Molecular characterization and genetic variability of Toxocara vitulorum from naturally infected buffalo calves for the first time in Bangladesh

Abstract

Toxocara vitulorum is one of the deadliest parasite of buffalo calves in Bangladesh. This study was conducted to explore genetic variability within and among the T. vitulorum populations in buffalo calves of Bangladesh. Genomic DNA was extracted, ITS2, COX1 and NAD1 gene were amplified and sequenced. Distinct 29 ITS2, 21 unique NAD1 and 24 COX1 genotypes were detected among the T. vitulorum of different geographic regions. These three gene genotypes similarities ranged from 97 to 99%, when these were compared to best hit scoring T. vitulorum sequences retrieved from GenBank. A total of 12 and 6 unique haplotypes were detected for COX1 and NAD1 gene sequences. The average nucleotide and haplotype diversity for COX1 and NAD1 were 0.0931 & 0.89493 and 0.00658 & 0.77895 respectively and the recorded values were more dispersed than previously published values. The pairwise Nst values ranged from −0.050 to 0.602 and Fst from −0.050 to 0.600 between all the T. vitulorum genotypes indicated huge genetic differentiation which were reportedly higher than other published reports Fst values. This is the first report of T. vitulorum on the basis of COX1 gene in Bangladesh. The study findings will be helpful for further extensive epidemiological studies regarding anthelmintic resistance, control and prevention of T. vitulorum infection in buffalo calves.

Full text

Parasitology
cambridge.org/par
Research Article
*Authors contributed equally to this paper.
Cite this article: Biswas H, Ahmed N, Roy BC,
Hasan MM, Rahman MDK, Talukder MH (2024).
Molecular characterization and genetic
variability of Toxocara vitulorum from naturally
infected buffalo calves for the first time in
Bangladesh. Parasitology 1–13. https://doi.org/
10.1017/S0031182024000842
Received: 12 February 2024
Revised: 28 May 2024
Accepted: 23 June 2024
Keywords:
Bangladesh; Buffalo calves; Genetic variability;
Phylogeny; SNP; T. vitulorum
Corresponding author:
Md. Hasanuzzaman Talukder;
Email: talukdermhasan@bau.edu.bd
© MD HASANUZZAMAN TALUKDER, 2024.
Published by Cambridge University Press. This
is an Open Access article, distributed under
the terms of the Creative Commons Attribution
licence (http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted re-use,
distribution and reproduction, provided the
original article is properly cited.
Molecular characterization and genetic
variability of Toxocara vitulorum from naturally
infected buffalo calves for the first time in
Bangladesh
Hiranmoy Biswas1,2*, Nurnabi Ahmed1* , Babul Chandra Roy1,
Mohammad Manjurul Hasan1,2, MD Khalilur Rahman1and
Md. Hasanuzzaman Talukder1
1
Department of Parasitology, Bangladesh Agricultural University, Mymensingh, Bangladesh and
2
Department of
Livestock Services, Dhaka, Bangladesh
Abstract
Toxocara vitulorum is one of the deadliest parasite of buffalo calves in Bangladesh. This study
was conducted to explore genetic variability within and among the T. vitulorum populations
in buffalo calves of Bangladesh. Genomic DNA was extracted, ITS2, COX1 and NAD1 gene
were amplified and sequenced. Distinct 29 ITS2, 21 unique NAD1 and 24 COX1 genotypes
were detected among the T. vitulorum of different geographic regions. These three gene
genotypes similarities ranged from 97 to 99%, when these were compared to best hit scoring
T. vitulorum sequences retrieved from GenBank. A total of 12 and 6 unique haplotypes were
detected for COX1 and NAD1 gene sequences. The average nucleotide and haplotype diversity
for COX1 and NAD1 were 0.0931 & 0.89493 and 0.00658 & 0.77895 respectively and the
recorded values were more dispersed than previously published values. The pairwise Nst
values ranged from −0.050 to 0.602 and Fst from −0.050 to 0.600 between all the T. vitulorum
genotypes indicated huge genetic differentiation which were reportedly higher than other pub-
lished reports Fst values. This is the first report of T. vitulorum on the basis of COX1 gene in
Bangladesh. The study findings will be helpful for further extensive epidemiological studies
regarding anthelmintic resistance, control and prevention of T. vitulorum infection in buffalo
calves.
Introduction
Toxocara vitulorum is one of the deadliest ascarid nematode that lives in the small intestine of
cattle, buffalo and other bovids (Biswas et al., 2022). It has worldwide distribution but mostly
found in bovids of tropical and subtropical regions (Dorny et al., 2015). This zoonotic ascarid
causes visceral larval migrans (VLM) in humans (Toochukwu, 2017). In the young buffalo
calves aged under 3 months, toxocariasis is one of the prime causes of morbidity and mortality
(Rahman et al., 2018; Biswas et al., 2022). The most common routes of receiving infection by
this buffalo calves are transmammary and transplacental route while they can also get infection
by consumption of larvated eggs contaminated forage (Rast et al., 2013). In buffalo, the preva-
lence of T. vitulorum varied from 2 to 32% while 11 to 57% for buffalo calves in Bangladesh for
last 12 years and it is still increasing with the course of time (Islam et al., 2005; Rahman et al.,
2018). Previous reports also unveiled that the buffalo calves had 3 times higher odds of getting
infection than dam buffalo (Rahman et al., 2018). It is assumed that if this parasitic infection is
not controlled properly, the prevalence and mortality can be reached up to 100 and 50% in
buffalo calves respectively (Radostits et al., 2006; Rast et al., 2013). Continuous reporting of
ineffectiveness of benzimidazole s against T. vitulorum from different buffalo rearing farms
and Bathan (a vast waking marshy green grass land on the river bed) regions of Bangladesh
have been recorded for last 12 years and developing resistance against some available benzimi-
dazoles such as albendazole and levamisole (Biswas et al., 2022). It is evident that parasitic
nematodes are incredibly diverse and treatments rely on a limited arsenal of anthelmintic
drugs with same drug classes, over-reliance and inappropriate use of these anthelmintics
have placed strong selective pressures on parasites and caused the evolution of anthelmintic
resistance (AR) to every drug class (Kotze et al., 2020). In many cases it has been proved
that the evolution of AR to every drug class depend on genetics of resistant nematode, there-
fore phenotypic variation in anthelmintic responses that can be explained by genetic variation
in a population (Evans et al., 2021). As of today, few studies were conducted to determine the
prevalence and associated factors of transmission of T. vitulorum in Bangladesh, but interest-
ingly no molecular based approach taken into consideration to characterize the ascarid which
will be a tool to unveil resources for understanding the pathogen ecology, epidemiology and
control (Halajian et al., 2010; Sultan et al., 2015). In pursuit of drawing the hidden relationship
between AR and genetic makeup of T. vitulorum, we ought to unveil molecular data from the
circulating T. vitulorum in Bangladesh as first step. Currently, a wide range of molecular

