Parasitology Molecular characterization of Moniezia

cambridge.org/par
denticulata (Rudolphi, 1810) and its distinction
from M. expansa infecting sheep and goats
raised in the north and north-western regions
Research Article of India
Cite this article: Kumar S, Kaur H (2023).
Molecular characterization of Moniezia
denticulata (Rudolphi, 1810) and its distinction Susheel Kumar and Harpreet Kaur
from M. expansa infecting sheep and goats
raised in the north and north-western regions Parasitology Laboratory, Department of Zoology, Panjab University, Chandigarh, India
of India. Parasitology 150, 831–841. https://
doi.org/10.1017/S003118202300063X
Abstract
Received: 22 February 2023 The tapeworms of Moniezia spp. are heteroxenous parasites and their adult forms occur in
Revised: 3 May 2023
Accepted: 26 June 2023
ruminants’ alimentary tract. They steal a significant portion of hosts’ nourishment initiating
First published online: 19 July 2023 monieziasis, thereby inflicting economic losses in animal rearing. Despite their high economic
importance, the molecular characterization and taxonomic status of these parasites have
Keywords: remained poorly understood. In the present study, cestodes were isolated from the sheep
cestode; domestic ruminants; India; M.
and goats’ intestines and were stained with Gower’s carmine. Upon careful evaluation of mor-
expansa; Moniezia denticulata; parasite
phological characters, 2 species Moniezia denticulata and Moniezia expansa were identified.
Corresponding author: The genomic DNA was extracted and polymerase chain reaction (PCR) amplified targeting
Harpreet Kaur; regions of mitochondrial cytochrome c oxidase subunit 1 (cox1), small subunit ribosomal
Email: [email protected] RNA (SSU rRNA) and internal transcribed spacer 1–5.8S rRNA (ITS1–5.8S rRNA) genes fol-
lowed by sequencing. The partial sequences of cox1, SSU rRNA and ITS1–5.8S rRNA genes of
M. denticulata generated in the present study revealed that even though they share high simi-
larities with M. benedeni (93.2% cox1; 92.6% SSU rRNA; 84.70% ITS1–5.8S rRNA) and M.
expansa (88.85% cox1; 92.27% SSU rRNA; 81.70% ITS1–5.8S rRNA), they are not identical
to them. In the maximum likelihood phylogenetic trees, M. denticulata and M. expansa con-
sistently appeared as distinct species from each other. The high values of pairwise divergence
between these 2 species collected in the present study confirmed their separate identity. The
present study reports the first molecular characterization of M. denticulata with reference to
M. expansa infecting sheep and goats in India.

Introduction
It is well recognized that livestock has many roles in the farm ecosystem and contributes
greatly to human livelihoods in developing economies. The small domestic ruminants,
sheep and goats, constitute an important part of livestock. They are raised to suffice the
need for meat, skin, wool, manure and milk. The incidences of monieziasis, a gastrointestinal
disorder, have been a potent cause of declined health of ruminants, thereby inflicting consid-
erable economic losses in the domestic ruminant raising (Bashtar et al., 2011; Diop et al., 2015;
Guo, 2017). The mild pathogenicity of monieziasis is associated with a moderate infection;
however, heavy infection often leads to adverse clinical manifestations such as pot-belly,
poor growth rate, diarrhoea, anaemia, intestinal pathology, poor quality of wool, fleshless
and even death of the ruminant host (Fagbemi and Dipeolu, 1983; Zhao et al., 2009; Yan
et al., 2013). The disease monieziasis is caused by the infection of parasitic anoplocephaline
tapeworms of Moniezia spp. belonging to the family Anoplocephalidae of the class Cestoda.
These tapeworms parasitize the intestine of definitive hosts belonging to the orders
Artiodactyla, Perissodactyl and Primates (Ohtori et al., 2015). These cestodes show the world-
wide distribution and have an indirect mode of life cycle involving oribatid mites as an inter-
mediate host (Nagarajan et al., 2022).
In 1891, Blanchard gave the first account of the genus Moniezia in which he included 11
species. The concept was elaborated by Moniez in 1878 by including 2 more members in the
group (Denegri et al., 1998). On the basis of the absence or presence of the interproglottidal
© The Author(s), 2023. Published by glands, Stiles and Hassall (1893) deducted the number of species to 8 and classified them into
Cambridge University Press. This is an Open 3 groups (i) denticulate group without interproglottidal glands, (ii) expansa group comprising
Access article, distributed under the terms of
sac-like interproglottidal glands and (iii) plannissina group in which the interproglottidal
the Creative Commons Attribution licence
(http://creativecommons.org/licenses/by/4.0/), glands show linear arrangement. Baer (1927) in his monographic study of the genus
which permits unrestricted re-use, distribution Moniezia included 6 species, M. denticulata (Rudolphi, 1810), M. expansa (Moniez, 1810),
and reproduction, provided the original article M. benedeni (Moniez, 1879), M. rugosa (Diesing, 1810), M. pallida (Moning, 1926) and M.
is properly cited. trigonomorpha (Stiles and Hassall, 1893). The genus Moniezia is divided into 3 subgenera
in the classical taxonomical scheme proposed by Skrjabin and Schulz in 1937 namely
Moniezia, Blanchariezia and Baeriezia differentiated by the presence and type of interproglot-
tidal glands (Haukisalmi et al., 2018). In 1982, a new species, Moniezia sichuanensis was
described from a specimen obtained from wild musk deer. The new species was distinct
832 Susheel Kumar and Harpreet Kaur

