cloth together with the leaked
blood that prevented further leakage. 30% of D. reticulatus attached, but 20% detached
again during the feeding period.
U1:
U2:
U3:
U4:
U5:
U6:
U7:
U8:
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Dermacentor reticulatus (5♂ + 5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Ixodes hexagonus (5♀) (cow perfume + small hairs)
Dermacentor reticulatus (5♂ + 5♀) (cow perfume + small hairs)
U1:
Time
(hours)
16:50
Attachment
(%)
0
Death
(%)
0
24:10
0
0
41:10
0
48:30
U2:
Time
(hours)
16:50
Attachment
(%)
0
Death
(%)
0
24:10
0
0
0
41:10
0
0
0
0
48:30
0
0
64:50
0
0
64:50
0
0
72:01
0
0
72:01
0
0
89:50
0
20
89:50
0
20
Attachment
(%)
0
Death
(%)
60
Attachment
(%)
0
Death
(%)
100
24:01
0
60
24:01
0
100
41:10
0
80
41:10
0
100
48:30
0
80
48:30
0
100
64:50
0
80
64:50
0
100
72:01
0
80
72:01
0
100
89:50
0
80
89:50
0
100
U3:
Time
(hours)
16:50
U4:
Time
(hours)
16:50
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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U5:
Time
(hours)
16:50
Attachment
(%)
0
Death
(%)
0
24:10
0
0
41:10
0
48:30
U6:
Time
(hours)
16:50
Attachment
(%)
0
Death
(%)
0
24:10
0
0
0
41:10
0
0
0
0
48:30
0
0
64:50
0
0
64:50
0
0
72:01
0
0
72:01
0
0
89:50
0
0
89:50
0
0
Attachment
(%)
0
Death
(%)
0
U7:
Time
(hours)
16:50
U8:
Time
(hours)
16:50
Attachment
(%)
0
Death
(%)
0
24:10
0
0
24:10
0
0
41:10
20
0
41:10
30
10
48:30
0
0
48:30
30
10
64:50
0
0
64:50
30
10
72:01
0
0
72:01
0
10
89:50
0
20
89:50
10
20
PCR and RLB
In order to determine transmission of pathogens PCR and RLB were
performed on the four I. hexagonus ticks that attached to the
membrane during the study.
From those units where I. hexagonus ticks attached to the
membrane a blood sample was taken from the sixwell plate every
time the blood was refreshed. The blood was also tested by PCR
and RLB for pathogens as well.
Unfortunately the ticks that attached tested negative for all the
pathogens tested.
Results are featured in figure 9 on the right site of the page, and also
in Appendix B.
Figure 9: The RLB results.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Discussion
Feeding 1
The purpose of this study was to determine the transmission dynamics of I. hexagonus
ticks in vitro. In order to determine this, I. hexagonus had to feed in the in vitro feeding
system. Unfortunately, the first feeding was no success, since none of the ticks attached.
Of the D. reticulatus ticks two ticks attached, whereas one of the ticks detached again.
Mortality in both D. reticulatus units went up to 60%. Whether this was due to lack of
viability of the ticks or to the conditions is difficult to determine.
No clear conclusions can be drawn, though there is a difference seen between attachment
en mortality rate between D. reticulatus and I. hexagonus. This could mean that conditions
favorable for D. reticulatus are not the same as for I. hexagonus. In feeding 2 three units
were filled with I. hexagonus using the same attachment stimulus of rubbing the
membrane on a cow, in order to get more data on how this may influence I. hexagonus
ticks attachment rate and mortality.
Feeding 2
To test the hypothesis that smaller ticks would attach more easily, the I. hexagonus ticks
were divided in different groups according to their weight.
This hypothesis was rejected, since only one tick attached to the membrane, and that was
one from the largest group.