techniques such as PCR, restriction fragment length polymorph-
ism, randomized amplified polymorphism DNA and sequencing
have been used widely to identify parasite species more precisely
(Prichard and Tait, 2001; Ahmed et al., 2023). The nuclear ribo-
somal DNA (rDNA) particularly internal transcribed region 2
(ITS2) is a potential marker for identification of species because
of its some distinct attributes such as easy amplification, integrity
of conserved regions, fast evolution of variable nuclear loci, good
number of rRNA clusters to unleash closely interlinked species (Li
et al., 2006;Chenet al., 2012; Ahmed et al., 2023). The mitochon-
drial nicotinamide dehydrogenase subunit 1 gene (NAD1) and
cytochrome oxidase 1 (COX1) genes have been used as candidates
for studying diversity and finding out population structure for a
long time (Jones et al., 2009; Wickramasinghe et al., 2009a,
2009b). Therefore, we selected conventional PCR technique to
amplify the ITS2,COX1, and NAD1 gene markers followed sanger
sequencing to detect Toxocara nematode at species level and find-
ing similarities and dissimilarities among the identified isolates
and with other isolates detected from other parts of the world
base on genetic P distance. The investigation also established
population structure and phylogenetic link between the detected
T. vitulorum isolate and additional T. vitulorum isolates, as well
as congenerics from distantly related species.
Materials and methods
Study area
The study was conducted on multiple sites of 7 divisions such as
Barishal (Charfassion, Bhola Sadar in Bhola district of Barishal),
Chattogram (Sandwip and Anowara upazila in Chattogram),
Khulna (Fakirhat, Mongla and Morelgonj in Bagerhat district),
Rajshahi (Godagari and Paba in Rajshahi district and Sariakandi
Figure 1. Map of Bangladesh showing the 7 study areas as Barishal, Chattogram, Khulna, Rajshahi, Rangpur, Mymensingh and Sylhet division.
2 Hiranmoy Biswas et al.

in Bogura district), Rangpur (Kurigram Sadar, Rawmari in
Kurigram district and Kaligonj in Lalmonirhat district),
Mymensingh (Trishal, Madargonj and Nokla in Myemsingh,
Jamalpur and Sherpur district, respectively) and Sylhet
(Gowainghat, Jaintapur and Kanaighat in Sylhet district) during
July 2018 to December 2020 (Fig. 1). These selecting areas were
highly prevalent for toxocariasis and the availability of different
age groups buffaloes (Rahman et al., 2018).
Parasite collection
Based on the coproscopic examination, we selected buffalo calves
harbouring Toxocara worms. We gave them anthelmintic treat-
ments such as Ivermectin @0.2 mg kg
−1
BWT (Biswas et al.,
2022) and following treatment adult Toxocara worms were
expelled with feces. Then these adult Toxocara worms were
washed with normal saline and transported to the Department
of Parasitology, Bangladesh Agricultural University for
Figure 2. (A, B) Toxocara vitulorum collected from calves. (C, D) Anterior end of T. vitulorum showed 3 lips of male and female worm (lip), (E) posterior end of male
showed coiled tail (black arrow) and showed spicules (sp) (F) posterior end of female showed short tail (st), and posterior end of female worm showed a straight tail
end (long white arrow), (G) 30 cm long female T. vitulorum.
Figure 3. Conventional PCR product showing a. T. vitulorum ITS2 gene (625 bp), b. COX1 (446 bp) and c. NAD1 genes (370 bp) in separate agarose gel. [Lane M
(Marker-1 kb), lane NC (negative control)].
Parasitology 3

