A new record of the occurrence of Trichuris skrjabini Baskakov, 1924 in goats of Pakistan

More than 23 Trichuroidea species have been identified in ruminants in different parts of the world. Most are pathogenic, causing trichurosis. Trichuris adults of most species within this family have a predilection for the ceca, where they may cause damage to the epithelial wall. In the present study, Trichuris spp. from large intestine of goats were analysed based on morphological and molecular characteristics. Fifty adult worms (male = 25 and female = 25) were selected for morphometric and molecular analysis. Male Trichuris were distinguished by their longer spicules, typical spicule sheaths, and small spicules that were always completely covered by the spicule sheath. The presence of an uneverted vulva in the vagina distinguished female worms. We have performed the molecular characterisation of adult warms to identify as Trichuris skrjabini. Genetic comparison of T. skrjabini rDNA ITS2 sequences with those from other Trichuris spp. was performed to assess within and between species variation and validate the use of ITS-2 rDNA as a robust species-specific marker for T. skrjabini identification. This work provides the first report of this parasite species from Pakistan and validated species-specific marker of T. skrjabini that reduces the production potential of goats in the country.

Different biometric and morphological traits have been employed in the taxonomic characterisation of the Trichuris species [5].Only a few characteristics have been described, and congeneric species have not been sufficiently compared [6].Although morphometric analysis provides keys for Trichuris identification, it is a more traditional approach that might lead to ambiguity due to overlapping morphological and morphometrical traits leading to taxonomic and nomenclatorial challenges [7].
In taxonomic groups of Trichuris with complex systematics and overlapping characterised by morphological convergence, using molecular methods to identify Trichuris at the species level is essential [8,9].Numerous helminth investigations have used the ITS-2 of the ribosomal DNA (rDNA) as their target locus.This region is considered an acceptable genetic marker for resolving links at the species level [10].Molecular methods have shown that not all species initially described by morphometric characteristics would remain fully identified [11,12].Therefore, it is essential to use rDNA regions encompassing the ITS2 to identify Trichuris from sympatric areas worldwide [10,13].Pakistan has an agriculture-based economy, with livestock being an integral part.The distribution of Trichuris in goats has been sporadically investigated in Pakistan.A few studies previously described Trichuris ovis through egg and adult morphology isolated from sheep and goats, whereas most simply identified at the genus level [14][15][16][17][18][19].Trichuris suis (pig whipworm) and Trichuris vulpis (canine whipworm) are considered zoonotic parasites which can threaten the human population [20] and require more reliable methodologies to improve the knowledge of the Trichuris species.However, much of the discussion around integrative taxonomy deals with the merits of applying morphological versus molecular characteristics [21][22][23].The present study is the first to confirm the species identity of Trichuris skrjabini from goats using the rDNA ITS-2 genetic marker.Our results suggest, in contrast to previous morphologically based studies, that Trichuris skrjabini is the predominant goats species in the Punjab province, Pakistan.There is no evidence of Trichuris ovis found in this study.

Ethical approval
The collection of worms from the slaughtered animals does not require ethical approval, the animals were slaughtered for other purposes to meet the high protein demand of population.The study was approved by the Animal Ethics Committee of the Quaid-i-Azam University, Islamabad.

Worm collection
In this cross-sectional study, 231 slaughtered goats were examined between February and August 2022 at the Sihala abattoir in the Rawalpindi division of the Punjab province.The sample size was determined by using the formula [24], where n was the sample size, Z was the desired confidence interval (95%), P was the expected proportion of infected animals in the population (0.35) and d was precision of estimation (5%).The calculated sample size was 188, but we increased it to 231 for greater precision.The animals were examined using a convenient random sample method.Fifty worms (2 to 3 worms per host) were recovered from the cecum or the cecal epithelial wall.The worms were rinsed in phosphate-buffered saline (PBS) before being preserved in 70% ethanol for morphometric and molecular analysis.

Morphological examination
50 adult warms (male = 25 and female = 25) were fixed for morphometric analysis.The worms were cleared in lactophenol and identified under the microscope [25].Standardised measurements were obtained among the male characters, and spicule length is regarded as the most important criterion for differentiating Trichuris [26].The main factors used to identify females are the vulvar morphology [27], the structure and lining of the vagina and, alternatively, the distance from the vulva to the uterine sphincter [3,8].The descriptive univariate statistics based on mean values, standard deviation and range for all parameters were determined for male and female worms [9].

