New Scanning Electron Microscopy Look of Ascaridia galli (Schrank,
1788) Adult Worm and its Biological Control
E.H. Abdel Rahman
The in vitro activity of three plant extracts; Artemisia cina, Peganum hormala and Calendula micrantha was investigated against Ascaridia galli adult worms. In vitro treatment of A. galli worms with different concentrations of each extract revealed potency of C. micrantha over the other two extracts as judged by the calculated LD50 values. These values were 2.66 ppm for C. micrantha, 33.7 ppm for P. harmala and 48.98 ppm for A. cina. The impact of the most potent extract, C. micrantha on A. galli worms was detected by scanning electron microscopy. C. micrantha treated worms showed enlargement and destruction of lips with subsequent damage of buccal cavity organelles. Moreover, papillae were disturbed and the amphids structures were lost. The cuticle showed wrinkled surface with loss of striations. Also, the cloacal structures were affected.
Aacaridia galli is one of the most common parasitic round worms of poultry,
occurs in chickens and turkeys. Adult worms are semitransparent and considered
the biggest nematode in poultry. Heavily infected birds may show droopiness,
emaciation and diarrhea. The primary damage reduced efficiency of feed utilization,
but death has been observed in severe infections. Control of A. galli infection
can be conducted through implementation of management practice and treatment
strategies. The management practice includes regular cleaning and use of disinfectants,
proper disposal of the used litter and farm garbage, alternate use of pens,
etc. Chemotherapy is the wide use approach to control the infection. Compared
to the other non-chemotherapeutic approaches to parasitic control, use of nematode
trapping fungi has shown promising results in different countries (De and Sanyal,
2009). Verma et al. (1991) studied the comparative efficacy of three
broad-spectrum anthelmintic chemotherapy against A. galli in poultry.
Although, ivermectin proved potency against A. galli, as observed by
Sharma et al. (1990), its use still limited due to its parenteral administration.
Moreover, difficulties associated with chemotherapy of parasitic diseases as
the ineffectiveness of some drugs, incomplete elimination of certain diseases
in addition to undesired side effects looked a problem. It is not surprising
therefore, to make a stress on the importance of biological control of helminth
infections. Larsen et al. (1997), Abdel-Rahman and Saleh (2006) and Behnke
et al. (2008) stated that biological control has the potential of becoming
an important part of a future integrated control strategy against parasitic
nematodes. The bioactivity directed fraction (Pinocembrine) of the acetone extract
of Teloxys graveolens exhibited ovicide activity on infective eggs of
A. galli (Comacho et al., 1991).
The antiascarid activity of Caesalpinia crista Linn seeds popularly known as Karanjwa was evaluated in chickens of the Fayomi breed, suffering from experimental infection with A. galli 50 mg kg-1 seed powder showed significant results based on excreted eggs/g faeces (Javed et al., 1994). The in vitro evaluation of the activity of root-tuber-peel extract of Flemigia vestita, an indigenous plant consumed by the natives in Northeast India against Heterakis gallinarum, Raillietina echinobothrida and Paramphistomum sp. was performed by Tandon et al. (1997). But unfortunately, only nematodes did not show any structural alternations in their tegumental architecture after exposure to F. vestita extract.
There are no available literatures dealing with the ultrastructure of Nematode parasite A. galli except Ashour (1994), who studied the scanning electron microscopy of the mentioned nematode. He described the lips, papillae, cuticle and cloaca with the prominent characteristic features for each part.
Fagerholm et al. (1998-2000) studied the scanning electron microscopy of Ascaris suum (Nematode, ascaridaidea). They described the cuticular annuli and splitting it into two subannuli during growth of the third-stage larva into adult worms. Also they stated that the increase in length of A. suum is due to lengthening of individual transverse annuli in the cuticle. They added that the increase in size also results in increased diameter of different cuticular sense organs. The researchers identified a two prominent asymmetrically placed cuticular sensilla, called centrids, are reported in A. suum. In adult worms, the centrids are plate like, asymmetrically placed. The name centrids was originally chosen to indicate the placement of the papillae in the midbody region of worms.