from the other described species of Moniezia by the saw tooth- min, dehydrated in ascending grades of the alcohol, i.e. 30, 50 and
shaped interproglottidal glands, the thick vagina and the absence 70% each for about 20 min, respectively, and differentiated in acid
of a cirrus spine (Xu et al., 2018). The species M. expansa is ethanol (100 mL of 70% ethanol + 2 mL of concentrated HCl)
known to infect goats and sheep, M. benedeni infects cattle and (Ndom et al., 2018). Further dehydration was done in 70, 90
M. denticulata occurs in sheep, goats and cattle. and 100% for 15–20 min each. Xylene was used to clear the spe-
In India, the incidence of M. denticulata infection was cimen for 30 min and clove oil (for 2 h) was also used. Then spe-
recorded by Bhalerao in (1935) from some localities. Saxena cimens were mounted in dibutylphthalate polystyrene xylene
and Deo (1964) also gave the morphological account of M. den- (DPX), examined and photographed under Jenoptik progress
ticulata based on the specimen recovered from sheep in Magnus photographic unit, Radical Stereo Zoom microscope
Bareilly, India. Other than India, Bashtar et al. (2011) reported and LAS version V4.1 (Leica Microsystems, CMS GmbH,
an 8.5% prevalence of M. denticulata in Cairo, Egypt. Wetzlar, Germany) photographic unit. Line drawings of the para-
In many recent reports on anoplocephalid cestodes, it has been sites were made using projection microscope and camera lucida
proposed that there is a need of molecular characterization and attached to the compound microscope. Measurements were
re-evaluation of the taxonomic status of species (Diop et al., taken in the software namely, LAS V4.1 using the microscope
2015; Guo, 2017; Ndom et al., 2018, 2019). Despite some recent Leica DM3000 (Leica Microsystems) and Radical, ProgRes
efforts which have been made to describe the Moniezia spp. at (JENOPTIK Optical System Cmbh, version 2.10.0.1). The stained
the molecular level (Chilton et al., 2007; Nguyen et al., 2012; proglottids and scolices have been deposited in the museum of the
Shalaby and Amer, 2012; Yan et al., 2013; Ohtori et al., 2015; Department of Zoology, Panjab University, Chandigarh, India
Thosar et al., 2015; Kaur and Gupta, 2019; Tam et al., 2020; (Catalogue No: Md/001/m1/2021–22; Md/001/m2/2021–22;
Nagarajan et al., 2022), but there is no molecular description of Md/001/m3/2021–22; Md/001/m4/2021–22; Me/001/m1/2021–
M. denticulata available. Although Shalaby and Amer (2012) in 22; Me/001/m2/2021–22; Me/001/m2/2021–22; Me/001/m3/
their attempt to identify Moniezia spp. from Saudi Arabia got 2021–22; Me/001/m4/2021–22).
some morphological indications of the occurrence of M. denticu-
lata, but could not successfully describe it due to lack of molecular
DNA isolation, amplification and sequencing
data. Therefore, keeping in mind the need for molecular evidence,
we provide the first molecular identification and phylogenetic The immature fragments of cestodes, which were fixed in ethanol
analyses of M. denticulata and its comparison with M. expansa (95–99.5%) at −20°C immediately following the enough normal
from the north and north-western region of India based on mito- saline wash, were used to extract the genomic DNA for molecular
chondrial cytochrome c oxidase subunit 1 (cox1) gene, small sub- analysis. The Qiagen’s DNeasy tissue kit was used to isolate the
unit ribosomal RNA (SSU rRNA) gene and internal transcribed genomic DNA following the tissue DNA extraction protocol pre-
spacer 1–5.8S rRNA (ITS1–5.8S rRNA) gene sequences accom- scribed by the manufacturer. To determine the quality and quan-
panying the morphological affinities. tity of the extracted genomic DNA, agarose gel electrophoresis
and nanodrop spectrophotometer analysis were performed,
respectively. The extracted genomic DNA which had desired qual-
Materials and methods ity and quantity was used as the template for polymerase chain
Sample collection reactions (PCRs). For the amplification of SSU rDNA gene,
primers were designed in the present study; left primer SSU
In the present study, fresh gut (n = 480) of slaughtered sheep (n = mbF: 5′ -ACGGGTCCTTCAAATGTCTG-3′ and right primer
67) and goat (n = 413) was availed from the abattoir located at SSU mbR: 3′ -GGTCTGACTCGTTGACAC-5′ with the aid of
Industrial Area, Chandigarh, India where slaughtering is done Integrated DNA Technologies’s (IDT’s) oligo analyser tool
for consumption purpose. On average, the sheep and goats (https://sg.idtdna.com/pages/tools/oligoanalyzer) and Primer 3 soft-
slaughtered were 2–5 years old. These flocks of sheep and goats ware (Untergasser et al., 2012) using the SSU rDNA gene sequences
were raised in various localities belonging to north-western and of Moniezia spp. available in the National Centre for Biotechnology
northern regions of India. During the period from October Information (NCBI). The newly designed pair of primes yielded the
2018 to September 2021, the gut infected with cestodes was care- PCR product which had a size of 1893 bp. To amplify a portion of
fully transported to laboratory in a biomedical-disposable bag and cox1 gene, the left and right primers MoCox1F (5′ -CTGAGT
dissected. The anoplocephalid cestodes were isolated from GTTTTCAAAACATTTAG-3′ ) and MoCox1R (5′ -AAGCATGAT
infected gut and were washed with saline solution (0.85%) to GCAAAAGGCA-3′ ) (Diop et al., 2015), respectively, were referred.
clean. The immature portion of strobila of each worm was cut The amplification of the ITS1–5.8S region of ribosomal DNA was
into small pieces and fixed in 95–99.5% alcohol at −20°C for achieved by using up the left primer ITS1-F (5′ -GCTGCTACCC
molecular studies. The remaining portion of worm’s body was GCATGATGTT-3′ ) and the right primer ITS1-R (5′ -GGC
relaxed in freshly prepared 4% formalin solution (hot). Then, AAGCCTATAGCCGCAAT-3′ ) (Nguyen et al., 2012) for conven-
relaxed strobila of each worm was gently intertwined on paper tional PCRs.
rolls ensuring the morphology was not altered. Subsequent pres- PCRs were carried out in a reaction mixture having a total vol-
ervation for morphological studies was done by putting the stro- ume of 25 μL containing 2.5 μL 10× PCR buffer including 2 mM
bila in 4% formalin solution till the use. Out of total infected gut MgCl2, 2 μL of each primer of 10 μM concentration (20 pmol of
dissected in laboratory, the infection of M. denticulata and each primer in final volume of reaction mixture), 2 μL of deoxy-
M. expansa was found in 6.7 and 24% gut, respectively. Other nucleotide triphosphates (dNTPs) of 0.2 mM, 2.5 μL DNA tem-
guts were having the infection of anoplocephalid tapeworms plate, 0.6 units of Taq polymerase and 13.8 μL molecular grade
other than Moniezia spp. In total, 16 M. denticulata and 51 nuclease-free water. PCR amplification’s 35 cycles, having steps
M. expansa were collected. of denaturation (at 94°C) for 30 s, annealing (at 55.7°C for cox1
gene, 57.3°C for SSU rRNA gene and ITS1–5.8s rRNA gene)
for 30 s and extension (at 72°C) for 60 s (90 s for SSU rRNA
Permanent stained preparations and line drawing
gene), were preceded by an initial denaturation step at 94°C for
The proglottids fixed in 4% formalin were washed in fresh water 5 min. A final extension was carried at 72° C for 5 min. PCR pro-
thoroughly, stained in Gower’s carmine (Gower, 1939) for 50–60 ducts were visualized by 1.7% agarose gel electrophoresis.
Parasitology 833