Table 1. Nucleotide details and distribution of 29 ITS2 of T. vitulorum isolated from buffalo with reference sequence retrieved from GenBank (MK100346.1)
Genotypes
Nucleotide distribution
60 66 116 124 130 132 133 135 150 156 158 164 177 192 205 206 234 274 275 277 298 301 308 317 319 331
MK100346.1T. vitulorum_ Nagaland C G T T T T T G A A T A A A T A A A A G T A G C A T
BDBSTV01 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDBSTV05 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDBSTV08 . . . . . . . . . C . . . . . C . . T . . . . . .
BDBSTV12 . . . . . . . . . C . . . . . T C G T T . . . . . .
BDCGTV01 . . . . . . . . T . . . . . A . . . . . . . . . . .
BDCGTV02 . . . . . . . . T . . . . . A . . . . . . . . . . .
BDCGTV05 . T . . . G T . . . . . A . . . . . . . . . . .
BDCGTV07 . . . . . . . . T . . . . . A . . . . . . . . . . .
BDKLTV08 . . . . . . . . . . . . . . . . . . . . . . . . .
BDKLTV01 . . . . . . . . . . . . . . . . . . . . . . . . .
BDKLTV02 . . . . . . . . . . . . T . . . . . . . . . . . . .
BDKLTV06 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDMSTV01 G . G C . . . C . . . . . . . . . . . . . G A A . .
BDMSTV03 G . G C . . . C . . . . . . . . . . . . . G A A . .
BDMSTV04 G . G C . . . C . . . . . . . . . . . . . G A A . .
BDMSTV08 G . G C . . . C . . . . . . . . . . . . . G A A . .
BDMSTV09 G . G C . . . C . . . . . . . . . . C G A A T
BDSLTV02 G . G C . . . C . . . . . . . . . . C . . G A A . G
BDSLTV03 G . G C . . . C . . . . . . . . . . C . . G A A . G
BDSLTV04 G . G C . . . C . . . . C . . . C . . G A A . G
BDSLTV05 G . G C . . . C . . G . . . . . . C . . G A A . G
BDRSTV02 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDRSTV03 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDRSTV05 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDRSTV06 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDRPTV01 . . . . . . . . . . . . . . . . . . G . . . . . . .
BDRPTV04 . . . . . . . . . . C . . . . . . . . . . . . . .
BDRPTV05 . . . . . . . . . . . . . . . . . . . . . . . . . .
BDRPTV06 . . . . A G . . . . . . . . . . . . . . . . . . .
BD, Bangladesh; RP, Rangpur; RS, Rajshahi; CG, Chattogram; KL, Khulna; BS, Barishal; SL, Sylhet; MS, Mymensingh; TV, Toxocara vitulorum, the sur number was representative of isolate number.
4 Hiranmoy Biswas et al.

microscopic detection and then stored at −20°C for molecular
investigation.
Isolation of genomic DNA
Genomic DNA was extracted from 84 adult Toxocara species (12
from each division) by using QIA amp mini kit (Original product
by Qiagen AG, Hombrechtikon, Switzerland and provided by
Qiagen India Pvt. Ltd. Jasola, Delhi) according to manufacturer’s
recommendations (Khademvatan et al., 2013). The eluted DNA
was stored at −20°C prior to PCR.
Amplification of ITS2,NAD1 and COX1 gene and gel
electrophoresis
ITS2 (∼625 bp)
ITS2 gene was amplified from genomic DNA by using the con-
served oligo-nucleotide primer pair: 3S (forward: 5′-CGGT
GGATCACTCGGCTCGT-3) and 28A (reverse: 5′-CCTGGTT
AGTTTCTTTTCCTCCGC-3′) (Wickramasinghe et al., 2009a,
2009b;Mahdyet al., 2020). PCR reaction with a final reaction
volume of 25 μL was conducted. 5 μL of DNA extract, 12.5 μL
of Taq® Green Master Mix, 5.5 μL of nuclease-free water, and
1μL of each forward and reverse primer were used in the ampli-
fications. The amplification programme was run in a
MyCyclerTM heat cycler (BioRad, USA) with a 3 minutes initial
denaturation at 94°C, 31 cycles of 30 s at 94°C, 30 s at 46°C, and
1 min at 72°C. For all primers, except the 12S and ITS2 primers,
which were annealed at 50 and 53°C, respectively, there was a
final 5 min of extension at 72°C (Mahdy et al., 2020). The
resultant gel was analysed and captured on camera using a
transilluminator.
NAD1 (∼370 bp)
During the conventional PCR, the primer pairs (forward: 5′-TT
CTTATGAGATTGCTTTT-3′and reverse: 5′-TATCATAAC
GAAAACGAGG-3′) were used (Li et al., 2016; El-Seify et al.,
2021). 9.75 μL autoclaved, distilled water, 5 μL PCR buffer
(10×), 0.25 μL Taq, 2 μL dNTPs (2.0 mM), 1 μL DNA, 3 μL MgC
l2 (25 mM) and 2 μL of each forward and reverse primer (working
concentration: 10 μmol L
−1
) were all included in the PCR mixture
ina25μL reaction volume. After a heated start of 94°C for 5 min
and concluding with 72°C for 5 min, each of the 40 PCR cycles
included 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min (Li
et al., 2016; El-Seify et al., 2021). The PCR products were observed
using a UV transilluminator after being separated on 1% agarose
gels and stained with ethidium bromide.
COX1 (∼446 bp)
A fragment of COX1 was amplified by PCR, yielding a 446 bp
sequence using primers JB3 (5′-T TTTTTGGGCATCCT
GAGGTTTAT-3′)andJB4.5(5
′-TAAAGAAAGAACATAAT
GAAAATG-3′), a final volume of 25 μLwasusedforthePCR,
which contained 7.5 μL of sterile distilled water that was free
of RNase and DNase, 10 μL of 5× MyTaq Reaction buffer,
1μL of each primer (20 pmol), 5 μLoftemplateDNA
(100–200 ng), and 0.5 μL of TaqDNA polymerase (1.25 IU)
4
(Wickramasinghe et al., 2009a,2009b;Oguz, 2018). The follow-
ing were the conditions for the PCR: 5 minutes at 94°C for ini-
tial denaturation, 35 cycles of 30 s at 94°C, 45 s at 50°C, 35 s at
72°C, and 10 min at 72°C for the final extension. The PCR pro-
ducts were observed using a UV transilluminator after being
separated on 1.5% agarose gels and stained with ethidium
bromide (Oguz, 2018).
Table 2. Nucleotide diversity and haplotype diversity of ITS2,COX1 and NAD1 gene sequences of T. vitulorum isolated from 7 different topographic regions of Bangladesh
Geographical
locations
ITS2 gene COX1 gene NAD1 gene
No of
sequences
No of
haplotypes
Haplotype
diversity
(Hd)
Nucleotide
diversity
(Nd)
No of
sequences
No of
haplotypes
Haplotype
diversity
(Hd)
Nucleotide
diversity
(Nd)
No of
sequences
No of
haplotypes
Haplotype
diversity
(Hd)
Haplotype
diversity
(Hd)
Khulna 12 2 0.50 0.00141 12 3 0.833 0.00749 10 1 0.00 0.00
Barishal 15 3 0.833 0.00992 14 1 0.00 0.00 13 1 0.00 0.00
Chattogram 12 2 0.500 0.00282 12 1 0.00 0.00 10 2 0.667 0.00208
Mymensingh 16 2 0.400 0.00226 15 2 0.50 0.0015 12 1 0.00 0.00
Sylhet 10 2 0.50 0.0028 10 2 0.667 0.00208 10 2 0.667 0.00208
Rajshahi 12 1 0.00 0.00 12 3 1.00 0.00499 11 1 0.00 0.00
Rangpur 12 4 0.833 0.00562 10 1 0.00 0.00 12 1 0.00 0.00
Overall 87 16 0.01530 0.83498 82 12 0.89493 0.01691 77 6 0.77895 0.00658
Parasitology 5

