Journal of Helminthology, page 1 of 5 doi:10.

1017/S0022149X13000266
q Cambridge University Press 2013

Regulatory role of adrenergic

neurotransmitters on the spontaneous
muscular activity in the ruminant trematode
Paramphistomum cervi
(Paramphistomatidae)
B. Saikia1, C.C. Barua1*, S. Hazarika1, L.C. Lahon1, D. Saikia2,
R.S. Borah3 and P.K Verma4
1
Department of Veterinary Pharmacology and Toxicology, College of
Veterinary Science, Assam Agricultural University, Khanapara, Guwahati
781022, India: 2Department of Animal Biotechnology, College of
Veterinary Science, Assam Agricultural University, Khanapara, Guwahati
781022, India: 3Department of Livestock Production and Management,
College of Veterinary Science, Assam Agricultural University, Khanapara,
Guwahati 781022, India: 4Department of Veterinary Pharmacology and
Toxicology, College of Veterinary Science and Animal Husbandry,
SKAUST, R.S. Pura, Jammu, India
(Received 30 July 2012; Accepted 26 March 2013)

Abstract

The neuromuscular system of helminths is an important area for target

identification and drug development. Many anthelmintics, namely ivermectin,
levamisole, piperazine, pyrantel, praziquantel and organophosphates, produce
paralysis of helminths by affecting their neuromuscular systems. The
neuromuscular system of helminths is also an important area of research to
identify some of the important differences between the neuromuscular
physiology of helminths and mammals. The identification of differences
would help in developing newer target-specific, safe and effective anthelmintics.
The present study was carried out to investigate the effects of different
adrenergic neurotransmitters (epinephrine, norepinephrine, dopamine, L -dopa)
and their antagonists (propranolol and haloperidol) on the spontaneous
muscular activity of isometrically mounted Paramphistomum cervi.

