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ORIGINAL PAPER
Development of a protocol testing the ability of Stomoxys
calcitrans (Linnaeus, 1758) (Diptera: Muscidae) to transmit
Besnoitia besnoiti (Henry, 1913) (Apicomplexa: Sarcocystidae)
E. Liénard &A. Salem &P. Jacquiet &C. Grisez &F. Prévot &
B. Blanchard &E. Bouhsira &M. Franc
Received: 20 September 2012 / Accepted: 27 September 2012 /Published online: 12 October 2012
#The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract Cattle besnoitiosis due to the cyst-forming cocci-
dian parasite Besnoitia besnoiti has recently been reported in
expansion in Europe since the end of the twentieth century.
The B. besnoiti life cycle and many epidemiological traits
are still poorly known. Hematophagous flies, including the
worldwide-distributed Stomoxys calcitrans, could be me-
chanical vectors in the contamination of mouthparts after
the puncture of cutaneous cysts or ingestion of infected
blood. In this study, a protocol is presented to assess more
deeply the role of S. calcitrans, reared in laboratory con-
ditions, in parasite transmission. A preliminary trial showed
that stable flies could transmit tachyzoites from bovine
artificially parasite-enriched blood to B. besnoiti-free blood
using glass feeders. Evidence of transmission was provided
by the detection of parasite DNA with Ct values ranging
between 32 and 37 in the blood recipient. In a second time, a
B. besnoiti-infected heifer harboring many cysts in its der-
mis was used as a donor of B. besnoiti. An interruption of
the blood meal taken by 300 stable flies from this heifer was
performed. Immediately after the blood meal was interrup-
ted, they were transferred to a glass feeder containing B.
besnoiti-free blood from a non-infected heifer. Quantitative
PCR and modified direct fluorescence antibody test (dFAT)
were used to detect B. besnoiti DNA and entire parasites,
respectively, in the blood recipient, the mouthparts, and the
gut contents of S. calcitrans at two time intervals: 1 and 24 h
after the interrupted blood meal. Parasite DNA was detected
at both time intervals (1 and 24 h) in all samples (blood
recipient, mouthparts, and gut contents of stable flies) while
entire parasites by dFAT were only found in the abdominal
compartment 1 h after the interrupted blood meal. Then, S.
calcitrans were able to carry B. besnoiti from chronically
infected cattle to an artificial recipient in the conditions of
the protocol.
Introduction
Besnoitia besnoiti (Henry, 1913), a cyst-forming apicom-
plexan parasite, is the causative agent of cattle besnoitiosis.
This disease, widely distributed in Africa, Asia, and in
southwestern Europe, has spread in Portugal (Cortes et al.
2005), in Spain (Fernández-García et al. 2009), from south-
ern France to central and western France since the end of the
twentieth century (Alzieu et al. 2007) with recent and lim-
ited outbreaks in Germany (Mehlhorn et al. 2009) and Italy
(Agosti et al. 1994) probably due to importation of infected
cattle (Olias et al. 2011). Since 2010, cattle besnoitiosis is
considered a reemerging disease according to the European
Food Safety Authority (http://www.efsa.europa.eu/en/
scdocs/doc/1499.pdf). Severe economic losses are observed
in infected farms due to definitive or transient sterility in
bulls, abortions, decline in milk production, mortality, and
low body conditions, resultingindepreciatingslaughter
values and skin quality (Pols 1960;Cortesetal.2005).
The course of the disease is well described (Jacquiet et al.
2010). During the acute stage of the disease, tachyzoites
E. Liénard (*):A. Salem :P. Jacquiet :C. Grisez :F. Prévot :
E. Bouhsira :M. Franc
Laboratoire de Parasitologie, Université de Toulouse,
INP, ENVT,
F-31076 Toulouse, France
e-mail: e.lienard@envt.fr
B. Blanchard
Adiagène, 38 rue de Paris,
22000 Saint-Brieuc, France
Parasitol Res (2013) 112:479–486
DOI 10.1007/s00436-012-3157-6
multiply quickly within bovine macrophages and endothe-
lial cells of blood vessels. The chronic stage is characterized
by the formation of numerous cysts containing thousands of
bradyzoites in various tissues including the skin. However,
the life cycle of B. besnoiti and the routes of contamination are
not yet clearly elucidated (Diesing et al. 1988;Kiehletal.2010;
Basso et al. 2011; Olias et al. 2011). The cattle-to-cattle trans-
cutaneous contamination is probably the most common way of
infection via hematophagous insects. Experimentally, tachy-
zoites in blood or cutaneous bradyzoites have been successfully
transmitted from an infected animal to a susceptible one by,
respectively, thetransfusion of large volumes of infected bovine
blood or through the mechanical transmission by biting flies
such as tabanids and Stomoxys calcitrans (Linnaeus, 1758)
commonly named stable fly (Cuillé et al. 1936;Pols1960;
Bigalke 1968). Since the survey of Bigalke (1968) showing
the mechanical vector role of those flies, these works, to our
knowledge, have not been repeated to assess the epidemiolog-
ical importance of S. calcitrans, one of the most serious pests of
herds (Taylor et al. 2012), in the transmission of besnoitiosis.