Table 3. Nucleotide details and distribution of 24 T. vitulorum COX1 gene isolated from buffalo with reference sequence retrieved from GenBank (AJ920062.1)
Genotypes
Nucleotide distribution
104 143 149 155 185 197 218 220 227 245 284 296 314 344 362 368 376 389
AJ920062.1Toxocara vitulorum Sri Lanka T G G A A G T A A T C A T G A G T C
BDBSTV01 . . . . . . . . . . . . . . . . . .
BDRPTV01 . C . . . . . . . . . . . . . . . .
BDRPTV04 . C . . . . . . . . . . . . . . . .
BDRPTV05 . C . . . . . . . . . . . . . . . .
BDCGTV01 . . . . . . . . . . . . . . . . . .
BDBSTV08 . . . . . . . . . . . . . . . . . .
BDRSTV05 . . . . . . . . . . . . . . . . G .
BDCGTV02 . . . G . . . . . . . . . . . . . .
BDRSTV02 . . . G . . . . . . . . . . . . . .
BDRSTV03 . . . . . . . . . . . . A . . . . .
BDBSTV05 . . . . . . . . . . . . . . . . . .
BDKLTV01 . . . . . . . . . . . . . . G . . .
BDKLTV08 . . . . . . . . . . . . . C G . . .
BDBSTV12 . . . . . . . . . . . . . . . . . .
BDSLTV03 . . A . G A C . G C T G . . . A . T
BDSLTV02 . . A . G A C . G C T G . . . A . T
BDMSTV03 . . A . G A C . G C T G . . . A . T
BDCGTV05 . . . . . . . . . . . . . . . . . .
BDKLTV02 A . . C . . . . . . . . . . . . . .
BDKLTV06 A . . C . . . . . . . . . . . . . .
BDMSTV01 . . A . G A C . G C T G . . . A . T
BDSLTV04 . . A . G C C . G C C G . . . A . T
BDMSTV09 . . A . G A C . G C C G . . . A . T
BDMSTV08 . . A . G A C T G C C G . . . A . T
BD, Bangladesh; RP, Rangpur; RS, Rajshahi; CG, Chattogram; KL, Khulna; BS, Barishal; SL, Sylhet; MS, Mymensingh; TV, Toxocara vitulorum, the sur number was representative of isolate number.
6 Hiranmoy Biswas et al.