Amplified products of expected size were purified by gel extrac- (Figs 1A–D and 2A–D; Table 1). Scolex small, spherical, distin-
tion and sequences were determined by chain termination guishable from neck, measuring 590–621 (525.8) × 538.3–558
method (Sanger et al., 1977). Internal primers, designed in the (538.1) μm, unarmed, 4 suckers of dimension 183–222.5
present study, MbF: 5′ -TGCGGGACTCATTTAAGAGG-3′ and (205.9) × 172–177 (174.7) μm. Scolex followed by short neck
MbIR: 3′ -AACTA CCACCACGGATCGTC-5′ were also used to which is continuous with strobila. Strobila craspedote but vel-
sequence the amplicons of SSU rRNA gene. The sequences lum indistinguishable, immature proglottids begin from the
were assembled, inspected for vague bases and contigs were gen- neck, 164–294 (229) × 2390–2995 (2692.5) μm, mature proglot-
erated from the multiple sequences for each molecular marker in tids 6–11 times broader than long, 1409.5–1594 (1523.7) ×
BioEdit and manually. The sequences determined in the study, for 8901–9225 (9023.2) μm.
each molecular marker, were deposited into GenBank of NCBI Mature proglottids with double male and female reproductive
and accession numbers assigned to sequences were obtained. sets, 290–340 (310) testes scattered in posterior half of medulla of
For the phylogenetic analyses, the sequences of the genus proglottids, nearly round in shape, measuring 62.2–90.1 (76.15)
Moniezia available in NCBI, for each molecular marker in the μm in diameter. Cirrus sac 169–195 (174.3) × 199.5–252 (226.4)
present study, were downloaded along with other comparable μm, anterior to the vagina, located near to lateral margin running
sequences of closely related species. Multiple sequence alignments through cup-shaped genital atrium 21.95–173.8 (139) × 107.4–
of respective molecular marker datasets were carried out using 253.4 (167.3) μm. Vas deferens narrow tube, convoluted having
MUSCLE (Multiple Sequence Comparison by Log-Expectation) diameter of 215–309 (254.1) μm. Cirrus slender, either somewhat
program in MEGA 11(Tamura et al., 2021) with parameters set curved or straight, protruding out occasionally, 43–67 (57.4) ×
to default and the alignments were further inspected for any non- 18–34 (23.8) μm.
conformity and were manually corrected, when needed. Each female reproductive set includes 1 ovary 981.4–1001.7
Maximum likelihood (ML) phylogenetic trees for sequences of (993.3) μm, a prominent receptaculum seminis 418–421
each molecular marker were rebuilt using the best-fit model stated (418.1) × 139–141 (139.3) μm, a ductus vagina 362.2–371.6
in results. The bootstrapping method, with 1000 replicates, was (367.2) × 21.9–24.92 (23.45) μm. Vitellaria lobate, 99–109
used to test the robustness of the trees. (104) × 106–124 (115.12) μm, uterus persistent, saccate or
reticulate. Interproglottidal glands absent in all proglottids,
common genital pores 126–199 (155.76) μm, occurring in geni-
Results tal atrium situated at the middle of each lateral margins. Most
of the internal organs disintegrate partially or fully in gravid
Morphological analysis of M. denticulata proglottids. Longitudinal osmoregulatory excretory canals
Worms whitish in appearance, 3–3.5 m in length and bulky stro- run through the entire length of strobila, occur on either
bila with numerous immature, mature and gravid proglottids side of the proglottids, narrow, 120–240 (180) μm diameter.

