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sa qa rT ve lo s mecn ierebaT a er ov nuli ak ad em ii s m oa mbe, t. 7, #1, 2013
BU LLE TIN OF TH E GE OR GI AN NATI ONAL A CA DE M Y OF SCI ENC ES , vol. 7,
no. 1, 2013
© 2013 Bul l. Georg. Natl. Acad. Sci.
Parasitology and Helminthology
Morphological and Biochemical Investigation
of the Bothridium pithonis Blainville,1824
(Cestoda: Diphyllobothriidae)
Lali Murvanidze*, Tsitsino Lomidze*, Ketevan Nikolaishvili*
* Institute of Zoology, Ilia State University, Tbilisi
(Presented by Academy Member Irakli Elia va)
ABSTRACT. Cestodes are the most prevalent helminths in reptiles and particularly in pythons. In
case of intensive invasion cestodes can cause death in pythons in the Zoological Parks.An African Rock
Python (Python sebae Gmelin, 1788) was brought to Tbilisi Zoological Park from Tanzania in 2008. A
month later the animal died. In a small intestine cestoda Bothridium pithonis Blainville, 1824 (one
specimen) was detected. Based on the morphological investigation and its comparison with literature data
its identity with Bothridium pithonis Blainville, 1824, was proved. The length of the python was 960 mm
and the width at its broadest part was 15 mm. These measurements exceed the data given in the literature.
The unarmed scolex was made of two spherical parts with a medial depression between them. Each part
was surrounded with semicircular muscle ridges. The maximum width of the scolex was – 6 mm,
height – 3 mm, the width of the neck in its narrow part – 1.5 mm. The tape-shaped strobila was clearly
segmented with 13-14 units per 1 cm of the length. Minimal width of the segments was 9-10 mm,
length – 0.5 mm. Maximal width of the segments was found in upper third of the body and equaled 15 mm
and the length – to 0.9 mm. Two kinds of eggs were found: immature with 0.066 mm length
and 0.044 mm width and mature – 0.13 mm in length and 0.09–0.11 mm in width. For the characteristic
of the nervous system of B. pithonis the activity of enzyme cholinestherase in homogenates of the
helminth was studied. In different parts of strobila the activity was different and fluctuated from 45.98 to
142.0 mkmol acetylcholine/mg protein/h.
Improved quarantine period of the reptiles and intensive parasitological monitoring are required. ©
2013 Bull. Georg. Natl. Acad. Sci.
Key words: African rock python, Bothridium pithonis (Cestoda), morphology, cholinesterase.
E. Wiesenhütter [1] described tapeworm-Bothridium
pithonis found in a midgut of the Python reticulates
which died in the Berlin Zoological Garden. He studied
the scolex histology of the cestode. The author gives
the pathologoanatomic picture of the injured portion of
the python intestine and mentions profound changes
in the mucous membrane; however, he could not state
the mentioned parasite as the cause of death in py-
thons. Sawada and Kugi [2] suppose that a mass inva-
sion of reptiles by cestodes and enterohepatitis induced
by endoparasites cause death in Japanese reptiles within
a few days. The authors described 8 species of the
Morp hological and Biochemical Investigation... 101
Bull. Georg. Natl. Acad. Sci., vol. 7, no. 1, 2013
Bothridium genus including Bothridium pithonis. They
note that the reptiles brought out of the natural habitats
have already been infected with helminths. During post
mortem examination of Indian Rock python (Python
molurus) in the Madras Snake Park (India) [3] and Na-
tional Zoological Garden of Sri Lanka [4] B. pithonis
was found. Jacobson [5] mentions the pathological ef-
fect of Bothridium and Bothriocephalus on constrictor
snakes.
Information about cestoda invasions in pythons
in Georgia (Caucasus) is represented in the work of
Kamalov [6], who studied 5 dead specimens of the
reticulated pyth ons (Python reticulatus) in the
Tbilisi Zoological Park. One species of cestodas
fou nd in small in test ines was identi fi ed as
Salenophorus megalocephalus (Creplin, 1839),
syn.: Bothriocephalus pythonis (Retzias, 1829). The
description of the helminth is not given in this work.
It is only mentioned that the length of the longest
specimen was about 50 cm.
In January 2008 African Rock Python (Python
sebae) died in the Tbilisi Zoological Park after some
months of conveyance from Tanzania. The length of
the python was 3m 70cm . In the anterior part of a
small intenstine one specimen of cestoda was found.