Genomic DNA extraction
Genomic DNA was extracted from 15 out of 50 individual worms of either sex.A small tissue sample of 2 mg was taken from each worm head and put in a petri dish with distilled water (dH2O) to prevent egg contamination.Each worm piece was rinsed twice for 5 min to remove all traces of ethanol.The worms were lysed in a 25 μl worm lysis solution made by mixing 50 μl of proteinase K (10 mg/ml, New England Bio Labs) in 1 ml of Direct PCR lysis reagent (Viagen) following the lysates were incubated for 2 hrs at 60˚C and then for 15 min at 85˚C [28].

Sequence and phylogenetic analysis of ITS-2 ribosomal DNA
PCR products were submitted for commercial sequencing (Macrogen, Korea), using the same amplification primers.Both strands of rDNA ITS-2 sequences from each worm were assembled, aligned, and edited to remove primers from both ends using the MUSCLE Alignment tool of the Geneious Pro 5.4 software [31].The CD-HIT Suite software grouped sequences showing 100% base pair similarity into single unique sequence variants [32].The unique sequence variants were further aligned with previously published NCBI GenBank rDNA ITS-2 of Trichuris species.All sequences of field samples and the GeneBank were trimmed to 388 bp, the length of the shortest sequence available that contained all the informative sites.The phylogenetic analysis was inferred using the Maximum Likelihood method and Kimura 2-parameter model [33].The tree with the highest log likelihood (-2414.08) is shown.The percentage of trees where the associated taxa clustered together is displayed next to the branches.Initial tree (s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value.A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter = 7.5222)].The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.All positions containing gaps and missing data were eliminated (complete deletion option).

General characteristics of Trichuris
Morphological characters revealed that the worms had a filiform anterior half and a broad handle-like posterior part (Fig 1).The narrow anterior part displays two different cuticular patterns.One side is heavily striated with transverse grooves, while the other is a delicately tuberculate band showing small circular elevated bodies uniformly spaced (Fig 1A).The anterior part of the body has a ventral side that tapers somewhat toward the cephalic end, revealing a broad longitudinally elongated "bacillary band" with typical cuticle inflations.A set of cephalic papillae surrounds the mouth, formed in two circles (an inner circle and a lateral circle), with a prominent organ, the stylet, emerging from the mouth cavity's central portion.

Male worms characteristics
The body is 25-49 mm (36.6±7.65)long.The ratio between anterior and posterior body length is 2:1.The thin anterior part is 0.66:1 of the entire body length.The length of the oesophagus is 18-38 (24.4±7.08).The body's posterior end is curved ventrally.The width at oesophageal region of body is 0.11-0.18(0.16±0.02), the width at the level of oesophagus-intestinal junction is 0.21-0.75(0.45±0.19), and the width of the posterior region of body is 0.57-0.96(0.7±0.14).
The distance from the head to the beginning of the bacillary stripes is 0.8-0.98 (0.86±0.06), and the length of the bacillary strips is 1.8-2 (1.92±0.07).
The reproductive system has a spicular tube with a length of 4.31-5.29 (5.0±0.43),diameter of spicule is 0.012-0.017(0.014± 0.001), and the width of the proximal end of the spicule is 0.02-0.04(0.027±0.007).The maximum length of the spicular sheath is 1.07-1.4(1.21±0.11), the width of the spicular sheath is 0.25-0.4(0.29±0.05),and the width of the spicular sheath at the tail end of body is 0.25-0.4(0.29±0.05).The distal bulb/expansion length measurement is 0.13-0.21(0.16±0.02), and the distal bulb/expansion width is 0.28-0.43(0.336±0.05).The testis is the first part of the genital apparatus, and it is very long and highly convoluted, starting in the posterior part of the male body, oriented anteriorly, and extending along the longitudinal axis of the body, finishing not far from the oesophagus transition into the intestine.The vas deferens run somewhat anteriorly along the intestine, connect the testis in the initial component of the genital apparatus and are pretty lengthy and convoluted to the ejaculatory duct via a tiny tube.The distance between posterior part of testis and tail end of body is 2.85-10.4(6.44 ±2.75).The length of the ejaculatory duct is 2.57-4.8(4.23±0.86),length of the cloaca is 3.51 ±0.48 (3.2-4.45).The spicule is in the distal cloacal tube, and protrudes from the sheath in a distinctive spherical expansion.The spines on distal expansion are longer than those on the spicular sheath (Fig 1B).