Figure 1. Photomicrograph of Moniezia denticulata. (A) Scolex, (B) mature proglottids, (C) magnified portion of a mature proglottid showing morphological details,
(D) eggs.
834 Susheel Kumar and Harpreet Kaur

Figure 2. Line drawings of Moniezia denticulata. (A) Scolex, (B) mature proglottid, (C) gravid proglottid, (D) egg. O, ovary; T, testis; EX, excretory canal; GN, gonopore;
SC, sucker; C, cirrus; V, vagina; RS, receptaculum seminis; GA, genital atrium; VD, vas deference; H, hooks; ONC, onchosphere; OM, onchosphere membrane; CAP,
capsule; PA, pyriform apparatus; OE, outer envelope; IE, inner envelope.

Table 1. Morphometric comparison of Moniezia denticulata and Moniezia expansa

Characters Moniezia denticulata (present study) Moniezia expansa (present study)

Scolex (μm) 590–621(525.8) × 538.3–558 (538.1) 523–565 (551.6) × 580–625 (613.8)

Sucker (μm) 183–222.5 (205.9) × 172–177 (174.7) 222–255 (236.8) × 178–202 (192.2)
Immature proglottids 164–294 (229) × 2390–2995 (2692.5) 160–258 (203.2) × 2105–2916 (2590.4)
Mature proglottids (μm) 1409.5–1594 (1523.7) × 8901–9225 (9023.2) 622–692 (674.2) × 3998–4201 (4072.8)
Gravid proglottids (mm) 0.7–1 (0.8) × 1.1–1.8 (1.5) 0.4–0.9 (0.7) × 0.9–1.7 (1.3)
Genital atrium (μm) 21.95–173.8 (139) × 107.4–253.4 (167.3) 19.2–160.5 (89.9) × 101.3–247.6 (174.5)
Cirrus pouch (μm) 169–195 (174.3) × 199.5–252 (226.4) 166–176.5 (168.5) × 199–202.6 (300.7)
Testes (diameter) (μm) 62.2–90.1 (76.15) 32.8–48 (40.4)
Vas deferens (μm) 215–309 (254.1) 260–308 (294)
Cirrus (μm) 43–67 (57.4) × 18–34 (23.8) 53–62.1 (57.5) × 34–53 (47.1)
Interproglottidal glands (μm) Absent 45–55.5 (50.3) × 42–79.2 (60.6)
Receptaculum seminis (μm) 418–421 (418.1) × 139–141. (139.3) 80.5–-90.1 (85.8) × 50.3–70.6 (60.45)
Vagina (μm) 362.2–371.6 (367.2) × 21.9–24.92 (23.45) 532–567 (172) × 40.1–57.6 (46.86)
Ovary (diameter) (μm) 981.4–1001.7 (993.3) 495–538 (513) × 292–372 (335.16)
Osmoregulatory canal (μm) 120–240 (180) 392–452 (410.4)
Egg (diameter) (μm) 68.01–75.2 (71.6) × 69–78.1 (74.2) 39.01–51.2 (48) × 47–59 (54.5)

Eggs cuboidal, about 68.01–75.2 (71.6) × 69–78.1 (74.2) μm, and 4A–D; Table 1). Scolex small, distinguishable but not
with thick capsule, contains an embryo, oncosphere with demarcating very clearly from neck, measuring 523–565
embryonic hooks 2.5–6 (4.25) μm, pyriform apparatus ending (551.6) × 580–625 (613.8) μm, unarmed, 4 wide suckers 222–255
in disc. (236.8) × 178–202 (192.2) μm. Short neck 287–312 (295.6) μm fol-
lowing the scolex, not marking off sharply from the strobila.
Strobila craspedote with very prominent vellum, mature proglottids
Morphological analysis of M. expansa
10–11 times broader than long, 622–692 (674.2) × 3998–4201
Worms milky white, 2–3.5 m in length, bulky strobila with (4072.8) μm, immature proglottids begin from the neck, 160–258
numerous immature, mature and gravid proglottids (Figs 3A–D (203.2) × 2105–2916 (2590.4) μm.
Parasitology 835

Figure 3. Stained whole mount of Moniezia expansa. (A) Scolex, (B) mature proglottids, (C) magnified view showing morphological details of a mature proglottid,
(D) eggs.

Figure 4. Line diagrams showing the morphological details of Moniezia expansa. (A) Scolex, (B) mature proglottid, (C) gravid proglottid, (D) egg. O, ovary; T, testis;
EX, excretory canal; GN, gonopore; SC, sucker; C, cirrus; V, vagina; RS, receptaculum seminis; GA, genital atrium; ING, interproglottidal glands; VD, vas deference; H,
hooks; ONC, onchosphere; OM, onchosphere membrane; CAP, capsule; PA, pyriform apparatus; OE, outer envelope; IE, inner envelope.

Double reproductive set, 210–260 (235) testes scattered in diameter 260–308 (294) μm. Cirrus very slender, slightly curved
most of the medullary region of mature proglottids, nearly or straight, 53–62.1 (57.5) × 34–53 (47.1) μm.
round in shape, measuring 32.8–48 (40.4) μm. Cirrus pouch Female reproductive set with 2 ovaries of 495–538 (513) ×
measuring 166–176.5 (168.5) × 199–202.6 (300.7) μm, cylindrical, 292–372 (335.16) μm, a receptaculum seminis 80.5–90.1
running through genital atrium, located anterior to the vagina. (85.8) × 50.3–70.6 (60.45) μm, a narrow ductus vagina measuring
Genital atrium, cup-shaped, 19.2–160.5 (89.9) × 101.3–247.6 532–567 (172) × 40.1–57.6 (46.86) μm, uterus persistent reticulate.
(174.5) μm. Vas deferens narrow tube, convoluted having Interproglottidal glands present in all proglottids, rosette-like,
836 Susheel Kumar and Harpreet Kaur