We present additional data on this helminth on the
basis of morphological and biochemical studies.
Materials and methods
Morphometric description is made after investi-
gation of both fresh and fixed slides. The cestoda
was fixed in 70° ethanol after rinsing for morphologi-
cal study. In order to study the composition of the
scolex it was cleaned by equal mixture of lactic acid
and glycerin.
For biochemical study live tissues of the cestoda
were homogenized in 1:20 diluted distilled water and
centrifuged. The supernatant was collected and used
as enzyme extract. According to the method of
Willgerodt et al. [7] cholinesterase activity was de-
termined spect rophotometricall y using acetyl-
choliniodide at a concentration 1×10–²M as substrate.
The results were expressed as mkmol acetylcholine
liberated /mg protein/h. The protein content was de-
termined by the method of Lowry et al. [8], using
bovine serum albumin as standard. The obtained re-
sults were treated statistically [9].
Results and discussion
Morphological description. The color of the
cestoda body is milk white. The length of the helminth
about 960 mm that noticeably exceeds literature data
[2,4]. Such a large size of parasite is a common envi-
ronmental phenomenon and is caused by the low-
intensity of the invasion (one specimen).
The scolex (Fig. A,B) is unarmed, quite thick, con-
sisting of two spherical parts. The scolex with a me-
dial depression, the surface is smooth. There are two
deep adhesions on two elevated upper parts of the
scolex. The edges of the adhesions are surrounded
with well-developed, comparatively darker semicir-
cular muscle ridges, the openings of which are di-
Fig. Bothrid ium pithon is Blainville, 1824 (Origi nal). A) scolex, lateral view; B) scolex, apica l view.
102 Lali Murvanidze, Tsitsino Lomidze, Ketevan Nikolaish vili
Bull. Georg. Natl. Acad. Sci., vol. 7, no. 1, 2013
rected outside. The maximum width of the scolex is 6
mm, height 3 mm, distance between the depression
and the neck 2.5 mm, diameter of each spherical part
3 mm. The morphological characteristics of the scolex
of the studied cestode are similar to the description
of B. pithonis scolex given by Sawada and Kugi [2].
The neck is narrow and fairly long. The width of
the neck at the narrowest side is about 1.5 mm and
widens gradually.
The strobila is tape-shaped. The body is dense,
strongly muscular. Opaque segmentation is sharply
defined. The segments are broader than long. In one
cm of the strobila 13-14 divisions are counted. The
margins of the segments are serrate. Minimum width
of the segments of strobila 9–10 mm, length 0.5mm;
segments reach a maximum width at the anterior third
of the body and are equal to15 mm, length 0.9 mm.
The average width of the strobila – about 12-13 mm,
length 0.76mm.
Depressions are found along the central part of
the body. The reproductive system is located in one
row on the full length and is filled with eggs in differ-
ent developmental stages.
Eggs are of two types. First: small size, defective,
unfertilized eggs: length – 0.066 mm, width – 0.044
mm. Second classic oval eggs, with two-layered,
transparent, reddish envelope, which has a well-de-
veloped valve at one pole and a small, dark, subter-
minal thickening at the opposite one. Most eggs are
large in size: length-0.13 mm, width – 0.09-0.11 mm.
Such egg-size variation in one specimen of the para-
site is known in literature [10], indicating the defec-
tive structure of the ootype.
By morphological features, especially by struc-
ture and shape of the scolex the above-mentioned
cestoda belongs to the order Pseudophyllidea Carus,
1863, family Diphyllobothriidae Lühe,1910, genus
Bothridium Blainville,1824, Bothridium pithonis
Blainville,1824.
Biochemical data. Together with morphometric
description, biochemical investigation of the species
was also carried out. The enzyme cholinesterase,
which represents an important index of the nervous
system of the helminths, was studied.
For the first time in cestoda Bothridium pithonis
activity of cholinesterase was found, which was equal
on average to 96.3±11.12 mkmol of acetylcholine/
mg protein/h (n=8). This level probably determines
realization of the main function of the enzyme during
nervous-muscular transmission and represents the
index of the total movement activity of the tapeworm.