Based on the limited research concerned with the control of A. galli by plant extracts and also on the medical importance of this helminth in poultry production, the objective of the study was to focus on the in vitro assessment of the potency of three plant extracts in the control of A. galli worms utilizing scanning electron microscopy. The selection of these plants A. cina, P. harmala and C. micrantha was attributed to their previous anthelmintic impact on other helminthes (Mehrota et al., 1990; Hassanain et al., 1991; Kang, 1994; Miraldi et al., 1998; Shuhua et al., 2000; El-Garhy and Mahmoud, 2002).
MATERIALS AND METHODS
This research project was conducted from 5-2007 to 5/2010 (3 years duration) and it is carried out at the Zoonotic Diseases Department, National Research Center, Cairo, Egypt.
Collection of A. galli Worms
Worms were collected from the small intestine of slaughtered domestic chickens
during the year of 2008. Worms were washed thoroughly with physiological saline
and subjected to the in vitro effect of the plant extracts.
Preparation of Plant Extracts
Preparation of A. cina Aqueous Extract
Artemisia cina was soaked in water for 4 days at room temperature
with a ratio of 1:6 weights/volume. The supernatant was filtered and lyophilized.
Preparation of P. harmala Ethanolic Extract
Crushed P. harmala seeds were soaked in ethyl alcohol for two days
at room temperature with a ratio of 1: 4 weights/volume. The suspension was
filtered and dried.
Preparation of C. micrantha Ethanolic Extract
C. micrantha flowers were soaked in ethyl alcohol at room temperature
for two days at a ratio of 1: 10 weight/volume. After filtration, the clear
supernatant was dried.
Treatment of A. galli Worms with Plant Extracts
Three concentrations (80, 160 and 320 ppm) of A. cina, six concentrations
(5, 10, 20 , 40, 80 and 160 ppm) of P. harmala and five concentrations
(0.5, 1, 1.5, 2 and 4 ppm) of C. micrantha in physiological saline were
added separately to three replicates each 10 worms each. Worms were mixed and
incubated at 37°C for 24 h. At the same time control untreated replicates
of worms were incubated in physiological saline for 24 h. Results of worm mortality
percentages were subjected to probate statistical analysis based on the work
of Polo-Pc (Robertson et al., 1980) to determine the LD50 values.
Scanning Electron Microscopic Examination (SEM)
Worms treated with 4 ppm of C. micrantha and with the LC50 value
2.66 was prepared for scanning electron microscopy as described by Ashour and
Lewis (1982). Specimens of A. galli were fixed in 2.5% glutraldehyde
after washing. The specimens were dehydrated through ascending ethanol series,
dried in CO2 critical point drier after which they were sputter coated
with gold at 20 nm. Finally, the specimens were examined and photomicrographed
with SEM (Jeol, JEM-1200 EX II, Japan).
RESULTS AND DISCUSSION
Scanning Electron Microscopy of A. galli Adult Worms
Mouth opening of the worm was surrounded by three main large lips; one dorsal
and two subventral. Each lip was not globular but it had a wide base with gentle
tapering towards apex. Each main lip was provided with two small accessory ones
at lateral sides. Three paired cephalic papillae and two amphids were found
on the outer surface of the lips. The amphidial surface was provided with several
pores. Small papillae were seen scattered on the cervical area of the worm (Fig.
The cuticular surface of the body was transversely striated (0.014 mm wide). These transverse annuli were well developed and appear darker in colour than the subannuli. In some sites, two successive transverse annuli might be fused at a short distance from lateral toward the middle of the cuticle with subsequent increasing the subannuli numbers and the width (0.021 mm) (Fig. 2).
Also, there were small scattered papillae with irregular pattern distribution (Fig. 1, 2). A narrow several discontinued median plate like centrids deeper to the cuticular transverse annuli extended through the whole length of the worm except the posterior one (Fig. 3, 4).