arranged in a linear row, 18–29 (23) in number, measuring 45– study showed more than 98% similarity (query coverage more
55.5 (50.3) × 42–79.2 (60.6) μm. Vitelaria lobate, 98–103 than 99%) with the respective gene sequences of M. expansa avail-
(98.5) × 97–114 (103.25) μm, common genital pores 116–189 able on online database of NCBI. To conduct the phylogenetic
(145) μm situated at the middle of each lateral margins. analyses, the sequences generated in the present study and com-
Longitudinal osmoregulatory excretory canals wide, twisted, occur parable sequences available from NCBI GenBank for respective
on either sides of the proglottids, run through the entire length molecular markers (cox1, SSU rRNA gene, ITS1–5.8S rRNA
of strobila, measuring 392–452 (410.4) μm in diameter. Eggs nearly gene) were used. In phylogenetic analysis, the sequences down-
triangular or pear-shaped measuring 39.01–51.2 (48) × 47–59 loaded from NCBI GenBank and sequence generated in the pre-
(54.5) μm with thick capsule, contains an oncosphere, embryonic sent study were aligned and non-comparable sequences flanking
hooks 2.3–6 (4.2) μm, pyriform apparatus present and ends in disc. the sequences of molecular marker under investigation were
removed. The ML trees were constructed using 1000 bootstrap
replications and Kimura 2-parameter model (Kimura, 1980)
Molecular and phylogenetic analysis
with G + I (γ distributed with Invariant sites) rate and pattern
The primers employed in the present study for the PCR amplifi- parameter, and all positions containing gaps and missing data
cation of SSU rDNA gene successfully amplified the amplicons were subjected to partial deletion. The phylogenetic analysis of
having a size of about 1893 bp (Fig. 5A) and the same were cox1 consisted of 22 nucleotide sequences and there were a total
sequenced by employing internal primers in addition to those pri- of 1590 positions in the final dataset. The codon positions
mers which were used for PCR amplification. After processing, included were 1st + 2nd + 3rd + non-coding, and an ML tree
the final sequence of size 1751 bp (M. expansa) and 1816 bp with the highest log-likelihood value of −5560.70 was generated.
(M. denticulata) was deposited in NCBI GenBank and accession In the ML tree of cox1 gene sequences (Fig. 6), the morpho-
numbers OM296991.1 (M. expansa) and OM296990.1 (M. denti- type identified as M. expansa in the present study nested within
culata) were obtained. the clade formed by sequences of M. expansa deposited in
The primers referred for cox1 and ITS1–5.8S rRNA gene GenBank from various regions of the world, while the sequence
yielded the amplicons of size about 1596 bp (Fig. 5B) and 749 of morphotype identified as M. denticulata formed a distinct
bp (Fig. 5C), respectively. After sequencing and processing, the branch with the clade of sequences of M. benedeni available
final sequences for cox1 and ITS1–5.8S rRNA gene of M. expansa from GenBank. The number of base substitutions per site
and M. denticulata have sizes of 1574 bp (accession number: between sequences of M. denticulata and M. expansa was high
OQ134466.1) and 1565 bp (accession number: OQ134465) and ranging from 0.12 to 0.13 and the same ranged from 0.7 to
731 bp (accession number: OQ133455.1) and 730 bp (accession 0.8 between M. denticulata and M. benedeni suggesting closer
number: OQ133454.1), respectively. relationship but distinct taxa. The overall rates of various transi-
The sequences of cox1, SSU rRNA, ITS1–5.8S rRNA genes of tional and transversionsal base substitutions were 17.37 and
M. denticulata generated in the present study showed 88.85, 92.27 3.82, respectively, with estimated 2.28 transition/transversion
and 81.70% similarity with cox1, SSU rRNA, ITS1–5.8S rRNA bias (R).
gene sequence of M. expansa (accession no. OQ134466.1) gener- The ML tree, computed including 15 SSU rRNA gene
ated in the same study with query coverage of 98, 92 and 78%, sequences having 1913 positions in the final dataset with highest
respectively, and, 93.2, 92.6 and 84.70% similarity with M. bene- log-likelihood value −4675.45, showed similar pattern wherein
deni sequences present on online database of NCBI with more M. denticulata formed a distinct branch (Fig. 7), but with the
than 99% query coverage. The sequence of M. expansa generated clade formed by M. expansa sequences. The number of base sub-
for cox1, ITS1–5.8S rRNA and SSU rRNA gene in the present stitutions per site between sequences of M. denticulata and

Figure 5. Agarose gel electrophoresis showing the PCR amplifications. (A) SSU rRNA gene (lanes 1–4: M. denticulata; lanes 5–7: M. expansa). (B) cox1 gene (lanes 2, 5
and 6: M. denticulata; lanes 8–11: M. expansa). (C) ITS1–5.8S rRNA gene (lanes 4 and 7: M. denticulata; lane 10: M. expansa). Letter ‘L’ represents DNA ladder, i.e. size
marker.
Parasitology 837

Figure 6. Phylogenetic tree of Moniezia spp. using cox1 gene sequences and generated by using the maximum likelihood (ML) method. Numbers preceding the
generic name are accession numbers in the GenBank database and the values of each node are bootstrap percentages (bootstrap percentages above 50% are
displayed). **Out-group; *species isolated in the present study.

Figure 7. Maximum likelihood (ML) phylogenetic tree of Moniezia spp. built using SSU rRNA gene sequences. Numbers preceding the generic name are accession
numbers in the GenBank database and the values of each node are bootstrap percentages (bootstrap percentages above 50% are displayed). **Out-group; *species
isolated in the present study.
838 Susheel Kumar and Harpreet Kaur

Figure 8. ITS1–5.8S rRNA gene sequence-based phylogenetic tree of Moniezia spp. generated by the maximum likelihood (ML) method. Numbers preceding the
generic name are accession numbers in the GenBank database and the values of each node are bootstrap percentages (bootstrap percentages above 50% are
displayed). **Out-group; *species isolated in the present study.