Mechanical transmission may occur when a stable fly is inter-
rupted during blood feeding by host defensive behavior or other
flies (Schofield and Torr 2002) and completes its blood meal on
nearby animals (Doyle et al. 2011). A large proportion of
animals exposed to and infected by B. besnoiti only become
seropositive without developing clinical signs (Bigalke 1968;
Jacquiet et al. 2010). Then, the use of an artificial host recipient,
such as a blood glass feeder, to estimate the parasite carriage in
S. calcitrans could eliminate the bias of cattle susceptibility
(Doyle et al. 2011). The aim of this study was to establish a
technical protocol to assess the role of S. calcitrans,fedwith
blood from a glass feeder, as a mechanical vector of B. besnoiti
from infected cattle harboring cutaneous cysts.
Material and methods
Source and maintenance of S. calcitrans
A colony of stable flies is established at the ENVT (France)
since May 2009. More details about rearing methods are
provided by Salem et al. (2012). Adult stable flies required
for the experimental purpose were 2 to 5 days old. They
were fed only with water and honey ad libitum after emer-
gence and before experiments. To study the ability of B.
besnoiti to be transmitted, one or two sterilized glass feeders
(according to the protocol used) were placed in contact with
the upper side of the mesh cages (30×30×30 cm). A syn-
thetic membrane (Parafilm 3M, Pechiney Plastic Packaging,
Chicago, IL) sealed the inner chamber containing bovine
blood of the glass feeder. Stable flies fed on blood by
piercing the membrane through the mesh of the cage with
their mouthparts.
Source of B. besnoiti-free blood
The uninfected blood was provided by a 14-month-old
heifer reared at the ENVT, which has not been treated with
insecticides within 3 months prior to the study. The blood
was collected in 4-ml tubes containing 60 USP U Lithium
Heparin (Terumo Europe N.V., Leuven, Belgium) to prevent
coagulation at the day of experiment.
Source of B. besnoiti-infected blood and skin
The source of bradyzoites, used in transmission experiments 3
and 4 (see below), was a second 14-month-old heifer with
chronic besnoitiosis. The infected heifer was reared at the
ENVT under the same conditions as the uninfected cow. It
came from an infected farm in Dordogne (France). Regarding
both heifers, the presence or absence of B. besnoiti cutaneous
cysts were checked clinically, and at least two biopsies
(Biopsy Punch 8 mm, Kruuse, Langeskov, Denmark) were
taken from the right forelimb and the neck. This was then
followed by direct observation and quantitative PCR (see
below). Their serological status against bovine besnoitiosis
was also evaluated by Western blot (see below).
Serological analysis of heifers
Purified B. besnoiti tachyzoites from a strain isolated in the
French Pyrenees were used as antigen for the Western blot
(WB) and to infect the blood in experiment 2 (see below).
Vero c ell s and B. besnoiti tachyzoites are maintained at the
ENVT since 2008 as described by Cortes et al. (2006a). WB
procedures were adapted from Cortes et al. (2006a)andwere
performed as described by Liénard et al. (2011). A serum was
considered positive if the three main antigenic domains (low,
medium, and high molecular weights) were observed with a set
of at least four bands within each of them (Cortes et al. 2006a).
Experimental design
For all experiments, the sex ratio of S. calcitrans was close to
1:1. Experiments were achieved in November 2011 and in
January 2012 when wild adult stable flies were not present in
cattle facilities.
Experiment 1: control group
It was firstly necessary to assess whether some organisms pres-
ent within S. calcitrans would interact with further analyses.