The posterior extremity of the male was slightly inflated forming ill developed
caudal alae with normal cloacal protrusion (Fig. 5, 6).
The ventral sucker was situated a short distance anterior to the cloacal opening,
it had an ill boundered circular rim. The cloacal opening was situated on the
top of a ventral conical protrusion (Fig. 5-7).
The cloacal papillae were arranged in two groups, the precloacal and postcloacal
ones. The precloacals papillae were arranged in the two sided-laterals of the
worm. The postcloacals papillae were arranged in one group.
||(a, b) Anteroposterior view showing the mouth opening of the
worm surrounded by three main large lips; one dorsal and two subventral.
Each lip is not globular but it has a wide base with gentle tapering towards
apex. Each main lip is provided with two small accessory ones at lateral
sides. Three paired cephalic papillae and two amphids are found on the outer
surface of the lips. The amphidial surface is provided with several pores.
Small papillae were seen scattered on the cervical area of the worm
||In vitro anthelmintic effect of the plant extracts
on galli worms
||Anteroposterior view showing the cuticular surface of the
body which is transversely striated (0.014 mm wide). These transverse annuli
are well developed and appear darker in colour than the subannuli. In some
sites, two successive transverse annuli may be fused at a short distance
from lateral toward the middle of the cuticle with subsequent increasing
the subannuli numbers and the width (0.021 mm)
||Anteroposterior view showing small scattered papillae with
irregular pattern distribution. Several narrow median centrids
The number of the cloacal papillae was several and could not be counted (Fig.
The in vitro impact of the three plant extracts on A. galli worms was depicted on Table 1 proving the advantage of C. micrantha over the other extracts. The impact was assessed by calculating the LD 50 which was 48.98 ppm for A. cina extract, 33.7 ppm for P. harmala and 2.66 ppm for C. micrantha.
The worms group that showed high mortality rate and potent LC50 was selected to be subjected to SEM directly.
In vitro Effect of C. micrantha Extract as Anthelmintics against
A. galli Adult Worms: The three lips guarded the mouth opening of the
worm were greatly affected together with the buccal cavity. There were morphological
disfigurements of the three lips (one dorsal and two subventral ones).
||Anteroposterior view showing several narrow discontinued median
centrids deeper to the cuticular transverse annuli which, extend through
the whole length of the worm except the posterior one
||Ventral view showing the posterior extremity of the male and
the ventral sucker is situated a short distance anterior to the cloacal
opening, it has an ill boundered circular rim
The lips became globular or quadrangular and the smaller accessory lips were
separated and lost from the two subventral large lips. But in the dorsal one,
was partially separated and still connected to the mother one (Fig.
8, 9). The mouth opening and cavity were occluded by granulation
tissues in the form of triangular plate with adhesion to the surrounding lips
which lead to widening of the lips (Fig. 8, 9).
Also, the amphids architectures were lost due to the compressed lips (Fig.
8, 9). The cuticular striations were lost due to its swelling
with longitudinal or transverse dividing masses (Fig. 8, 9).
Concerning the cloacal area, the precloacal papillae distribution were lost
and the ventral sucker lost its demarcation with area of hemorrhage together
with the surrounding tissues and the cloacal opening was still patent opened
with intact edges (Fig. 10a, b). The cloacal
protrusion was severely destructed and its tip was protruded to outside with
herniation of its internal content (Fig. 11).