Female worms characteristics
The body is 36-69 mm (50.4±12.46)long.The ratio between anterior and posterior body length is 1.75:1.The thin anterior part is 0.63:1 on the entire body length (Fig 1C).The total length of the oesophagus is 18-52 (32±12.37).The width of the oesophageal region of the body is 0.15-0.22(0.19±0.02), the body width at the level of the oesophagus-intestinal junction is 0.31-0.87(0.47±0.20), the width of the posterior region of body is 0.7-1.1 (0.81±0.14).The distance from the head to the beginning of the bacillary stripes is 0.96-1.37(1.26±0.15),and the length of bacillary stripes is 1.3-2.3(1.68±0.37).
A single uterus and vulva are non-protrusive and with a length of 0.02-0.49(0.24±0.15) at the confluence of the oesophagus and the intestine.The length of muscular zone of the oesophagus is 0.71-2 (1.00±0.49).The vagina is lengthy, about 2.3-4.45(0.24±0.15), with circumvolutions close to the uterus, a brief straight zone, an extended middle part, and one gently coiled straight portion at the vulva.The distance of the uterus's posterior loop from the body's tail end is 0.14-1.24(0.92±0.41).The ovary is lengthy and connects to the oviduct in the back of the body.The distance of tail end of body and posterior fold of seminal receptacle is 0.34-0.5 (0.41±0.05).At the end of the tail, the anus is sub-terminally located (Fig 1C).The eggs found in the gravid uterus were barrel-shaped and had clear mucoid-appearing polar plugs with a brown outer covering (Fig 1C

Discussion
It is assumed that T. ovis were the predominant species in the Punjab province of Pakistan [14][15][16][17][18][19].However, all these reports were based on egg and or adult morphology without genetic confirmation of species identity.In the present study, adult Trichuris infecting goats were characterised by the adult morphology at the genus level and the sequences of rDNA ITS-2 region, providing the first documented report of T. skrjabini in the Rawalpindi division of the Punjab province.
First, we have performed the morphological characteristics of Trichuris genus infecting goats.The male of the genus Trichuris displayed a similar morphological pattern in the reproductive system.Additionally, the characteristic of the spicule sheath, spicular length, and the fact that the spicule sheath covers a short spicule was identified in male [34].The presence of vagina with a noneverted vulva allows for distinguishing between the females [35].Trichuris skrjabini morphological characters were compared with T. ovis [Baylis (1932), Ortlepp (1937) and Sarwar (1937)].Morphological characters used for the routine separation of T. ovis from T. skrjabin without the necessity for measurement in the male are the size, shape and degree of eversion of the spicule, and in the female, the type of vulval expansion, nature of the vagina and straight portion of the ovary [36][37][38].However, the length and breadth of egg range overlap between the two species.All the measurements of T. skrjabini recorded in the present study are within the range described by Baskakov [39], Magomedbekov [40], Knight [36] and Hinks and Thomas [38].The speciation of the Trichuris genus is challenging because of the Trichuris phenotypic plasticity, lack of morphological features, and the substantial overlap in morphometric traits among species [41].

Table 1. Estimates of evolutionary divergence between sequences of
Trichuris species.This analysis involved 25 nucleotide sequences.The percentage of base substitutions per site between sequences is shown.The analyses were conducted using the Maximum Composite Likelihood model.All positions containing gaps and missing data were eliminated.There was a total of 318 nucleotide positions in the final dataset.

Conclusions
In conclusion, the molecular confirmation and the phylogenetic analysis of the intestinal nematode of goats confirm, for the first time, the presence of Trichuris skrjabini in the Rawalpindi division of Punjab.Furthermore, Trichuris skrjabini was the only nematode identified in 11 worms from goats.The results of our study have implications for the diagnosis and control of Trichuris in the region and the need for accurate species identification to understand parasite distribution and population genetics.There is a need to identify new genetic markers for molecular analysis of a wide range of Trichuris isolates from different host species and geographical areas to improve our understanding of parasite population genetic structures and transmission dynamics.
/doi.org/10.1371/journal.pone.0290906.t001Therefore, 11 Trichuris rDNA ITS-2 sequences from the present study and 14 Trichuris sequences from NCBI GenBank were examined.The comparison between Trichuris of the present study yielded 99-100% similarity to a previously identified rDNA ITS2 sequence of Trichuris skrjabini (KT630825, AJ489248) isolated from the sheep in Czech Republic and goats in Spain and Trichuris spp.(KJ507245) isolated from the black goat in China.Similarly, phylogenetic analysis indicates that Trichuris from Pakistan revealed a strong genetic link between

Fig 2 .
Fig 2. Maximum-likelihood tree was obtained from the rDNA ITS2 sequences of Trichuris.11 Three unique sequence variants of Trichuris were aligned with 14 NCBI GeneBank sequences of different Trichuris species.The sequences of each species were identified with the name and color circles.The adjacent table indicates the accession number, host, and country of origin.https://doi.org/10.1371/journal.pone.0290906.g002