M. expansa was 0.05 and 0.07–0.08 between M. denticulata and molecular level. To date, the molecular description and validation
M. benedeni. The rates of various transitional and transversionsal of M. denticulata has remained very obscure.
base substitutions were 16.55 and 4.23, respectively, and estimated In the present study, the description of 2 anoplocephaline ces-
transition/transversion bias (R) was 1.96. todes, M. denticulata and M. expansa isolated from sheep and
In the evolutionary analysis based on ITS1–5.8S rRNA nucleo- goats originating from various areas of north and north-western
tide sequences, the ML tree (Fig. 8) having highest log-likelihood India, is attempted at the molecular level using phylogenetic
value of −2281.86 was generated by including 18 nucleotide approach. Upon careful evaluation of morphological characters,
sequences with a total of 510 positions in the final dataset. The 2 types of materials (morphotype) were identified in the present
number of base substitutions per site between sequences of M. den- study. One morphotype, which showed the presence of a row of
ticulata and M. expansa ranged from 0.14 to 18 and 0.15 to 0.16 rosette-like interproglottidal glands along the posterior margin
between M. denticulata and M. benedeni. The rates of various tran- of proglottids, was identified as M. expansa and the other mor-
sitional and transversionsal base substitutions were 18.80 and 3.10, photype in which interproglottidal glands were completely absent
respectively, and estimated transition/transversion bias (R) was 3.03. throughout the strobila was identified as M. denticulata. Other
An ML phylogenetic tree (Fig. 9) of Moniezia spp. and other evident differences in morphological characters were the uni-
anoplocephalid genera was also built using cox1 genes sequences formly scattered testes anterior and posterior to ovary in paren-
available from NCBI database and cox1 gene sequences of chyma of the specimens which possessed rosette-type
Moniezia spp. generated in the present study. This analysis interproglottidal glands. The testes were distributed in posterior
involved 36 nucleotide sequences. All positions containing gaps half of proglottids of the specimen which showed complete lack-
and missing data were eliminated. There were a total of 323 posi- ing of interproglottidal glands. The receptaculum semini were
tions in the final dataset. more prominent, scolex very distinguishable advocating clear
demarcation from neck and even though proglottids were craspe-
tode, vellum was hardly visible in the proglottids of the specimen
Discussion
who lacked interproglottidal glands. Similarly, there were marked
The tapeworms of Moniezia spp. are very common gastrointes- differences in the osmoregulatory canals which were wider and
tinal helminth parasites in domestic ruminants and have excep- twisted in the specimens possessing interproglottidal glands.
tionally bulky strobila reaching up to 4 m in length. The Besides the structural differences, there were morphometric differ-
continuously proliferating neck adds new proglottids to the ever ences as well (Table 1). The eggs also showed the dissimilarities;
increasing strobila, and for this, it needs continuous supply of they were triangular or pyriform and smaller [39.01–51.2 (48) ×
nutrients. To meet its demand for nutrients, it steels host’s food 47–59 (54.5) μm] in the case of specimen possessing interproglot-
and certainly deprives the host from much needed nutrient. Till tidal glands and cuboidal and larger [68.01–75.2 (71.6) × 69–78.1
now, as many as 12 species have been reported from various (74.2) μm] in the case of specimen lacking interproglottidal
hosts (Ohtori et al., 2015), and in domestic ruminants, only 2 spe- glands. The receptaculum seminis were more prominent in the
cies M. benedeni and M expansa have been described widely at specimens without interproglottidal glands. These dissimilarities
Parasitology 839

Figure 9. ML phylogenetic tree of anoplocephalid tapeworms showing evolutionary relationship of Moniezia spp. with other related genera. The tree was built using
cox1 gene sequences. Numbers preceding the generic name are accession numbers in the GenBank database and the values of each node are bootstrap percen-
tages (bootstrap percentages above 50% are displayed). **Out-group; *species isolated in the present study.

depict evolutionary events from ancestral condition to the derived interproglottidal glands are same. They depicted that the speci-
condition. The present results contradict the assumption of mens lacking the interproglottidal glands (designated as
Chilton et al. (2007), inferred from multi-enzyme electrophoresis ‘unknown’ in their study) shared alleles at 13 loci with M. bene-
study, that the specimens of Moniezia with or without deni (designated as ‘Bt6–9’ in their study) and 15 loci with
840 Susheel Kumar and Harpreet Kaur