Moreover, it was also necessary to confirm that laboratory-bred
S. calcitrans were free from B. besnoiti contamination. Two
control groups were then defined. A first group of 50 S. calci-
trans were fed on 8 ml uninfected blood from the B. besnoiti-
free heifer contained in a glass feeder during 1 h while a second
480 Parasitol Res (2013) 112:479–486
group was exposed to the same blood for 24 h (Fig. 1a). At the
end of the exposure, they were immediately knocked down at
−20 °C for 1 h and dissected. For each group, mouthparts were
manually removed under a magnifying glass with a needle and
pooled into a grinding tube containing 1.4 ml of PBS (Bio-Rad,
Marnes-la-Coquette, France). Abdomens of engorged flies were
also opened with a needle, and abdominal contents were col-
lected and pooled into the same sterile 4-ml tube containing 3 ml
of PBS. Two tubes were eventually available for each control
group. After mixing, both 3-ml tubes were equally divided to
assess the presence of B. besnoiti by direct fluorescent antibody
test (dFAT, entire parasites) and quantitative PCR (qPCR, para-
site DNA). Blood samples of the B. besnoiti-free heifer before
and after exposures (1 and 24 h) to S. calcitrans in glass feeders
were also analyzed with both of these methods.
Experiment 2: experimental transmissions
with tachyzoite-enriched blood as a source of parasites
This step was a preliminary trial of transmission before
using a chronically infected heifer as a source of parasites.
The aim was to assess the opportunity of B. besnoiti trans-
mission from infected blood to uninfected blood by S.
calcitrans using artificial tachyzoite-enriched blood as a
source of parasites and recipient. A group of 100 stable flies
was maintained in mesh cages (30× 30 × 30 cm). A first
double-chambered glass feeder was placed in contact with
the upper side of the cage for 3 h. The inner chamber of this
glass feeder contained 8 ml of blood from the uninfected
heifer mixed with 10
8
B. besnoiti culture tachyzoites. After
3 h, the first glass feeder was removed, and a second glass
feeder with B. besnoiti-free blood from the same heifer was
set up for 24 h (Fig. 1b). Quantitative PCR was used to
detect parasite DNA in 2 ml of blood from both glass
feeders (donor and recipient). This experiment was replicat-
ed three times.
Experiment 3: experimental transmissions with long exposure
time to a chronically infected heifer as a source of parasites
The aim of this trial was to assess the ability of S. calcitrans
to ingest B. besnoiti bradyzoites contained in cutaneous
cysts via bites. A group of 100 stable flies were transferred
into a 15×15×15-cm cage enclosed by thin wire mesh
allowing them to bite after being placed on bovine skin.
Sedative xylazine hydrochloride (0.1 mg/kg) (Rompun 2 %,
Bayer Inc., Puteaux, France) was administered to the chron-
ically infected heifer via intramuscular injection. A 20 × 20-
cm
2
area of skin on the right side of the rump was washed
with water and shaved. An external antiseptic and antifungal
solution of 10 % povidone iodine (Vétoquinol Vétédine
Solution, Vétoquinol, Tarare, France) was applied and rinsed.
The cage was manually maintained for 1 h (Fig. 1c). At the
end, all stable flies were treated as previously described for the
control groups (dFAT and qPCR). The test was replicated once
more.
Experiment 4: experimental transmissions with short exposure
time to a chronically infected heifer as a source of parasites
The aim of this trial was to assess the ability of S. calcitrans
to transfer parasites from a chronically infected heifer to B.
besnoiti-free bovine blood contained in an artificial glass
feeder. A group of 300 stable flies was placed on the rump
of the chronically infected heifer according to the same
conditions as experiment 3. The duration of the heifer ex-
posure was interrupted after 5 min (Dougherty et al. 1995).
Immediately, all stable flies were allowed to achieve their
blood meal on a glass feeder providing the B. besnoiti-free
blood for 1 h (Fig. 1d). One hour later, 100 stable flies and
2 ml blood from the glass feeder recipient were removed to
be treated as previously described for the control group. The
remaining 200 stable flies were left to feed on the same
bovine blood for 24 h before analyses already presented.
Dead or non-blood-engorged flies were discarded. This trial
was replicated once again with 400 S. calcitrans (150 flies
after 1 h and 250 after 24 h).