||Lateral view showing the posterior extremity of the male which
is slightly inflated forming ill developed candal alae with normal cloacal
protrusion. The cloacal opening is situated on the top of a ventral conical
protrusion, the cloacal papillae that are arranged in two groups, the precloacal
and postcloacal ones
||Lateral view showing the cloacal papillae which are arranged
in two groups, the precloacal and postcloacal ones. The precloacals papillae
are arranged in the two sided-laterals of the worm. The postcloacals papillae
are arranged in one group
The in vitro study, performed in the current research, revealed the potent lethal effect of C. micrantha extract against A. galli adult worm recording LD50 value of 2.66 ppm. While P. harmala showed lesser effect with LD50 of 33.7 ppm followed by A. cina with LD50 of 48.98 ppm. The potent lethal effect of C. micrantha against nematodes was previously recorded against larvae of Haomonch contortus, Trichostrongylus colubriformis, Bunostomum spp. and Toxocara vitulorum eggs as observed by Hassanain et al. (1991). In contrary to the recorded weak effect of A. cina observed in the present study, A. santonica proved ascaridial efficacy in vitro against eggs and larvae of Ascaris lumbricoides (El-Garhy and Mahmoud, 2002). The difference between both observations could be attributed to the difference of the species of the used plant and to the developmental stages of both worms.
||Anteroposterior view showing the great affection of three
lips guarded the mouth opening of the worm together with the buccal cavity.
There are morphological disFig. ments of the three lips (one dorsal and
two subventral ones). The lips became globular or quadrangular and the smaller
accessory lips were separated and lost from the two subventral large lips.
The dorsal one is separated and still connected to the mother one
||Anteroposterior view showing the mouth opening and cavity
occluded by granulation tissues in the form of triangular plate with adhesion
to the surrounding lips which lead to widening of the lips. Also the amphids
architectures were lost due to the compressed lips. The cuticular striations
were lost due to its swelling with longitudinal or transverse dividing masses
Scanning electron microscopical examination was used to assess the effect of
C. micrantha, the most potent extract against A. galli worm compared
with normal structure. Scanning electron microscopy of normal A. galli
was previously described by Ashour (1994) except in some detailed morphological
features as observed in the current research. Ashour (1994) stated that the
lips of the A. galli worm has a wide base and narrow extremity with trilobed
appearance and the inner surface of each lip is provided with a thick smooth
cuticular plate. The cuticular surface of the body is transversely striated
with smaller diameter (0.006 mm wide) than proved in the present study (0.014
||(a, b) Face view showing the loss of precloacal papillae distribution
and the ventral sucker loss its demarcation with area of hemorrhage together
with the surrounding tissues and the cloacal opening is still patent opened
with intact edges
||Face view showing the severe destruction of cloacal protrusion
and its tip is protruded to outside with herniation of its internal content
In addition no subannuli were detected in his study, while the subannuli were
observed, for the first time, in the current research. The cloacal papillae
are arranged as follows: 3 pairs of precloacales, 2 pairs of lateral and 2 pairs
of ventral postcloacal and one pair of lateral and one pair of ventral subterminal
papillae. This pattern of distribution is different from that currently described.
For the first time, the present study revealed new morphological characteristics
in A. galli as those shown by Fagerholm et al. (1998-2000) and
Fagerholm et al. (1999) in scanning electron microscopy of A. suum.
There are circular or transverse annuli which were split into subannuuli during
embryonic development of A. galli larvae to adult and also its
lengthening in size to adult due to the cuticular development of worm. The presence
of centrids in A. galli, which is described currently for the first time,
is very important and aids in its morphological discrimination from A. suum.
Treatment of A. galli worms with C. micrantha resulted in dramatic changes in the mouth parts and cuticle. The changes in the mouth parts are associated mainly with the lips which showed enlargement and damage of the sensory papillae. These changes unable the worms to fix themselves to intestinal mucosa and led to their expulsion. Consequently, it may be postulated that, the mode of action of C. micrantha plant extract is worm fuge. On the other hand, the effect of both plants on the cuticular structure probably affects its function such as nutritional uptake which resulted in worm mortality and in this case may be act as wormicide. Unfortunately, no literatures are available regarding the assessment of the impact of plant extracts on A. galli worm using scanning electron microscopy which could support our postulations or not.
The present study introduced a biological control model of A. galli adult worms, in vitro, using plant extracts and clarified, at the level of ultra structural examination, the damages associated with worm organelles.
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