PCR positive electrophoresis product purification
Purification of the ITS2, COX1 and NAD1 PCR electrophoresis
products was accomplished with the use of SV Gel and PCR
Clean Up System (Cat. No. A9281; Origin: Promega, USA).
Purified products were sequenced using an Applied Biosystems
automated DNA sequencer (3730 XL; Applied Biosystems,
Foster City, USA) in accordance with manufacturer instructions
from a commercial source (DNA Laboratories Sdn Bhd
(736763-T), UKM-MTDC Technology Centre, Selangor,
Malaysia through Invent Technology Ltd. Banani, Dhaka). The
forward and reverse PCR primers orientation was same for both
the forward and reverse reads.
Intra-population diversity parameters and phylogeny
Using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), unique
sequences for each marker (29 ITS2,21NAD1 and 24 COX1) gen-
erated in this study were compared with best hit scoring
sequences available in GenBank (Supplementary Table S4). The
Mega11 software was used to aligned the sequences using
ClustalW program by using a gap opening penalty of 15.00 and
gap extension penalty of 6.66 for both pairwise and multiple
alignments as described by Nehra et al.(2022). Pairwise compar-
isons were made using the GenBank-retrieved sequences, and the
BioEdit program (version 7.0.5.3) (https://bioedit.software.
informer.com/7.2/) was utilized to determine similarities (%)
(Ahmed et al., 2023). Haplotype diversity, the average number
of nucleotide change, and nucleotide diversity were among the
characteristics linked to intra-population diversity that were mea-
sured using DnaSP version 5.1 (Rozas, 2017). After trimming
every sequence at both ends, the phylogenetic analysis was carried
out using the neighbor-joining method with the Tamura Nei par-
ameter of evolution based on lowest BIC score (Bayesian
Information Criteria) and AICc value (Akaike Information
Criteria) in the Mega11 programme (Tamura et al., 2021). While
MEGA v.11.0’s default values were used to acquire the other set-
tings, 1000 replicates were used to determine the bootstrap para-
meters for the definition of nodes statistical support (Tamura
et al., 2013).
Population genetic structure by using mitochondrial COX1
sequences
Genetic differences were estimated using statistics based on
haplotypes (Hs), nucleotide sequences (Ks) and some other
parameters such as average number of nucleotide differences
in pairs (Kxy), genetic differentiation index based on the fre-
quency of haplotypes (Gst), nucleotide-based statistics (Nst),
nucleotide substitutions per site (Dxy) and net nucleotide sub-
stitutions per site (Da) using DnaSP ver. 6.12.03 (Rozas et al.,
2017) the population pairwise genetic difference (Fst) to
determine the genetic differentiation and population genetic
structure.
Results
Morphological findings
Toxocara vitulorum is a large, robust worm up to 30 cm long with
three large, prominent lips. The body was soft and translucent
with clearly cuticle (Fig. 2A and B). The mean length of male
and female parasite was 18.5 (±1.2 cm) and 24.20 (±6.2 cm)
29.11 cm) and mean width were 0.4 and 0.6 mm in respectively.
The three well defined lips; two sub ventral and one dorsal lip
were determined from the worms (Fig. 2C and D). The male
worms had a posterior end curved ventrally and posterior end
exhibited two spicules (Fig. 2E). While, in female posterior end
was distinguishable a straight-tailed (Fig. 2F).
Species identification and genotyping
To validate the species of T. vitulorum, the ITS2, COX1 and NAD1
gene were amplified from 84 samples from seven divisions of
Bangladesh and conventional PCR product showing T. vitulorum
ITS2 gene (625 bp), COX1 (446 bp) and NAD1 genes (370 bp) in
separate agarose gel (Fig. 3).
From 84 positive PCR products, 84 ITS2, 79 for COX1 and 74
for NAD1 sequences were produced. Unfortunately, sequencing of
5T. vitulorum isolates for COX1 and NAD1 was failed to generate.
On the contrary, 5 sequenced data for NAD1 gene failed to gen-
erate due to numerous ambiguous nucleotides positioning.
Among 84 ITS2 sequences, 29 distinct genotypes were identified
while 79 COX1 and 74 NAD1 sequences, 24 and 21 distinct gen-
otypes were found.
When compared to five ITS2 reference sequences of T. vitu-
lorum (GenBank accession nos. MK100346.1, MG214152.1,
FJ418784.1, MG214151.1 and KY442062.1), the newly generated
ITS2 genotype similarities ranged from 97 to 100%. Pairwise
nucleotidic genetic distances (p-distance model) were measured
for the partial ITS2 sequences of T. vitulorum isolates in the
Table 4. Nucleotide details and distribution of 21 T. vitulorum NADI gene
isolated from buffalo with reference sequence retrieved from GenBank
(AJ920062.1)
Genotypes
Nucleotide distribution
40 83 121 157 265
AJ937266.1 T. vitulorum Sri
Lanka
GTG T T
BDKHTV01 . . . . .
BDKHTV02 . . . . .
BDMSTV03 . A . G .
BDKHTV06 . . . . .
BDMSTV05 . A . G .
BDSLTV06 . A . G A
BDCTTV01 . . . . .
BDCGTV02 . . . . .
BDCGTV05 . . . . .
BDSLTV03 . A A G A
BDSLTV02 . A A G A
BDBSTV12 . . . . .
BDRSTV02 A G . . .
BDRSTV03 A G . . .
BDRSTV04 A G . . .
BDBSTV05 . . . . .
BDRPTV05 A G . . .
BDRPTV01 A G . . .
BDBSTV01 . . . . .
BDKHTV01 . A . G .
BDKHTV02 A G . . .
BD, Bangladesh; RP, Rangpur; RS, Rajshahi; CG, Chattogram; KL, Khulna; BS, Barishal; SL,
Sylhet; MS, Mymensingh; TV, Toxocara vitulorum, the sur number was representative of
isolate number.
Parasitology 7

present study with best hit scoring reference sequences of differ-
ent countries retrieved from GenBank and genetic distances ran-
ging from 0.000 to 0.0326 (Supplementary Table S1).
Twenty-six single nucleotide polymorphisms (SNPs) were
found when 29 ITS2 genotypes were aligned with the reference
sequence (MK100346.1 in India). These SNPs resulted from sub-
stitutions at nucleotide positions 60, 66, 116, 124, 130, 132, 133,
135, 150, 156, 158, 164, 177, 192, 205, 206, 234, 274, 275, 277,
298, 301, 308, 317, 319 and 331. In such substitutions, there
were six transitions (three T˂-˃C and three A<->G) and twenty
transversions (seven A˂->T, two G<->C, five A˂-˃C, and six
G<->T) (Table 1). Table 2 shows that the total haplotype diversity
and nucleotide diversity of T. vitulorum among its ITS2 sequences
from seven divisions in Bangladesh were 0.83498 and 0.01530,
respectively. Comparing the newly generated COX1 and NAD1
genotypes of T. vitulorum to the best hit scoring sequences
from GenBank revealed matches ranging from 98 to 100%
(Supplementary Tables S2 and S3). For the COX1 and NAD1
genes, respectively, 79 and 74 amplicons yielded 12 and 6 distinct
haplotypes. When compared to the reference sequence
(AJ920062.1) in the COX1 gene, 18 SNPs were observed at posi-
tions 104, 14, 149, 155, 185, 197, 218, 220, 227, 245, 284, 296, 314,
344, 362, 368, 376 and 389 (Table 3). Five SNPs were found in the
NAD1 gene (T<->G, A<->T, and G<->A) (Table 4). SNP’s were
both transitions (A<->G, C<->T) and translations (<A->T,
C<->G and G<->T) type in nature (Table 3). The T. vitulorum
isolates from Bangladesh showed a high degree of diversity in
the COX1 and NAD1 genes; for COX1, the average nucleotide
Figure 4. Neighbour-joining phylogenetic tree was constructed using partial ITS2 gene of T. vitulorum isolates from different hosts and geographical regions.
Strongyloides stercoralis was used as an out group. Red dots were study generated sequences. Scale bar indicates the proportion of sites changing along each
branch. [BD, Bangladesh; RP, Rangpur; RS, Rajshahi; CG, Chattogram; KL, Khulna; BS, Barishal; SL, Sylhet; MS, Mymensingh; TV, Toxocara vitulorum, the sur number
was representative of isolate number].
8 Hiranmoy Biswas et al.