Mortality in the D. reticulatus unit decreased to 40% compared to feeding 1. All conditions
were kept the same as the first feeding, so it is plausible that these ticks were more viable
then those used the previous week. Even though a difference of two ticks is too small to
draw conclusions. Attachment rates of D. reticulatus did increase, maybe also because of
the viability of the ticks. But it could also have been due to membranes of better quality..
Feeding 3
To collect more data on previous experiments the same set up was used again. In the I.
hexagonus group and in the D. reticulatus group there was no attachment. Mortality in the
D. reticulatus group did decrease to 10%. It is most likely this was due to the viability of
the ticks, since the conditions were the same as the two previous feedings.
Possibly, ticks with a mean weight of 1,036mg would be too large to put into the in vitro
feeding assay. They may have had enough blood. The mortality of 20% in the unit with the
smallest ticks and of 0% in the other groups seems to indicate that a mean weight of
1,036mg isn’t too big jet. But to draw real conclusions, more data is needed.
Feeding 4
To investigate the transmission dynamics, higher attachment rates are needed. For that
purpose, new attachment stimuli were tested. Attachment rates of D. reticulatus did
increase, but for I. hexagonus there was no attachment. Attachment rates for D.
reticulatus were higher with cow perfume then with repeated rubbing, mortality rates were
the same. In the I. hexagonus unit with the cow perfume one tick died in the unit with the
cow perfume versus none in the cow rubbing unit. This is such a small difference that no
conclusions can be drawn on mortality for I. hexagonus.
For D. reticulatus cow perfume seemed better or at least an equal feeding stimulus
compared to rubbing the membrane on a cow.
Feeding 5
Since perfume seemed to be a good feeding stimulus for D. reticulatus, the hypothesis was
tested that I. hexagonus has a different host preference and might therefore show a higher
attachment rate on membrane with dog perfume.
There was no noticeable difference in attachment rates using cow hair or dog perfume.
However, there a high mortality rate of 50% in unit 2. Whether the dog perfume was toxic
for D. reticulatus or whether the ticks were less viable is not clear.
It could be that I.hexagonus did not prefer the combination of dog perfume and cow blood,
but would have better to use membranes sprayed with dog perfume on top of a well of dog
blood.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Feeding 6
Turning up the temperature of the water bath had the disadvantage that there could not
be a control group at a lower temperature. However, compared to the weeks before,
attachment of one I. hexagonus tick was a success. Mortality of I. hexagonus did increase
by one tick in both unit 3 and 4. The units with D. reticulatus respectively had a 10 and a
30% mortality. In conclusion, the higher temperature made it harder to survive, although
the numbers are too low.
Feeding 7
Spraying cow perfume onto the membrane and covering it with small hair increased the
attachment rate of I. hexagonus in two units to 20%. Compared to rubbing the
membranes on cows the attachment was higher, even though in week 2 this gave the
same result. Attachment and mortality rate for D. reticulatus using cow perfume and hair
did not differ much from the results obtained with the membrane rubbed on a cow.
Viewing this in perspective, no conclusions can be drawn. Therefore, in the next feeding all
of the units were sprayed with cow perfume and covered with small hairs in order to collect
more data for this attachment stimulus.
Feeding 8
None of the I. hexagonus ticks attached, except for one in unit 7, but this tick detached
before the next checking round. 30% of D. reticulatus attached, but 20% detached again
during the feeding period.
Therefore can be concluded that in this study attachment rates using cow hairs and cow
perfume are comparable to rubbing the membranes on cows.
During week 7 and 8 it is noted that in two of the units containing I. hexagonus eggs were
present after a few days. This means that the ticks used here were already too big. At
least they fed enough prior to th experiment. Therefore it is advisable to do further
research with smaller ticks, knowing a weight around 0,608mg is small enough. A picture
of I. hexagonus with a few eggs is shown in figure 10.
Figure 10: I. hexagonus female with eggs.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Conclusion
Transmission of pathogens could not be confirmed, because of a lack of attachment from I.
hexagonus ticks in the in vitro feeding system.