Introduction sequently, there is an urgent need to develop newer,

The continuous indiscriminate use of anthelmintics
has caused a growing problem of parasite resistance to
conventional treatments (Jackson & Coop, 2000).
Adverse reactions and high costs are some problems
associated with currently available anthelmintics. Con-
*Fax: 91-361-2337700
E-mail: chanacin@gmail.com
selective and ecofriendly plant-based anthelmintics to
overcome some problems encountered in helminth
infections. The common pharmacological basis of
treatment of helminths generally involves either disrup-
tion of the energy generation process and subsequent
starvation of the parasite; or a break in neuromuscular
coordination, causing paralysis of helminths and
subsequent expulsion; or an adverse effect on reproduc-
tive processes (Robertson, 1982).
2 B. Saikia et al.
Neurotransmitters are endogenous substances in
mammals and helminths which are released selectively
from nerve terminals at synapses and neuromuscular
junctions. These neurotransmitters regulate muscle con-
traction and help in attachment, feeding and reproduc-
tion of helminth parasites (Prichard, 2005). Apart from
their effects on energy generation and reproduction,
many selective anthelmintics affect the functioning of the
neuromuscular system of helminths (Geary et al., 1992).
Anthelmintics such as levamisol, piperazine and iver-
mectin induce paralysis of nematodes by their action on
nicotinic receptors (Harrow & Gration, 1985), g-amino-
butyric acid (GABA) receptors (Martin & Humphrey,
1994) and glutamate-gated chloride ion channels (Cully
et al., 1996). Various studies have revealed the functional
presence of adrenergic and dopaminergic systems in
trematodes. Neurotransmitters such as epinephrine,
norepinephrine and dopamine have also been demon-
strated in flatworms (Maule et al., 1990). In both the
central and peripheral nervous system of trematodes,
neurotransmitters play an important role (Bennett &
Gianutsos, 1977; Maule et al., 1990) in regulating
spontaneous muscular contractions by interacting with
either adrenergic or dopaminergic receptors in the nerve
fibres of mammals (Bylund, 1994). The presence of
adrenergic and dopaminergic receptors have also been
observed in the muscle of Gastrothylax crumenifer (Verma
et al., 2010). Dopamine has been reported to elicit an
excitatory effect, whereas epinephrine and norepi-
nephrine produce an inhibitory effect, on the frequency
and amplitude of rhythmic activity of isometrically
mounted Schistosoma mansoni (Mellin et al., 1983), Fasciola
hepatica (Holmes & Fairweather, 1984) and F. gigantica
(Kumar & Tripathi, 1996). However, dopamine (100 mM )
had no observable effect on rhythmic contractions of
isolated muscle fibres of Bdelloura candida (Blair &
Anderson, 1994). Haloperidol, a dopaminergic antagonist,
completely blocked the excitatory effect of dopamine on
the spontaneous muscular activity of S. mansoni (Mellin
et al., 1983). However, a literature review revealed that
no such study has been carried out so far to investigate
the type of neurohumoral transmission of Paramphisto-
mum cervi. Therefore, the present study was undertaken
to investigate the role of adrenergic neurotransmitters
and their antagonists on the spontaneous muscular
activity of P. cervi.
Materials and methods
Parasitological and experimental procedures
Mature amphistomes identified as P. cervi were
collected from the rumen of freshly slaughtered cattle at
a local abattoir in warm (37 ^ 18C) Hank’s Balanced Salt
Solution (HBSS) in an insulated container and brought to
the laboratory. The amphistomes were maintained in a
Biological Oxygen Demand (BOD) incubator at 37 ^ 18C,
until further use.
Epinephrine (molecular weight (Mwt) 183.2), norepi-
nephrine (Mwt 169.2), dopamine (Mwt 189.6) and L -dopa
(Mwt 107.19) were obtained from Sigma-Aldrich (St Louis,
Missouri, USA), haloperidol (Mwt 425.91) from RPG Life
Sciences (Mumbai, India) and propranolol (Mwt 295.81)
from Merck India Ltd (Mumbai, India). Other chemicals
used in the study were of analytical grade.
Mature worms of P. cervi were mounted isometrically in
HBSS solution at 37 ^ 18C (Ahmed & Nizami, 1990). Each
worm was mounted with the help of two fine hooks. One
hook was inserted 1 – 2 mm below the anterior sucker and
fixed to the tip of an aeration tube and another hook was
pierced through the surface of the acetabulum and con-
nected to an isometric force transducer (Powerlab, 4/35,
4-Channel Data Acquisition System, Model: ML866/P,
AD Instruments, Delhi, India) and a tension of 200 mg
was applied to the parasite. After setting up, the
preparations were allowed to equilibrate in bath fluid
for 30 min and standard time intervals of 20 min were
allowed between drug exposures. After equilibration,
adrenergic neurotransmitters, epinephrine, norepi-
nephrine, dopamine, L -dopa, and their antagonists
propranolol and haloperidol, were added in a molar
concentration of 1 log unit in ascending order (1027 to
1023 M ) in the tissue bath to study their effects on the
spontaneous muscular activity of P. cervi. Three par-
ameters – amplitude (average of all peaks per 5 min or
average tension), baseline tension (average of all
minimum levels of contractions used for measuring
amplitude) and frequency (total number of contractions
in 5 min) – of the isometrically mounted P. cervi were
measured for a period of 10 min, and these measurements
were recorded using the software Omega Chart Windows
7 (url=http://www.windows7download.com/win7-omega
Chart/pkaunqyx.html). Control recordings were made for
15 min before the addition of neuropharmacological agents.
Data analysis
Three parameters – frequency, amplitude of rhythmic
contractions and baseline tension of the isometrically
mounted P. cervi – were measured and compared with
controls to elucidate the effects of various neuropharma-
cological agents. The results are presented as standard
error, and the levels of significance were measured by
paired t-test (Snedecor & Cochran, 1989).
Results
Isometrically mounted worms of P. cervi exhibited
spontaneous muscular activity (SMA) for several hours
without any significant change in amplitude, baseline
tension and frequency of the rhythmicity. The control
amplitude, baseline tension and frequency of SMA were
0.52 ^ 0.03 g, 0.19 ^ 0.01 g and 55.00 ^ 5.00 Hz, respect-
ively. The amplitude (0.48 ^ 0.02 g), baseline tension
(0.19 ^ 0.03 g) and frequency (51.00 ^ 4.71 Hz) of spon-
taneous contractions recorded after a period of 2 h, were
not significantly different from those recorded 15 min
after applying the tension to P. cervi (table 1).
Following the cumulative administration of 1 log unit
in ascending order (1027 to 1023 M ), epinephrine elicited a
concentration-dependent reduction of amplitude at 1025
to 1023 M , of baseline tension at 1024 to 1023 M and of
frequency at 1023 M concentration as compared to
control SMA. Similarly, with the administration of graded
(1027 to 1023 M ) molar concentration of 1 log unit in
 Adrenergic neurotransmitters and their role on P. cervi 3