diversity was 0.01691 and the haplotype diversity was 0.89493,
while for NAD1, the average nucleotide diversity was 0.00658
and the haplotype diversity was 0.77895 (Table 2).
Phylogeny
The phylogenetic tree has been constructed by 29 ITS2 genotypes
was divided into two clades: A and B. Furthermore, clade A was
divided into subclades I and II, where Strongyloides stercoralis
was used as outgroup. The neighbour-joining (NJ) phylogenetic
tree demonstrated that T. vitulorum isolates clustered together
with the reference sequences of Sri Lanka, USA, Canada, Egypt
& Germany that belong to the subclade I under the clade A with-
out any distinct geographical boundaries (Fig. 4).
In subclade II under clade A, the reference sequence of T. cati
from China, India, Japan, Italy was clustered in same position of
the tree, whereas, the reference sequences of T. canis from Egypt,
India, Sri Lanka, Iran under clade B were grouped in same pos-
ition. These findings unveiled that different species of Toxocara
nematode has different genomic background and thus Toxocara
species with same genomic background were clustered.
The phylogenetic trees were constructed using 24 COX1 and
21 NAD1 gene sequences of T. vitulorum collected from 7 divi-
sions of Bangladesh (Figs 5 and 6). Neighbour-Joining (NJ),
Maximum Parsimony (MP) and Maximum Likelihood (ML)
were used to produce phylogenetic tree that illustrate same
findings. Readers better understanding, only the neighbour-
joining trees have been incorporated, visualized and explained
here. The NJ phylogeny for COX1 gene generated with 1000 repli-
cates showed two distinct clades A and B where clade A was fur-
ther divided into three subclades. In subclade I, T. vitulorum
isolates produced in this study were grouped together without
any distinct boundary with Sri Lankan (AJ920062.1), German
(KY313642.1), Turkish (MG911730.1) isolates and supported by
strong bootstrap value (92%) (Fig. 5).
In case of NJ phylogeny for NAD1 gene, two main clades A
and B were also generated. In subclades I of clade A, T. vitulorum
isolates were clustered with Japanese (FJ664617.1), Sri Lankan
(AJ937266.1) and Chinese (KY825180.1 & KY825181.1) T. vitu-
lorum isolates. The bootstrap value was 90% (Fig. 6).
Population genetic structure
To evaluate genetic divergence among the examined populations,
Fst and Nst values of T. vitulorum populations were calculated in
various topographic zones of Bangladesh. When evaluating the
COX1 gene, genetic differentiation was observed and the pairwise
Nst values ranged from −0.050 to 0.602 and Fst from −0.050 to
0.600. When compared populations from different topographic
zones in Bangladesh, the T. vitulorum population of Barishal,
Khulna and Rangpur exhibited rather substantial genetic diver-
gence, with the greatest levels of Nst (0.602, 0.575, 0.559 to
0.9211) and Fst (0.600, 0.571 and 0.556) (Table 5).
Figure 5. Neighbour-joining phylogenetic tree was constructed using partial COX1 gene of T. vitulorum isolates from different hosts and geographical regions.
Strongyloides stercoralis was used as an out group. Scale bar indicates the proportion of sites changing along each branch. [BD, Bangladesh; RP, Rangpur; RS,
Rajshahi; CG, Chattogram; KL, Khulna; BS, Barishal; SL, Sylhet; MS, Mymensingh; TV, Toxocara vitulorum, the sur number was representative of isolate number].
Parasitology 9