It became clear that both the environmental conditions and the condition of the tick itself
during in vitro feeding have to be optimized. In this study environmental conditions were
varied and different feeding stimuli were tested. Unfortunately no clear difference in
attachment rates and mortality could be seen using different attachment stimuli. More
research is needed to determent the specific preferences of I. hexagonus ticks in an in
vitro feeding system. After this is done, the in vitro feeding system can subsequently be
used to study the transmission dynamics of pathogens by I. hexagonus ticks
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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References
1. A. Conijn, J. Lenssen, M. Wijnveld, A. Luijten, F. Jongejan, Het
"tickbusters" survey, Tijdschrift voor diergeneeskunde (2011) 136, 508511
2. A. Nijhof et al. Ticks and Associated Pathogens collected from Domestic
Animals in the Netherlands. Vector Borne Zoonotic Dis (2007) 7, 585-595
3. Josephus J. Fourie, Dorothee Stanneck, Herman G. Luus, Frederick
Beugnet, Michiel Wijnveld, Frans Jongejan, Transmission of Ehrlichia canis
by Rhipicephalus sanguineus ticks feeding on dogs and on artificial
membranes, Veterinary parasitology (2013) Volume 197, Issues 3–4, Pages 595–
603
4. C. Louly, S. Soares, D. da Nobrega Silveira, M. Guimaraes, L. Borges,
Differences in the behaviour of Rhipicephalus sanguineus tested against
resistand and susceptible dogs, Exp Appl Acarol (2010) 51:353-362
5. C. Lawrie, S. Randolph, P. Nuttall, Ixodes Ticks: Serum Species
Sensititvity of Anticomplement Activity, Experimental Parasitology (1999)
93, 207-214
6. L. Bell-Sakyi, H. Attoui, Endogenous tick viruses and modulation of tickborne pathogen growth, Frontiers in Cellular and Infection Microbiology
(2013) 3: artikel 25
7. L. Toutoungi, L. Gern, A. Aeschlimann, Biology of Ixodes (Pholeoisodes)
hexagonus under laboratory conditions Part II. Effect of mating on feeding
and fecundity of females, Experimental & Applied Acarology (1995), 19:
233-245
8. L. Gern, L. Toutoungi, C. Min Hu, A. Aeschlimann, Ixodes (Pholeoixodes)
hexagonus, an efficient vector of Borrelia burgdorferi in the laboratory,
Medical and Veterinary Entomology 5, (1991), 4: 431-435
9. D. Sonenshine, Biology of Ticks, volume 1, Oxford University Press, 1991
10. www.uctd.nl
11. T. Krober, P. Guerin, An in vitro feeding assay to test acaricides for control
of hard ticks, Pest Management Science 63 (2007): 17-22
12. A. Ullman, J. Stuart, C. Hill, Tick Genome Mapping, Public Health resources
(2008), paper 108 chapter 8
13. J. Oliver, K. Tanaka, M. Sawada, Cytogenetics of Ticks (Acari: Ixodoidea),
Chromosoma (1973), 42, 269-288
14. T. Burnell, K. Hanisch, J. Hardege, T. Breithaupt, The Fecal Odor of Sick
Hedgehogs (Erinaceus europeaus) Mediates Olfactory Attraction of the Tick
Ixodes hexagonus, (2011), J Chem Ecol 37: 340-347
15. F. Ruiz-Fons, P. Acevedo, R. Sobrino, J. Vincente, Y. Fierro, I. Fernandezde-Mera, Sex-biased differences in the effects of host individual, host
population and environmental traits driving tick parasitism in red deer,
(2013), frontiers in cellular and infection microbiology 3, article 23
16. A.J. Ullmann, J.J. Stuart, C.A. Hill, Tick Genome Mapping (2008), Public
Health Resources.Paper 108, chapter 8
17. http://bio-ggs.blogspot.nl
18. B. Alberts, D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts,
P. Walter, Essential Cell Biology, second edition, Garland Science, (2004)
19. F. Kong, G. Gilbert, Multiplex PCR-based reverse line blot hybridization
assay (mPCR/RLB)--a practical epidemiological and diagnostic tool, Nat.