Table 1. The effect of 1023 M to 1027 M epinephrine (EPI), norepinephrine (NOR), propranolol (PRO), dopamine (DOPA), L -dopa and
haloperidol (HALO) on amplitude (g), baseline tension (g) and frequency (Hz) of spontaneous muscular activity of six worms of
Paramphistomum cervi; mean ^ standard error (n ¼ 6) with P values significant at , 0.05* and , 0.01**.

Concentration
27 26
Treatment Parameters 10 10 1025 1024 1023 Control

EPI Amplitude 0.42 ^ 0.02 0.43 ^ 0.02 0.37 ^ 0.01* 0.33 ^ 0.01** 0.32 ^ 0.02** 0.49 ^ 0.03
Baseline tension 0.20 ^ 0.02 0.19 ^ 0.02 0.18 ^ 0.01 0.12 ^ 0.02* 0.12 ^ 0.01* 0.22 ^ 0.02
Frequency 60.00 ^ 4.27 61.00 ^ 3.08 59.00 ^ 2.63 57.33 ^ 4.91 53.00 ^ 4.70* 62.00 ^ 4.21
NOR Amplitude 0.48 ^ 0.02 0.43 ^ 0.03* 0.31 ^ 0.03** 0.37 ^ 0.02* 0.28 ^ 0.01** 0.51 ^ 0.03
Baseline tension 0.21 ^ 0.02 0.15 ^ 0.01* 0.13 ^ 0.02* 0.11 ^ 0.02** 0.09 ^ 0.01** 0.24 ^ 0.02
Frequency 57.00 ^ 4.25 55.00 ^ 5.22 54.33 ^ 4.03 53.00 ^ 5.72 52.00 ^ 5.16 59.00 ^ 5.78
PRO Amplitude 0.40 ^ 0.03 0.44 ^ 0.05 0.50 ^ 0.02* 0.55 ^ 0.02** 0.62 ^ 0.01** 0.38 ^ 0.05
Baseline tension 0.18 ^ 0.03 0.20 ^ 0.03 0.21 ^ 0.03* 0.23 ^ 0.03** 0.26 ^ 0.01** 0.17 ^ 0.03
Frequency 44.00 ^ 2.65 46.00 ^ 2.76 50.00 ^ 3.86* 56.50 ^ 4.21** 61.50 ^ 5.20** 41.00 ^ 3.70
DOPA Amplitude 0.59 ^ 0.02 0.61 ^ 0.04* 0.69 ^ 0.05** 0.75 ^ 0.07** 0.82 ^ 0.09** 0.53 ^ 0.01
Baseline tension 0.18 ^ 0.02 0.22 ^ 0.02* 0.27 ^ 0.03** 0.33 ^ 0.05** 0.38 ^ 0.09** 0.15 ^ 0.01
Frequency 41.00 ^ 3.70 44.00 ^ 2.65 47.00 ^ 4.57* 57.33 ^ 4.91** 62.0 ^ 4.21** 37.00 ^ 5.31
L -dopa Amplitude 0.35 ^ 0.03 0.40 ^ 0.03** 0.39 ^ 0.02 0.37 ^ 0.01 0.36 ^ 0.02 0.33 ^ 0.02
Baseline tension 0.10 ^ 0.02 0.12 ^ 0.01 0.12 ^ 0.01 0.13 ^ 0.02* 0.14 ^ 0.02* 0.09 ^ 0.01
Frequency 54.00 ^ 6.38 54.00 ^ 5.36 54.50 ^ 6.17 53.00 ^ 5.32 51.00 ^ 3.73 55.00 ^ 5.31
HALO Amplitude 0.55 ^ 0.07 0.47 ^ 0.02* 0.39 ^ 0.03* 0.28 ^ 0.05** 0.20 ^ 0.02** 0.58 ^ 0.09
Baseline tension 0.16 ^ 0.03 0.13 ^ 0.01 0.11 ^ 0.01* 0.09 ^ 0.01** 0.08 ^ 0.01** 0.19 ^ 0.02
Frequency 51.50 ^ 4.48 44.00 ^ 4.95* 39.00 ^ 5.15** 33.60 ^ 5.77** 23.0 ^ 4.57** 58.50 ^ 3.45