M. expansa (designated as ‘Ov2–4’ in their study). While ques- is a taxonomically valid species and certainly exhibits very close
tioning the reliability of interproglottidal glands as a diagnostic sister relationship with the species M. expansa and M. benedeni
character, they suggested assigning of the individuals without infecting the sheep and goats and cattle, respectively. The genetic
interproglottidal glands to one of the morphospecies (Bt6-9 or disparity involving partial sequences of 3 molecular markers
Ov2-4). However, in the present study, it was observed that the (cox1, SSU rDNA and ITS1–5.8S rDNA) was consistently
conditions of the absence and presence of interproglottidal glands observed between the 2 anoplocephaline species collected in the
showed synchrony with other features such as difference in egg present study. In the phylogenetic analysis, these tapeworms
shape, pattern of testes distribution, etc., as discussed earlier. clearly appeared as distinct species within the same genus.
The differences were also reflected in molecular results as dis- Undoubtedly, molecular markers are a powerful tool for distin-
cussed ahead. The present observations are in agreement with guishing closely resembling individuals or isolates with overlap-
the key to the species of Moniezia furnished by Bhalerao (1935) ping morphological features, but the careful evaluation of
in a monographic record of helminth parasites from India, morphological characters revealed disparity between the cestodes
wherein he depicted Moniezia tapeworms without interproglotti- identified as M. denticulata and M. expansa in the present study
dal glands, with linear interproglottidal glands and with saccular advocating that they are separate species, as traditionally acknowl-
or rosette interproglottidal glands as M. denticulata, M. benedeni edged, of the same genus. Meanwhile, the phylogenetic trees and
and M. expansa, respectively. Similarly, Saxena and Deo (1964) morphological observations show their clear genetic distinctive-
recorded the occurrence of M. denticulata in sheep from ness, their mode of parasitizing domestic ruminants also suggests
Bareilly, India, during a survey and mentioned that its eggs that these species may have diverged as a consequence of a trans-
were cuboidal and larger than the eggs of M. expansa, and inter- fer from caprine and ovine to bovine or vice versa. The present
proglottidal glands were lacking. The same are in agreement with study is the first report on molecular characterization of M. den-
the findings of the present study. The monographic study done by ticulata with reference to M. expansa infecting sheep and goats
Baer (1927) also mentioned the M. denticulata as one of the spe- raised in north and north-western regions of India. The study
cies of the genus Moniezia (Denegri et al., 1998). would certainly, directly or indirectly, facilitate future identifica-
The molecular analysis of the present study revealed that even tion of these species and improved control of monieziasis.
though sequences of M. denticulata share high similarities with
M. benedeni (93.2% cox1; 92.6% SSU rRNA; 84.70% ITS1–5.8S Data availability statement. The dataset generated in this study is available
from corresponding author upon reasonable request.
rRNA) and M. expansa (88.85% cox1; 92.27% SSU rRNA;
81.70% ITS1–5.8S rRNA), they certainly are not identical to Acknowledgements. The authors are thankful to the Department of Zoology,
sequences of either species. The phylogeny of cox1 gene clearly Panjab University, Chandigarh, India where the research work was done.
stated that M. denticulata is a distinct lineage existing somewhere
between the closely related M. expansa and M. benedeni lineages. Author contributions. Research work and writing the research paper: ori-
ginal draft preparation was done by Susheel Kumar. The research work was
The high values of pairwise divergence between M. denticulata
supervised, draft reviewed and edited by Harpreet Kaur.
and M. expansa (12–13%) collected in the present study depict
that they are different taxa of the same genus and this disparity Financial support. The present study was supported by financial assistance
is comparable to the genetic divergence between M. expansa from the University Grants Commission (UGC), Govt. of India as a fellowship
and M. benedeni (12.8–13.2%) reported by Diop et al. (2015). (JRF and SRF) under grant no. F.16-6(DEC.2016)/2017(NET). Recipient:
Furthermore, Ndom et al. (2018a) recorded 2.9% maximum pair- Susheel Kumar.
wise divergence between sequences of SSU rDNA of 2 taxonom-
Competing interests. The authors declare that they have no known compet-
ically valid species of anoplocephalid cestodes namely Thysaniezia
ing financial interests or personal relationships that could have appeared to
ovilla and Thysaniezia connochaeti, which is again comparable influence the work reported in this paper.
even less than the values of maximum pairwise divergence in
SSU rDNA (5% between M. denticulata and M. expansa; 7–8% Ethical standards. Ethical approval was obtained from the Institutional
between M. denticulata and M. benedeni) computed in the pre- Animal Ethics Committee (IAEC) of Panjab University, Chandigarh, India
sent study. To confirm the outcomes of cox1 and SSU rDNA, (approval No. PU/45/ 99/CPCSEA/IAEC/481).
third molecular marker ITS1–5.8S rDNA was also employed in
the present study. The values of pairwise divergence involving References
ITS1–5.8S rDNA sequences were also very high; 14–18% between
M. denticulata and M. expansa and 15–16% between M. denticu- Baer JG (1927) Monographie des cestodes de la famille des Anoplocephalidae.
lata and M. benedeni suggesting that the variations were not intra- Paris: Les Presses universitaires de France; Londres: Dulau.
specific but interspecific. Bashtar A-R, Hassanein M, Abdel-Ghaffar F, Al-Rasheid K, Hassan S,
Mehlhorn H, Al-Mahdi M, Morsy K and Al-Ghamdi A (2011) Studies
The ML trees of SSU rRNA and ITS1–5.8S rRNA genes con-
on monieziasis of sheep I. Prevalence and antihelminthic effects of some
firmed the phylogenetic differences between M. denticulata and plant extracts, a light and electron microscopic study. Parasitology
M. expansa found in the present study. However, there were Research 108, 177–186.
some variations in topology among ML trees of cox1, SSU Bhalerao GD (1935) Helminth Parasites of the Domesticated Animals in India.
rRNA and ITS1–5.8S rRNA genes which could be attributed to Delhi: Manager of Publications.
the fact that some of the species of Moniezia included in the ana- Chilton NB, O’Callaghan MG, Beveridge I and Andrews RH (2007) Genetic
lysis do not have comparable sequences on online database of markers to distinguish Moniezia expansa from M. benedeni (Cestoda:
NCBI for all 3 molecular markers employed in the present inves- Anoplocephalidae) and evidence of the existence of cryptic species in
tigation. But M. denticulata consistently appeared as a distinct Australia. Parasitology Research 100, 1187–1192.
taxon from M. benedeni and M. expansa in all the cases. Denegri G, Bernadinaz W, Perez-Serrano J and Rodriguez-Caabeiro F
(1998) Anoplocephalid cestodes of veterinary and medical significance: a
Apparently, from the topology of these evolutionary trees, the
review. Folia Parasitologica 45, 1–8.
taxonomical validity is assured and the position of M. denticulata Diop G, Yanagida T, Hailemariam Z, Menkir S, Nakao M, Sako Y, Ba CT
may be deemed to be a connecting lineage infecting the ovine, and Ito A (2015) Genetic characterization of Moniezia species in Senegal
caprine and bovine hosts. and Ethiopia. Parasitology International 64, 256–260.
In conclusion, the molecular phylogeny and morphological Fagbemi BO and Dipeolu OO (1983) Moniezia infection in the dwarf breeds
results of the present study profoundly assert that M. denticulata of small ruminants in Southern Nigeria. Veterinary Quarterly 5, 75–80.
Parasitology 841