Detection of B. besnoiti
The dFAT was essentially achieved as described by Schares et
al. (2010) and was adapted to detect entire parasites from the
blood glass feeder, as well as the mouthparts and abdominal
contents of stable flies. Briefly, before the suspensionin a 4 %
formaldehyde solution in PBS and the fixation on slides, red
blood cells were hemolyzed by adding nine volumes of sterile
water to one volume of blood during 5 min. Parasites were not
crushed throughout this step. For each test, one immunofluo-
rescence glass slide, to which were added eight drops, was
prepared. Tachyzoites from culture were used as positive
control slides. The parasite suspension was distributed in 15-
μl drops on slides, dried at 37 °C, and fixed in ice-cold acetone
(−20 °C) for 10 min. Moreover, a highly positive serum
sample from a chronically infected cow (seven drops
per slide) and a negative serum (one drop per slide)
were used at 1:200 dilution. This dilution (1:200) was
used as a positive cutoff value (Gentile et al. 2012).
Rabbit anti-bovine IgG (whole molecule)–FITC (Sigma-
Aldrich, Saint-Quentin Fallavier, France) was used as a
conjugate at a 1:300 dilution according to the manufac-
turer's recommendations. The reading was performed
under a fluorescence microscope at ×400 magnification
(Axio Scope.A1, Carl Zeiss, Le Pecq, France). The presence
of an entire parasite was confirmed when a complete and
peripheral bright fluorescence of the parasite membrane was
observed (Schares et al. 2010).
Parasitol Res (2013) 112:479–486 481
Quantitative PCR was used to detect B. besnoiti
DNA from bovine skin and blood samples of the two
heifers, from mouthparts and abdominal contents of S.
calcitrans and from blood of glass feeders before and
after transmission trials. Before DNA extraction with the
QIAmp® DNA Mini Kit (Qiagen, Courtabœuf, France)
commercial kit, mouthparts of stable flies were previ-
ously ground with the TeSeE™Purification Kit (Bio-
Rad, Marnes-la-Coquette, France) according to the man-
ufacturer's recommendation. B. besnoiti ITS-1 amplifica-
tion was performed with the commercial PCR kit
AdiaVet™Besnoitia (AES Chemunex, Bruz, France). The
quantitative PCR was performed with the Stratagene
MX3005P thermal cycler (Agilent Technologies, La Jolla,
CA), and results were analyzed using the MxPro QPCR
version 4.10 software (Agilent Technologies, La Jolla, CA).
A threshold cycle (Ct) value of 40 corresponded to a negative
result.
Results
Experiment 1. The serum of the B. besnoiti-free heifer was
negative for B. besnoiti infection as determined by WB. Its
blood and skin biopsies were qPCR-negative. None of the S.
calcitrans from both control groups that fed on the blood of
Mesh cage
Glass feeder Cattle B. besnoiti-free blood
Stable flies
(n=100)
Exposure time = 1 hour (n=50), 24 hours (n=50)
1 replicate
A– Experiment 1: control group
Mesh cage Stable flies (n=100)
Glass feeder B. besnoiti-enriched blood
(12.5.10 tachyzoites per ml)
6
Step1
Exposure time = 3 hours
3 replicates
B– Experiment 2: experimental transmission with tachyzoite-enriched blood as a source of
parasites
Stable flies (n=100)
B. besnoiti-free blood
Step 2
Exposure time = 24 hours
Bradyzoite cysts
Stable flies
(n=100)
Mesh cage
C – Experiment 3: experimental transmissions with long exposure time to a chronically
infected heifer as a source of parasites
Chronically infected heifer
Exposure time = 1hour
2 re
p
licates
Fig. 1 Diagrams of the
experimental design. a
Experiment 1: control group.
bExperiment 2: experimental
transmission with tachyzoite-
enriched blood as a source of
parasites. cExperiment 3:
experimental transmissions
with long exposure time to a
chronically infected heifer as
a source of parasites. dEx-
periment 4: experimental
transmissions with short
exposure time to a chronical-
ly infected heifer as a
source of parasites
482 Parasitol Res (2013) 112:479–486
this heifer were found positive by the B. besnoiti-specific
qPCR (Table 1)ordFAT.
Experiment 2. This trial showed that S. calcitrans trans-
mitted B. besnoiti DNA from highly infected blood to
uninfected blood. Indeed, Ct values for the glass feeder
containing tachyzoite-enriched blood were very low in
all three replicates (16 and twice 17), which was to be
expected. Parasite DNA was detected in the glass feeder
recipient with Ct values of 32 and twice 37 after a 24-
h exposure. This checking step allowed the following
experiments involving a living heifer acting as a source
donor.