ascending order, norepinephrine produced a significant cumulative administration of 1 log unit in ascending
reduction in amplitude and baseline tension at 1026 order (1027 to 1023 M ), L -dopa produced a significant
to 1023 M concentrations, compared with controls. increase in the amplitude of spontaneous muscular
Subsequently, propranolol (1027 to 1023 M ), an adrenergic contractions at 1026 and 1025 M . Furthermore, there was
antagonist, exhibited a significant increase in amplitude a dose-dependent (1027 to 1023 M ) increase in baseline
and baseline tension at 1025 to 1023 M concentrations and tension at 1024 and 1023 M concentration. L -Dopa did not
frequency at 1025 to 1023 M as compared with controls evoke any significant alteration in the frequency of the
(table 1). On the other hand, epinephrine induced an contractile activity of P. cervi when compared with
inhibitory effect which was completely blocked by controls. Haloperidol (1027 to 1023 M ), an antagonist
propranolol (1023 M ) and followed by an excitation of of dopamine, produced a significant increase in ampli-
SMA of P. cervi (table 2, fig. 1a). tude at the concentrations of 1026 to 1023 M , baseline
The addition of dopamine (1027 to 1023 M ) to the bath tension at 1025 to 1023 M and frequency at 1026 to 1023 M ,
fluid containing isometrically mounted mature P. cervi as compared to control observations (table 1). Dopamine-
caused an excitation of SMA in a dose-dependent manner. induced (1023 M ) excitatory activity on the amplitude,
Significant excitatory activities of dopamine on amplitude baseline tension and frequency of the contractile activity
and baseline tension were observed at 1026 to 1023 M of P. cervi was found to be completely blocked by
concentrations and on frequency at 1025 to 1023 M , as haloperidol, showing inhibition of SMA (table 2, fig. 1b).
compared to the control values. Likewise, following the
Discussion
Table 2. The effect of 1023 M concentration of propranolol,
epinephrine, haloperidol and dopamine on amplitude (g), Adrenergic neurotransmitters are important in the
baseline tension (g) and frequency (Hz) of spontaneous muscular functioning of the central and peripheral nervous systems
activity of six worms of Paramphistomum cervi; mean ^ standard of trematodes and their animal hosts (Maule et al., 1990).
error (n ¼ 6) with P values significant at , 0.01**. In vitro isometrically mounted P. cervi exhibited a uniform
pattern of SMA, which is apparently similar to that of
Parameters S. mansoni (Mellin et al., 1983), F. hepatica (Fairweather
Treatment/ Baseline et al., 1983), Gigantocotyle explanatum (Ahmed & Nizami,
control Amplitude tension Frequency 1990), F. gigantica (Tripathi et al., 2000) and G. crumenifer
(Verma et al., 2010). In the present study, both epinephrine
Control 0.36 ^ 0.02 0.23 ^ 0.01 47.00 ^ 3.84 and norepinephrine elicited inhibitory responses on the
Epinephrine 0.21 ^ 0.03 0.11 ^ 0.02 35.47 ^ 4.71 SMA of P. cervi. Similar inhibitory effects have been
Propranolol þ 0.42 ^ 0.04** 0.34 ^ 0.05** 55.00 ^ 6.32** reported in F. hepatica (Holmes & Fairweather, 1984),
epinephrine
F. gigantica (Kumar & Tripathi, 1996) and G. crumenifer
Control 0.42 ^ 0.02 0.18 ^ 0.01 48.00 ^ 4.23
Dopamine 0.67 ^ 0.05 0.29 ^ 0.04 63.37 ^ 3.37 (Verma et al., 2010) with epinephrine and norepinephrine,
Haloperidol þ 0.36 ^ 0.04** 0.15 ^ 0.05** 49.00 ^ 5.12** but the latter did not elicit the motor activity of S. mansoni
dopamine (Mellin et al., 1983). In the present experiment, the