Gower WC (1939) A modified stain and procedure for trematodes. Stain Sanger F, Nicklen S and Coulson AR (1977) DNA sequencing with chain-
Technology 14, 31–32. terminating inhibitors. Proceedings of the National Academy of Sciences
Guo A (2017) Moniezia benedeni and Moniezia expansa are distinct cestode spe- 74, 5463–5467.
cies based on complete mitochondrial genomes. Acta Tropica 166, 287–292. Saxena SN and Deo PG (1964) Studies on the morphology of Moniezia den-
Haukisalmi V, Laaksonen S, Oksanen A, Beckmen K, Halajian A, Yanagida ticulata (Rudolphi, 1810) Blanchard, 1891, with a note on the morphology
T and Nakao M (2018) Molecular taxonomy and subgeneric classification of Moniezia expansa (Rudolphi, 1810) Blanchard, 1891. Indian Journal of
of tapeworms of the genus Moniezia Blanchard, 1891 (Cestoda, Helminthology 16, 56–62.
Anoplocephalidae) in northern cervids (Alces and Rangifer). Parasitology Shalaby IMI and Amer SAM (2012) Preliminary molecular identification of
International 67, 218–224. two helminthes (Moniezia sp. and Paramphistomum sp.) in the province
Kaur H and Gupta A (2019) Morphological and molecular characterization of of Taif, Saudi Arabia. World Applied Sciences Journal 17, 986–991.
an anoplocephaline cestode, Moniezia expansa Rudolphi, 1810 infecting Stiles CW and Hassall A (1893) A revision of the adult cestodes of cattle, sheep
Capra hircus in Chandigarh (India). Advances in Applied Research 11, 39–49. and allied animals. US Department of Agriculture, Bureau of Animal
Kimura M (1980) A simple method for estimating evolutionary rates of base Industry.
substitutions through comparative studies of nucleotide sequences. Journal Tam TT, Lan NTK and Doanh PN (2020) Morphological differences and
of Molecular Evolution 16, 111–120. molecular phylogenetic relationship of two tapeworm species, Moniezia
Nagarajan G, Thirumaran SMK, Pachaiyappan K, Thirumurugan P, expansa and Moniezia benedeni, collected from domestic ruminants in nor-
Rajapandai S, Rajendiran AS, Velusamy R, Vannish MR and thern Vietnam. Parasitology International 74, 101998.
Kanagarajadurai K (2022) First report on molecular identification of Tamura K, Stecher G and Kumar S (2021) MEGA11: molecular evolutionary
Moniezia expansa in sheep from Mannavanur, Palani Hills, Tamil Nadu, genetics analysis version 11. Molecular Biology and Evolution 38,
India. Acta Parasitologica 67, 1626–1633. 3022–3027.
Ndom M, Yanagida T, Diop G, Quilichini Y, Ba A, Sako Y, Nakao M, Thosar A, Misal G, Gaware A and Borde S (2015) Morphological and
Marchand B, Dieye A, Ba CT and Ito A (2018) Genetic and morphological molecular studies of Moniezia sp. (Cestoda: Anoplocephalidea) a parasite
characterization of Thysaniezia tapeworms from cattle and sheep in Senegal. of the domestic goat Capra hircus (L.) in Aurangabad district (MS),
Veterinary Parasitology: Regional Studies and Reports 11, 27–31. India. Indian Journal of Applied Research 8, 10–14.
Ndom M, Diop G, Yanagida T, Quilichini Y, Dieye A, Marchand B, Nakao Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and
M, Ito A and Cheikh TBA (2019) Morphological and genetic characteriza- Rozen SG (2012) Primer3 – new capabilities and interfaces. Nucleic Acids
tions of Avitellina tapeworms from domestic ruminants in Senegal: an evi- Research 40, e115.
dence of specificity among sheep and cattle host. Veterinary Parasitology: Xu J, Lin H, Chen J, Cai Y, Fu W, Wang H, Gu X, Lai W, Peng X and Yang
Regional Studies and Reports 18, 100337. G (2018) Molecular characterization of Moniezia sichuanensis in captive
Nguyen TD, Le QD, Huynh VV, Nguyen ST, Nguyen TV and Vu-Khac H musk deer (Moschus berezovskii). Journal of Helminthology 92, 477–483.
(2012) The development of PCR methodology for the identification of spe- Yan H, Bo X, Liu Y, Lou Z, Ni X, Shi W, Zhan F, Ooi H and Jia W (2013)
cies of the tapeworm Moniezia from cattle, goats and sheep in central Differential diagnosis of Moniezia benedeni and M. expansa
Vietnam. Journal of Helminthology 86, 426–429. (Anoplocephalidae) by PCR using markers in small ribosomal DNA (18S
Ohtori M, Aoki M and Itagaki T (2015) Sequence differences in the internal rDNA). Acta Veterinaria Hungarica 61, 463–472.
transcribed spacer 1 and 5.8 S ribosomal RNA among three Moniezia spe- Zhao WJ, Zhang H, Bo X, Li Y and Fu X (2009) Generation and analysis of
cies isolated from ruminants in Japan. Journal of Veterinary Medical Science expressed sequence tags from a cDNA library of Moniezia expansa.
77, 105–107. Molecular and Biochemical Parasitology 164, 80–85.