Discussions
Parasite genomics is essential for determining epidemiology and
controlling parasitic infections in humans and animals.
Toxocara vitulorum is one of the most prevalent gastrointestinal
helminths infecting ruminants, especially in tropical areas.
Toxocara vitulorum has been found in several places in
Bangladesh; however, there has never been an investigation into
its molecular makeup and evolutionary status (Islam et al.,
2005; Rahman et al., 2018). In this study, the population genetic
structure and phylogenetic of T. vitulorum was investigated for
the first time in Bangladesh. The findings obtained from the iso-
lates of 7 divisions in Bangladesh and other global locations were
evaluated, and their correlation was ascertained.
The present findings by morphological and morphometric
measurements of body length and width, presence of 3 lips, spi-
cules in male and finger like projections and straight posterior
end in female T. vitulorum parasite are strongly supported by pre-
vious reports (Mahdy et al., 2020).
The variation of sequence identities among the T. vitulorum
isolates was 3.0% in ITS2 gene sequences. The degree of variation
(3.0%) is comparable to that of isolates of with the variation of T.
vitulorum from India, Sri Lanka, Egypt and Germany (Sultan
et al., 2015; Venjakob et al., 2017). Twenty-nine distinct ITS2 gen-
otypes were detected among T. vitulorum isolates in present study,
but the number of ITS2 genotypes of T. vitulorum isolates were
higher than described in previously reported (Sultan et al.,
2015; Mahdy et al., 2020). The number of polymorphic loci of
T. vitulorum isolates also differed between countries for as (Rast
et al., 2013). Data obtained from this study confirmed the species
as T. vitulorum.
The ITS2, NAD1 and COX1 genes were amplified from the
genomic DNA of T. vitulorum species found in 7 divisions of
Bangladesh in order to confirm the species’existence and investi-
gate its molecular composition. The nucleotide BLAST search was
used to retrieve best hit scoring ITS2,COX1 and NAD1 of T. vitu-
lorum sequences with high identities (97–99%) from the GenBank
and ClustalW program in Mega 11 was used to align all the
sequences (Ahmed et al., 2023). For the COX1 and NAD1 gene
sequences, a total of 12 and 6 distinct haplotypes were found
respectively. Five SNPs were discovered in the NAD1 gene,
while 18 SNPs were found in the COX1 gene. A great amount
of gene flow of COX1 and NAD1 gene were observed among
T. vitulorum species of Bangladesh and for COX1, the average
nucleotide diversity was 0.01691 and haplotype diversity was
0.89493 for COX1 and for NAD1 average nucleotide diversity
was 0.00658 and haplotype diversity was 0.77895 respectively.
In comparison to previously published estimates of nucleotide
diversity from T. vitulorum isolates in Sri Lanka, the recorded
values for both cases are more scattered. (Wickramasinghe
et al., 2009a,2009b), India (Mahdy et al., 2020), Egypt (Sultan
et al., 2015) and Turkey (Oguz, 2018).
The topology of the ITS2 phylogenetic tree showed two dis-
tinct clades, clade A and B. Clade A was again divided into sub-
clade I and subclade II when compared with the isolates of this
study with those of other countries. Subclade I representing all
the 29 T. vitulorum genotypes showed little resolution and
belonged to samples isolated from 7 districts of Bangladesh
Figure 6. Neighbour-joining phylogenetic tree was constructed using partial NAD1 gene of T. vitulorum isolates from different hosts and geographical regions. H.
contortus was used as an out group. Scale bar indicates the proportion of sites changing along each branch. [BD, Bangladesh; RP, Rangpur; RS, Rajshahi; CG,
Chattogram; KL, Khulna; BS, Barishal; SL, Sylhet; MS, Mymensingh; TV, Toxocara vitulorum, the sur number was representative of isolate number].
10 Hiranmoy Biswas et al.

Table 5. Gene flow and genetic differentiation indices between T. vitulorum genotypes based on ITS2 region
Population 1 Population 2 Distance (km) Hs Ks Kxy Gst DeltaSt GammaSt Nst Fst Dxy Da
Mymensingh Chattogram 400 0.889 3.714 7.667 0.089 0.007 0.479 0.567 0.565 0.021 0.012
Mymensingh Barishal 292 0.889 3.714 8.333 0.089 0.008 0.510 0.602 0.600 0.023 0.014
Mymensingh Khulna 330 1.000 4.286 9.000 0.003 0.008 0.487 0.559 0.556 0.025 0.014
Mymensingh Rangpur 293 1.000 4.286 9.333 0.003 0.009 0.500 0.575 0.571 0.026 0.015
Mymensingh Rajshahi 452 1.000 4.286 8.833 0.003 0.008 0.480 0.551 0.547 0.025 0.014
Mymensingh Sylhet 279 1.000 4.571 9.167 0.003 0.008 0.470 0.531 0.527 0.026 0.014
Chattogram Barishal 241 0.667 0.667 1.000 0.077 0.001 0.385 0.334 0.333 0.003 0.001
Chattogram Khulna 442 0.833 1.333 2.000 0.032 0.002 0.385 0.334 0.333 0.006 0.002
Chattogram Rangpur 545 0.833 1.333 2.000 0.032 0.002 0.385 0.334 0.333 0.006 0.002
Chattogram Rajshahi 498 0.833 1.333 2.000 0.032 0.002 0.385 0.334 0.333 0.006 0.002
Chattogram Sylhet 358 0.833 1.667 2.000 0.032 0.001 0.286 0.167 0.167 0.006 0.001
Barishal Khulna 118 0.833 1.333 1.667 −0.034 0.001 0.304 0.201 0.200 0.005 0.001
Barishal Rangpur 475 0.833 1.333 1.667 −0.034 0.001 0.304 0.201 0.200 0.005 0.001
Barishal Rajshahi 323 0.833 1.333 1.667 −0.034 0.001 0.304 0.201 0.200 0.005 0.001
Barishal Sylhet 400 0.833 1.667 1.444 −0.034 0.000 0.130 −0.155 −0.154 0.004 −0.001
Khulna Rangpur 396 1.000 2.000 2.667 −0.059 0.002 0.333 0.252 0.250 0.007 0.002
Khulna Rajshahi 258 1.000 2.000 2.667 −0.059 0.002 0.333 0.252 0.250 0.007 0.002
Khulna Sylhet 424 1.000 2.333 2.222 −0.059 0.001 0.176 −0.050 −0.050 0.006 0.000
Rangpur Rajshahi 227 1.000 2.000 2.667 −0.059 0.002 0.333 0.252 0.250 0.007 0.002
Rangpur Sylhet 499 1.000 2.333 2.667 −0.059 0.002 0.263 0.126 0.125 0.007 0.001
Rajshahi Sylhet 536 1.000 2.333 2.667 −0.059 0.002 0.263 0.126 0.125 0.007 0.001
Hs, Hudson’s haplotype-based statistics; Ks, Hudson’s nucleotide sequence-based statistics (Hudson et al., 1992); Kxy, Average proportion of nucleotide differences between T. vitulorum genotypes; Gst, Genetic differentiation index based on the frequency of
haplotypes; Nst, Nucleotide-based statistics (Lynch and Crease, 1990); Fst, Tajima and Nei, pairwise genetic distance; Dxy, the average number of nucleotide substitutions per site between T. vitulorum genotypes; Da, the number of net nucleotide substitutions per site
between T. vitulorum genotypes.
Parasitology 11