Experiment 3. The serum of the chronically infected heifer
was clearly positive by WB. Parasite DNA was detected in
blood (Ct value of 33)and in two skin biopsy punches from the
neck and the forelimb with Ct values of 16 and 18, respective-
ly. Experiment 3 showed that stable flies could ingest parasites
from cutaneous cysts after 1 h of exposure. qPCR tests
(Table 1) were positive in both replicates with a higher Ct
value for the mouthparts than that of the abdominal contents.
The dFATwas only positive for the gut contents of stable flies.
Experiment 4. DNA was detected in the intestinal contents
and mouthparts of S. calcitrans 1 h after the blood meal was
interrupted. The Ct values of abdominal contents were lower
Table 1 Cycle threshold values (Ct) of B. besnoiti ITS-1 in S. calcitrans or in blood glass feeders according to experiments
Experiment Replicate Duration of exposure to Number of stable
flies used per
replicate and per
experiment
Ct of B. besnoiti ITS-1
Skin of B. besnoiti-
infected heifer
Blood of B. besnoiti-
free heifer
In S. calcitrans In recipient blood
from glass feeder
Mouthparts Abdominal
contents
Experiment 1 1 None 1 h 50 No Ct No Ct No Ct
24h 50 NoCt NoCt NoCt
Experiment 3 1 1 h None 100 39 34 N.D.
2 100 36 24 N.D.
Experiment 4 1 5 min 1 h 100 35 29 34
24 h 200 39 34 32
2 1 h 150 39 27 No Ct
24 h 250 No Ct 33 39
Results of experiment 2 were reported directly in the text
No Ct negative Ct values are ≥40; N.D. not determined
D – Experiment 4: experimental transmissions with short exposure time to a chronically
infected heifer as a source of parasites
Stable flies
Step 1
Exposure time = 5 minutes
Replicate 1: n = 300 stable flies
Replicate 2: n = 400 stable flies
Cattle B.besnoiti-free blood
Step 2
Exposure time
Replicate 1: 1 hour (n = 100)
24 hours (n = 200)
Replicate 2: 1 hour (n = 150)
24 hours (n = 250)
Interruption of blood meal
Fig. 1 (continued)
Parasitol Res (2013) 112:479–486 483
than Ct values of mouthparts (Table 1). Twenty-four hours
after feeding on the infected heifer, the Ct values of the
abdominal contents of stable flies were higher (34 and 33
according to the replicates) than those obtained after 1-
h feeding (29 and 27, respectively, Table 1). No Ct and a
very high value (39) were recorded from mouthparts 24 h
after feeding according to replicates 1 and 2. The blood in
the glass feeder was positive in the first replicate only, at 1 h
(Ct034) and 24 h (Ct032) of exposure. Results of dFAT
revealed some discrepancy with qPCR. B. besnoiti tachy-
zoites isolated from Vero cell culture were clearly observed
on the control slides (Fig. 2a). Entire parasites were clearly
identified in the abdominal contents of the stable flies
(Fig. 2b). No entire B. besnoiti could be observed by dFAT
on the slides in the mouthparts nor in the blood from the
glass feeder after any of the exposure times (experiment 3
and 4).
Discussion
Cattle besnoitiosis has been neglected until its recent geo-
graphic expansion in Europe (Olias et al. 2011). Some
strains have been isolated in various European countries:
Portugal (Cortes et al. 2006b), Germany (Schares et al.
2009), Spain (Fernández-García et al. 2009), Italy (Gentile
et al. 2012), and for the first time in France.
The contamination of mouthparts and abdominal con-
tents of stable flies was achieved with our protocol using a
living and chronically infected heifer as a source of B.
besnoiti. The use of laboratory colonies of stable flies has
the advantage of preventing previous B. besnoiti infections,
as also demonstrated by Bigalke (1968). It was about the
15th generation of stable flies that was used in November
2011 (Salem et al. 2012), and no evidence has been collect-
ed showing that B. besnoiti could pass through the eggs of
stable flies (Bigalke 1968).