Protoc. (2006) 1, 2668-2680
20. www.nature.com
21. T. Krober, P. Guerin, In vitro feedin assays for hard ticks, Trends in
Parasitology (2007) Vol. 23 No.9, 445-449
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22. A. Pierce, M. Pierce, a note on the cultivation of Boophilus miroplus
(Oanesteini, 1887) Ixodidae: acarina) on the embryonated hen egg, Aust
Vet J (1956); 32: 144-146
23. B. Habdank, T. Hiepe, In-vitro-Futterun von Zecken, Dermacentor nuttalli,
Olenev 1928 (Acari: Ixodidae) Uver eine Silikonmembran, Dermatol
Monatsschr (1993); 179: 292
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Appendix A
IN VITRO FEEDING PREPARATION
Room
Tick species
Number of membranes
Tick strain
Number of units
Smell/perfume
Tick race track ID
Wear gloves
Membrane preparation
Done
1 Clean workspace with 70% ethanol.
2 Clean a glass plate with 70% ethanol and cover it with foil. Tighten the foil with tape.
3 Paste 8 lens papers on the foil of equidistant.
Prepare the silicon mix; 30g silicon glue, 9g silicon oil, 5.8g hexane and 0.5g white color glue. (This
4 mix is sufficient for 4 6-wells plates.)
5 Paste a piece of carton on the table with the smooth side up.
6 Cover the lens papers with an equal layer the silicon mix.
7 Dry the membrane for 24 hours.
Cut the membranes from the glass plate and measure the membrane with the micro calipers at 6
8 different spots. The layer should be between 87 and 117µm tick (including the foil (17µm)). Mark the
spots where the membrane is too thick or too thin.
9 Paste the membrane on the lid of a 6-wells plate.
10 Apply a smell to the membrane by rubbing it on a dog or spying a perfume.
11 Clean workspace with 70% ethanol
Unit preparation
Done
1 Clean workspace with 70% ethanol.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Clean whole Plexiglas tubules with 70% ethanol. When re-using tubules, remove all glue residues and
2 check for cracks.
3 Place all tubules on the membrane.
4 Paste a piece of carton on the table and add a small amount of glue.
5 Smooth the glue to a thin layer.
Place a tubule with a rotating movement in the glue and remove it from the glue with a rotating
6 movement. Check if the ring is completely covered in glue.
Place the tubule on the membrane and press it. Once on the membrane, the tubule should not be
7 moved. Excess glue on the inside of the unit can be removed with a small brush. Continue with the
next tubule until the whole membrane is used.
8 Dry the units for 3 hours at room temperature.
9 Cut the units from the lid and remove the foil with tweezers.
10 Place the units at least 15 minutes on a 6-wells plate filled with distilled water.
11 Check all units thoroughly for leakage.
12 Rinse the underside of the membrane with 70% ethanol and let it dry.
13 Place the units in a sterile 6-wells plate.
14 Cut the sides off several lids, tie a 6x6cm piece of voile around the lid and fasten it. (A bonbon)
15 Place the bonbon on the unit.
16 Clean workspace with 70% ethanol.
Tick preparation
Done
1 Turn on the tick race track for 30 minutes.
2 Place 5 males and 5 females in a small pot per unit.
3 Place the ticks in the incubator until usage or incubate them in a water bath at 37°C if needed.
4 Clean the race track with 70% ethanol and turn it off.
In vitro feeding membrane preparation done:
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Comments:
Appendix A
IN VITRO FEEDING MEAL PREPARATION
Cow number
Room
Blood volume
Wear gloves
Glucose weight
Meal preparation
Done
1 Take a sterile Erlenmeyer (with a large enough volume) and 2 10ml pipettes.
2 Go to the department of Farm Animals, dress appropriately and ask someone for help.
3 Take a needle, tube and some cotton wool with ethanol from the cart in the diffco room.
4 Go to cow 9751, 4104 or 0142 (rotate) and bind her head to the side.
5 Connect the needle with the tube.
Clean a spot on the cow with cotton wool and tap as sterile as possible blood from the V. jugularis.