b-adrenergic blocker, propranolol, was found to antagon-
4 B. Saikia et al.
(a)
SMA
Epinephrine 10–3
Control Washing
Control Epinephrine 10–3 Wash
(b)
SMA
Control Dopamine 10–3
Washing
Control Dopamine 10–3 Wash Haloperidol 10–3
Fig. 1. (colour online) The effect of 1023 M concentrations of (a) propranolol and epinephrine (b) haloperidol and dopamine on
spontaneous muscular activity (SMA) of Paramphistomum cervi.
ize the epinephrine-induced inhibitory response and
increased the amplitude, baseline tension and frequency
of the contractile activity of P. cervi.
The gross visual motility of G. crumenifer was also
inhibited by pargyline and other monoamine oxidase inhi-
bitors, thereby influencing the metabolism of norepinephrine
and epinephrine (Abidi & Nizami, 2000). Dopamine is
another adrenergic agent used as an important classical
neurotransmitter. Previous investigators have shown that
dopamine produces an excitatory effect on rhythmic
contractions in F. hepatica (Holmes & Fairweather, 1984)
and F. gigantica (Kumar & Tripathi, 1996). In the present
study dopamine also produced significant excitatory
effects on the amplitude, baseline tension and frequency
of SMA of P. cervi. In contrast, dopamine (0.1 mM )
produced a slight decrease in motor activity of S. mansoni
and the effect was blocked by haloperidol (Mellin et al.,
1983). Yet again, L -dopa, a precursor molecule of
dopamine, produced excitatory effects on amplitude
and baseline tension, without significant changes in the
frequency of contractions of P. cervi, which is in agree-
ment with the earlier reports on G. crumenifer (Verma et al.,
2010). In the present study, haloperidol (1023 M concen-
tration) completely blocked the excitatory activity of
dopamine (1023 M concentration) and thereby confirms
the antagonistic role of haloperidol on dopaminergic
receptors of the SMA of P. cervi. These observations
suggest that two types of receptors – adrenergic and
dopaminergic – in P. cervi, and their activities, were
antagonized by their classical blockers. Similarly, the
presence of serotonergic, cholinergic and peptidergic
neurons have been demonstrated immunocytochemically
in S. mansoni (Pax et al., 1984; Gustafsson, 1987), and
Propranolol 10–3 + 200 mg
Propranolol 10–3 Washing
Epinephrine 10–3 2 min
Propranolol 10–3 Epinephrine 10–3 Wash
Haloperidol 10–3 +
Haloperidol 10–3
200 mg
Dopamine 10–3 2 min
Dopamine 10–3 Wash
serotonergic, cholinergic and adrenergic receptors have
been shown functionally in G. crumenifer (Verma et al.,
2007, 2009, 2010). The present investigation also suggests
the involvement of adrenergic and dopaminergic recep-
tors in the muscle of P. cervi.
Furthermore, both epinephrine and norepinephrine
produced inhibitory effects, whereas dopamine and
L -dopa produced excitatory effects on the spontaneous
muscular contraction of P. cervi. Antagonistic reactions to
the inhibitory effect of epinephrine and norepinephrine
and excitatory effect of dopamine and L -dopa by their
respective blockers propranolol and haloperidol have
also corroborated their role on the spontaneous motility
of P. cervi.
Acknowledgements
The authors are grateful to the Indian Council of
Agricultural Research (ICAR), Government of India, New
Delhi, for providing financial assistance, and Director of
Research (Vety), CVSc, Khanapara for providing necess-
ary facilities to carry out the research work. The authors
also confirmed that there is no conflict of interest in
the study.
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