The use of glass feeders with a thin membrane was also
efficient for studying the transmission ability of S. calcitrans
by using an artificial and highly tachyzoite-enriched blood
or chronically infected cattle as a source of parasites. In
experiment 2, the parasite burden in the blood was signifi-
cantly high, which increases the probability of ingestion by
S. calcitrans since no data were previously available.
Moreover, this level of infection most likely did not occur
naturally. As shown with experiments 3 and 4, stable flies
were able to ingest parasites while blood feeding on chron-
ically infected cattle, which has never been demonstrated
since the survey of Bigalke (1968). Using qPCR for exper-
iment 4, we did not observe a decrease in Ct values between
1 and 24 h after exposure in the abdominal contents of stable
flies, suggesting that no effective parasite multiplication had
occurred within the gut of stable flies. Conversely, the
increase of Ct values could imply the death of parasites
and destruction of parasite DNA. One possible explanation
could be the presence of stomoxyn, a defense helical peptide
of 42 amino acids secreted by the anterior part of the gut
after the blood meal, which demonstrated a trypanolytic
activity (Boulanger et al. 2002). Stomoxyn probably had a
wide range of antimicrobial activities (Boulanger et al.
2002) that could also include B. besnoiti,giventhatno
entire parasite was observed by dFAT after 24 h in the
abdominal contents of stable flies.
The parasite burden in the mouthparts was probably very
low. The volume of blood retained in the mouthparts of S.
calcitrans was close to 0.4 nl (Scoles et al. 2005), which is
smaller than the volume of blood contained in the tabanids'
mouthparts evaluated at 12.5 nl (Scoles et al. 2008). Bigalke
(1968) estimated that 52,000 to 292,500 stable fly bites were
required to cause infection in cattle whereas only three horse
fly bites were sufficient. Ct values of mouthparts for both
experiments 3 and 4 were very high, which highlighted the
presence of a low burden of parasite DNA. Indeed, dFAT
analysis on this stable fly compartment remained negative in
both experiments. The survival time of a parasite in the
mouthparts of S. calcitrans is probably very short, as sug-
gested by Bigalke (1968), who did not obtain successful
contaminations with stable flies 3 h after their interrupted
and infected blood meal. Firstly, some B. besnoiti parasites
could have been destroyed by some components of S. calci-
trans saliva (Scoles et al. 2005). Secondly, the adapted dFAT
10 µm 10 µm
AB
Fig. 2 Positive dFAT for
tachyzoite culture (a) and for
abdominal contents of S.
calcitrans (b) after 1-h exposure
on blood glass feeder preceded
by 5 min of exposure to the
heifer's skin (experiment 4).
White rows indicate
B.besnoiti parasites
484 Parasitol Res (2013) 112:479–486
probably suffered from low sensitivity. Those facts could also
explain why no entire parasite was detected by dFAT in the
blood of the glass feeder in experiment 4. However, parasite
DNA was detected by qPCR and turned out to be positive in
recipient blood, suggesting that it was possible to transfer a
parasite from a living donor to another host, either by probos-
cis flushing or by regurgitation (Doyle et al. 2011).
Non-hematophagous arthropods, e.g., Musca domestica
and Musca autumnalis, which feed on lachrymal secretions,
on blood from skin injuries or on other exudative liquids,
have been suspected to transmit B. besnoiti mechanically.
This has never been confirmed (Bigalke 1968). In this case,
potential routes of mechanical transmission may include
parasite transfer from wounds of infected animals to wounds
of susceptible ones, for example by direct contact of legs,
exoskeleton, and licking mouthparts of insects, but this
could be probably extremely rare in field conditions.
To conclude, since the unique survey of Bigalke (1968), we
have collected some evidence with help of modern tools of
parasite detection and proved that S. calcitrans could ingest B.
besnoiti parasites from a chronically infected heifer harboring
cutaneous cysts and could mechanically transmit these para-
sites to a uniform recipient substrate. Several improvements
can be done on our protocol. For example, the increase of the
number of stable flies implied in these transmission experi-
ments to 500 or 1,000 could allow the detection of viable
parasites by dFAT and decrease Ct values. Moreover, in epi-
demiological surveys, this protocol could also be applied to
discriminate cattle that can be “suitable donors”of B. besnoiti
(Bigalke 1968) among a population of seropositive but
asymptomatic animals. In other words, it would be interesting
to estimate the correlation between the Ct values obtained
from the skin biopsy and the presence or absence of parasite
and/or parasite DNA in the artificial recipient after the insects'
interrupted blood meals.
Acknowledgments A. Salem benefits from a Ph.D. grant of the
Ministry of Higher Education (Damascus, Syria) and the ENVT. The
authors wish to thank Martine Roques, Solange Vermot, and Sonia
Gounaud for their technical assistance.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
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