6 Collect the blood in Erlenmeyer.
7 Stir 20 minutes with the 10ml pipet to remove all agglutination factors.
8 Remove the pipette gently and dispose in the yellow bio-waste bin.
9 Fill in the welfare diary in the office of Thijmen (date, cow number, action).
10 Take the Erlenmeyer to the UCTD, add glucose to a concentration of 2g/L and swerve the jar.
Pour the blood in the sterile tubes. Sterilize the edge of the Erlenmeyer and the tubes by putting it
11 through a blue flame before pouring.
12 Label the tubes accordingly and store at 4°C.
13 Turn off the Bunsen burner and clean workspace with 70% ethanol.
In vitro feeding meal preparation done:
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Comments:
Appendix A
IN VITRO FEEDING SETTING UP
Room
Tick species
Number of units
Tick strain
Number of ticks
Blood ID
Smell/perfume
Wear gloves
Water bath ID
Flow cabinet ID
Tick race track ID
done
1 Turn on the ventilation, clean the flow cabinet with 70% ethanol and place a 50ml Falcon tube in it.
2 Turn on the UV-light for 15 minutes.
3 Turn on a water bath at 37°C.
If needed, fill the outer water bath with distilled water until the upper line and the inner water bath with
4 120g/L KSO2 just below the water level of the outer water bath.
5 Turn on the tick race track.
6 Vortex a tube with blood and place it in the flow cabinet together with a sterile 6-wells plate.
7 Pipette 3.1ml blood to the 4 outer wells of a 6-wells plate. Cover the other wells, when pipetting.
8 Cover the plate with the lid and incubate it in the outer water bath at 37°C for 15 minutes.
9 Clean the flow cabinet with 70% ethanol.
10 Place the sorted ticks from the pots to the units on the tick race track.
Place the bonbon onto the unit until 0.5cm above the membrane. Be aware that no ticks are stuck
11 between the bonbon and the side of the unit.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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12 Turn off the race track and place the units in the flow cabinet.
13 Take the 6-wells plate from the water bath and dry the outside before placing it in the flow cabinet.
14 Place the unit side-ways onto the blood. Avoid air bubbles between the membrane the blood.
15 Place the 6-wells plate with the units in the inner water bath.
16 Close the water bath and cover it with a black cloth.
17 Clean the flow cabinet with 70% ethanol.
18 Turn on the UV-light for 15 minutes
19 Turn off the flow cabinet.
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The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Appendix A
IN VITRO FEEDING MAINTENANCE
Room
Tick species
Number of units
Tick strain
Number of ticks
Blood ID
Smell/perfume
Wear gloves
Hair
Water bath ID
Flow cabinet ID
Maintenance
Done
Turn on the ventilation, clean the flow cabinet with 70% ethanol and place a 50ml Falcon
1 tube and a glass petri dish in it.
2 Turn on the UV-light for 15 minutes.
If needed, fill the outer water bath with distilled water until the upper line and the inner
3 water bath with 120g/L KSO just below the water level of the outer water bath.
2
4
Vortex a tube with blood and place it in the flow cabinet together with a sterile 6-wells
plate.
5
Pipette 3.1ml blood to the 4 outer wells of a 6-wells plate. Cover the other wells, when
pipetting.
Cover the plate with the lid and incubate it in a water bath together with a tube of PBS at
6 37°C for 15 minutes.
7 Clean the flow cabinet with 70% ethanol.
Take both 6-wells plates (with and without units) and the PBS from the water bath and
8 dry the outside before placing it in the flow cabinet.