along with other T. vitulorum genotypes including India
(MK100346.1 &KJ777159.1), Egypt (MG214151.1), Germany
(KY442062.1), Canada (JQ083352.1), Sri Lanka (FJ418784.1),
USA (KT3738.1) and T. cati from India (KJ777179), China
(KY003088), Italy (MZ59634.1) and Japan (AB571303) that had
been consistent with previously published reports (Wickramasinghe
et al., 2009a,2009b).
The NJ dendrogram for COX1 gene generated with 1000 re-
plicates illustrates 2 definite clades A and B where clade A was
further divided into 3 subclades. In subclade I, T. vitulorum iso-
lates produced in this study grouped together without any distinct
boundary with Sri Lankan (AJ920062.1), German (KY313642.1),
Turkey (MG911730.1) isolates, were supported by the strong
bootstrap value (92%) and documented the previously published
reports (Oguz, 2018; Mahdy et al., 2020). In case of NJ phylogeny
for NAD1 gene, 2 main clades A and B were also generated.
In subclades I of clade A, T. vitulorum isolates were clustered
with Japanese (FJ664617.1), Sri Lankan (AJ937266.1) and
Chinese (KY825180.1 & KY825181.1) T. vitulorum isolates. The
results coincide with the same attributes that previously published
(Li et al., 2016).
In order to ascertain genetic variation within the examined
Toxocara populations, T. vitulorum populations’Fst and Nst
values were calculated across various Bangladeshi topography
zones. The pairwise FST values were recorded more than 0.5
when the population of Mymensingh compared with populations
from all other 6 divisions. The highest Fst were seen between T.
vitulorum populations of Mymensingh and Barishal zone fol-
lowed by Mymensingh-Rangpur, Mymensingh-Khulna and
Mymensingh-Chattogram zone. It was further bolstered by the
presence of a very low level of gene flow between them. The
low level of gene flow may in part be due to lack of prenatal
and transmammary transmission in case of T. vitulorum infec-
tion. The horizontal gene flow occurs due to the movement of
infected felids, whereas the vertical gene flow occurs due to trans-
mammary transmission and limited gene flow between popula-
tions can expedite the process of genetic differentiation (Choy
et al., 2015). The results are consistent with previously published
data for Nst (Oguz, 2018) whereas much higher than T. canis
population isolated from different regions of Iran (Ozlati et al.,
2016) and lower than T. cati populations in China (Venkatesan
et al., 2022). All the above results suggest that high genetic differ-
entiation and low gene flow without clear geographical barriers
and cross-infection between population of this certain area is
not frequently occurred. We hypothesize that random movement
of the buffalo calves act as the medium for genetic exchange.
Thus, further investigations are warranted to take into account
in the design of an effective control strategy.
Supplementary material. The supplementary material for this article can
be found at https://doi.org/10.1017/S0031182024000842.
Data availability statement. Data will be available based on request.
Acknowledgements. The authors would like to convey their sincere grati-
tude to the Bangabandhu Science and Technology Fellowship Trust, the
Ministry of Science and Technology, Govt. of Bangladesh and the officials of
the local DLS, and buffalo owners, who willingly allowed the collection of sam-
ples from their buffalo calves. The abstract P-1041 (Poster session) was pre-
sented in the 15th International Congress of Parasitology (ICOPA 2022)
through the DFC fellowship of travel grants, held in Copenhagen, Denmark
during 21–26 August 2022.
Authors’contributions. Hiranmoy Biswas: Review of literature, Resources,
Methodology, Original Draft Preparation, Investigation, Writing-Review &
Editing; Nurnabi Ahmed: Review of literature, Methodology, Formal analysis,
Resources, Visualization, Original Draft Preparation, Validation, Software,
Writing-Review and Editing; Mohammad Manjurul Hasan: Methodology,
Investigation, Formal analysis, Writing-Review and Editing; Babul Chandra
Roy: Co-supervision, Resources, Methodology, Formal analysis, Writing-Review
and Editing; Md. Hasanuzzaman Talukder: Conceptualization, Methodology,
Project Administration, Supervision, Validation, Visualization, Investigation,
Resources, Writing-Review and Editing. Hiranmoy Biswas and Nurnabi
Ahmed contributed equally to this work.
Financial support. This study was funded by the Bangabandhu Science and
Technology Fellowship Trust, Ministry of Science and Technology, Bangladesh
and partially funded by a project (BAU/586/2018) from Bangladesh
Agricultural University Research System (BAURES), Bangladesh Agricultural
University, Mymensingh.
Competing interests. The authors declare that they have no conflict of
interests related to this work. They are solely accountable for the content
and writing of the report.
Ethical standards. The study protocol was approved by the animal welfare
and experimental ethical committee of Bangladesh Agricultural University
(AWEEC/BAU/2018-11).
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Parasitology 13