We have found differentiations in enzyme activity in
different fragments of the helminth. Activity fluctu-
ated from 45.98 to 142.0 mkmol of acetylcholine/mg
protein/ h. The obtained data show unequal distribu-
tion of the enzyme in strobila, which is proved by
literature data for other species of cestodes, particu-
larly for Raillietina tetragona from the chicken [11],
Bothriocephalus opsariichtydis from fishes [12] and
Ophiotaenia europaea from Natrix tesselata [13].
According to the above-mentioned data, the choline-
sterase, found in B. pithonis, indicates normally formed
nervous system and reflects the general regularity of
cestoda cholinesterase.
Conclusion
The cestoda B. pithonis could be one of the rea-
sons of P. sebae death at the Tbilisi Zoo. At the same
time, we agree with the reviews of the forequoted
authors [1,2] that reptiles being brought from the
natural habitats to the Zoos have already been in-
fected with parasites that cause their death or clear
the way for secondary infections. Improved quaran-
tine period of the reptiles and intensive parasitological
monitoring are required.
Suppression of the activity of the enzyme choline-
sterase during the screening of antihelminth drugs,
may be important for the prevention and healing of
snakes living in captivity.
The cestoda is preserved in the collection of the
Institute of Zoology of Ilia State University of Tbilisi.
Acknowledgement. The authors express their
gratitude to the veterinary service of Tbilisi Zoologi-
cal Park for giving us access to the cestoda.
Morp hological and Biochemical Investigation... 103
Bull. Georg. Natl. Acad. Sci., vol. 7, no. 1, 2013
parazitologia da helminTologia
cestoda Bothridium pithonis Blainville,1824
(Cestoda: Diphyllobothriidae)-is morfologiuri
da bioqimiuri Seswavla
l. murvaniZe*, c. lomiZe*, q. nikolaiSvili*
*
ilias saxelmwifo universiteti, zoologiis instituti, Tbilisi
(warmodgenilia akademiis wevris i. eliavas mier)
2008 wels, Tbilisis zooparkSi tanzaniidan Semoyvanil iqna afrikuli kldis piToni
(Python sebae Gmelin, 1788
)
, romelic erTi Tvis Semdeg daiRupa. mis wvril nawlavSi
aRmoCenili iqna cestoda (1 egz.). Bothridium pithonis Blainville, 1824. cestodis sigrZea
960 mm, maqsimaluri sigane-15 mm, rac mniSvnelovnad aRemateba aRniSnuli saxeobis
ganazomebs literaturuli monacemebis mixedviT. amis safuZvelze Catarda axali
morfometriuli aRwera. cestodis SeuiaraRebeli skoleqsi Sedgeba ori sferuli
nawilisagan, romelTa Soris aRiniSna mniSvnelovani CaRrmaveba. TiToeuli nawili mTavrdeba
boTriiT. skoleqsis maqsimaluri sigane aRwevs 6 mm, sigrZe _ 3 mm. kisris sigane yvelaze
viwro adgilas aris 1.5 mm. mkvrivi lentiseburi sxeuli mkafiodaa segmentirebuli.
sigrZis erTi santimetri moicavs 13-14 segments. segmentebis minimaluri sigane 9-10 mm,
sigrZe _ 0.5 mm. maTi maqsimaluri sigane modis strobilas pirvel mesamedze da aRwevs
15 mm, xolo sigrZe _ 0.9 mm. parazitis kvercxebi ori saxisaa: moumwifebeli _ sigrZe
0.066 mm, sigane 0.044 mm da momwifebuli, ganviTarebuli _ sigrZe 0.13 mm, sigane _ 0.09-0.11 mm.
masalis morfologiuri Seswavlis safuZvelze da literaturul monacemebTan
SedarebiT dadginda cestodis saxeobrivi kuTvnileba.
cestoda
B. pithonis-is
nervuli sistemis daxasiaTebisaTvis misi sxeulis homogenatebSi
bioqimiurad ganisazRvra fermenti qolinesTeraza, romlis aqtivoba gansxvavebuli aRmoCnda
cestodis sxeulis sxvadasxva fragmentSi da Seadgenda 45,98-dan 142,0-mde mkmol
acetilqolini/mg cilaze/sT. bunebrivi habitatebidan zooparkebSi Semoyvanili reptiliebi
saWiroeben karantins da parazitologiur monitorings.
104 Lali Murvanidze, Tsitsino Lomidze, Ketevan Nikolaish vili
Bull. Georg. Natl. Acad. Sci., vol. 7, no. 1, 2013
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Received October, 2012