9 Check if the ticks are attached to the membrane in each unit.
10 Rinse the membranes with the PBS above the glass petri dish.
11
Place the unit side-ways onto the fresh blood. Avoid air bubbles between the membrane the
blood.
Remove the blood from the 6-wells plate with the units by pipetting and dispose it in the
12 50ml Falcon tube.
13 Place the 6-wells plate with the units in the inner water bath.
14 Close the water bath and cover it with a black cloth.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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15 Check the temperature of the water and fill in the log.
16 Clean the flow cabinet with 70% ethanol.
17 Turn on the UV-light for 15 minutes
18 Turn the flow cabinet off.
Unit
#
Tick species
Tick strain
Total ticks
Number of
Number of
attached ticks dead ticks
1
2
3
4
5
6
7
8
In vitro feeding maintenance procedure done:
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Appendix A
IN VITRO FEEDING MAINTENANCE AND SAMPLING
Room
Tick species
Number of units
Tick strain
Number of ticks
Blood ID
Smell/perfume
Wear gloves
Water bath ID
Flow cabinet ID
Maintenance and sampling
Done
Turn on the ventilation, clean the flow cabinet with 70% ethanol and place a 50ml Falcon
1 tube, sterile 500µl tubes and a glass petri dish in it.
2 Turn on the UV-light for 15 minutes.
If needed, fill the outer water bath with distilled water until the upper line and the inner
3 water bath with 120g/L KSO just below the water level of the outer water bath.
2
4
Vortex a tube with blood and place it in the flow cabinet together with a sterile 6-wells
plate.
Pipette 3.1ml blood to the 4 outer wells of a 6-wells plate. Cover the other wells, when
5 pipetting. If needed, pipette 2.5ml blood to a control well.
Cover the plate with the lid and incubate it in a water bath together with a tube of PBS at
6 37°C for 15 minutes.
7 Clean the flow cabinet with 70% ethanol.
Take both 6-wells plates (with and without units) and the PBS from the water bath and
8 dry the outside before placing it in the flow cabinet.
9 Check if the ticks are attached to the membrane in each unit.
10 Rinse the membranes with the PBS above the glass petri dish.
11
Place the units side-ways onto the fresh blood. Avoid air bubbles between the membrane the
blood.
If needed, pipette the blood from the 6-wells with the old blood up and down and take a
12 sample of 500µl each into sterile 500µl tubes. Label the tubes accordingly.
Remove the blood from the 6-wells plate with the units by pipetting and dispose it in the
13 50ml Falcon tube.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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14 Place the 6-wells plate with the units in the inner water bath.
15 Close the water bath and cover it with a black cloth.
16 Check the temperature of the water and fill in the log.
17
Store the blood samples at 4°C for usage within the next few days or at -20°C for long
term storage.
18 Clean the flow cabinet with 70% ethanol.
19 Turn on the UV-light for 15 minutes.
20 Turn the flow cabinet off.
Unit
#
Tick species
Tick strain
Total ticks
Number of
attached ticks
1
2
3
4
5
6
7
8
In vitro feeding maintenance and sampling procedure done:
by
on
time
Signature
Comments:
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Appendix A
IN VITRO FEEDING FINISHING
Room
Tick species
Number of units
Tick strain
Number of ticks
Blood ID
Smell/perfume
Wear gloves
Hair
Water bath ID
Flow cabinet ID
Tick track ID
Maintenance
Done
1 Turn on the ventilation, clean the flow cabinet with 70% ethanol and place a 50ml Falcon tube in it.
2 Turn on the UV-light for 15 minutes.
3 Turn on the tick track in the acaridarium.
4 Incubate a tube with PBS in the outer water bath at 37˚C for 15 minutes.
Take the 6-wells plate with the units and the PBS from the water bath and dry the outside before
5 placing it in the flow cabinet.
Pipette the blood from the 6-wells with the old blood up and down and take a sample of 500µl each
6 into sterile 500µl tubes. Label the tubes accordingly.
7 Rinse the membranes of the units with the PBS above the glass petri dish..
8 Place the units in a new 6-wells plate.
9 Remove the blood from the old 6-wells plate by pipetting and dispose it in the 50ml Falcon tube..
10 Take the 6-wells plate with the ticks to the acaridarium,
11 Remove the bonbons and take pictures for documentation.
Put the ticks that are attached in a sterile 1.5ml tube with 70% ethanol, store them at room
12 temperature. Label the tubes accordingly.
13 Put dead ticks in a jar with 70% ethanol and place it in the fridge.
14 Inform what has to be done with the rest of the ticks.
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Unit
#
Tick strain
Tick species
Total ticks
Number of
attached ticks
Number of
dead ticks
1
2
3
4
5
6
7
8
In vitro feeding finishing done:
by
on
Signature
Comments:
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Appendix A
DNA extraction protocol
DNA EXTRACTION FROM TICKS
Room
Water bath ID
Number of
samples
Sonification bath
ID
Sample
description
TissueLyser LT ID
Heating block ID
Wear gloves and use filter pipet tips
Centrifuge ID
Strictly follow the one-way route: Clean room  Dirty room  PCR room
Done
1 Clean workspace with 70% ethanol.
2 Turn on a water bath at 56°C.
3 Take the proteinase K solution from the freezer and store at 4°C.
Wash the ticks in a sonofication bath with demineralized water for up to 30
seconds.
Put the ticks, with cleaned forceps, in 1.5ml tubes with 70% ethanol and
5
vortex for several seconds.
Wash the forceps in 70% ethanol followed by washing in demineralized water
6
after each tick.
Take the ticks from the tubes and let it dry on a clean tissue paper and place
7
the dried ticks in a sterile 2ml tube with 180µl T1 lysis buffer.
4
8 Freeze the samples at -80°C for 15 minutes.
9 Add a 5 or 7mm (depending on tick size) metal bead to the frozen samples.
10
Disrupt the ticks in the TissueLyser LT at 50 oscillations per second for 3
minutes.
11 Briefly spin down the tubes. 1000x g maximum!
12 Add 25µl proteinase K and vortex.
13
Prelyse the samples at 56°C in a water bath for 3 hours and vortex every
hour.
14 During the incubation; empty and clean the sonification bath.
15
During the last incubation hour ; turn on the heating block at 70°C and
preheat the BE buffer.
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16 Briefly spin down the tubes. 1000x g maximum!
17 Add 200µl B3 buffer and vortex.
18 Incubate the tubes at 70°C for 15 minutes.
19 Briefly spin down the tubes. 1000x g maximum!
Add 210µl 96% ethanol, vortex and briefly spin down the tubes. 1000x g
maximum!
Transfer the supernatant to new sterile 1.5ml tubes. (Tick parts are allowed to
21
be transferred.)
20
22 Centrifuge the tubes at 11,000x g for 2 minutes.
23
Transfer the supernatant to spin columns. Avoid pipetting tick parts, as it can
block the spin column.
24 Centrifuge the columns at 11,000x g for 1 minute. Discard the flow through.
Add 500µl BW buffer and centrifuge the columns at 11,000x g for 1 minute.
Discard the flow through.
Add 600µl B5 buffer and centrifuge the columns at 11,000x g for 1 minute.
26
Discard the flow through.
25
27 Centrifuge the columns at 11,000x g for 1 minute.
28 Place the spin columns in sterile 1.5ml tubes. Label the tubes accordingly.
29
Add 100µl preheated BE buffer directly on the membrane of the spin columns
and incubate at room temperature for 1 minute.
30 Centrifuge the columns at 11,000x g for 1 minute. Discard the spin columns.
31
Store the DNA samples at 4°C for use within the next few days or store at -20°C
for long term preservation.
32 Turn off all equipment and clean working space with 70% ethanol.
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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Appendix B
Results of RLB
The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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The transmission dynamics of pathogens by Ixodes hexagonus ticks using an in vitro feeding system.
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