New Genetic Lineage of Whipworm Present in Bactrian Camel (Camelus Bactrianus)

Veterinary Parasitology 315 (2023) 109886 Available online 21 January 2023 0304-4017/Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). New genetic lineage of whipworm present in Bactrian camel (Camelus bactrianus) Julia Rivero, Cristina Cutillas * , 1 , Rocío Callej´ on Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain ARTICLE INFO Keywords: Whipworm Trichuris Camel Camelus bactrianus Phylogeny ABSTRACT With a global population of around 35 million in 47 countries, camels play a crucial role in the economy of many marginal and desert areas of the world where they survive in harsh conditions. Nonetheless, there is insufficient knowledge regarding camels’ parasite fauna which can reduce their milk and meat production. A molecular study for the Trichuris population of Camelus bactrianus from Spain is presented based on sequences of mitochondrial (cox1, cob, rrnL) and ribosomal (ITS1 and ITS2) DNA regions. Bayesian Inference and Maximum Likelihood methods were used to infer phylogenies for (i) each gene separately, (ii) the combined mitochondrial data, and (iii) the combined mitochondrial and ribosomal dataset. Molecular analyses revealed the existence of two different genetic lineages in the Trichuris parasites populations of C. bactrianus. Future studies should focus on whether there is a coevolution process corresponding to the wild or domestic character of C. bactrianus and Camelus dromedarius. Furthermore, it is necessary to increase integrative taxonomic studies on Trichuris spp. based on morphological, biometric, and molecular data, which will inevitably contribute to our knowledge of the etiology of trichuriasis. 1. Introduction Camels are an important livestock species that have adapted to hot and arid environments. The global camel population is estimated to be around 35 million animals in 47 countries. Approximately 85% of the population is found in Africa and the rest in the Indian subcontinent and Middle East countries (Demelash et al., 2014). The genus Camelus has two species, the one-humped camel (Camelus dromedarius) and the two-humped camel (Camelus bactrianus). Camels have been an important component of the desert ecosystem since time immemorial; humans depend on this animal not only for meat, milk and hide, but also as one of the most important means of transport in the desert and is recognized as the “Ship of the desert”. Camel production is practiced by herding communities under diverse constraints in dry and marginal areas. It is known that parasitic diseases of the gastrointestinal tract are the primary health problem of camels worldwide (Kinne et al., 2002; Radfar and Aminzadeh, 2013). Parasitic infections in camels may reduce milk and meat production, affect fertility and decrease calving rates (Sazmand and Joachim, 2017). They can also decrease work efficiency or even cause death and, consequently, significant economic loss. Camels play an important role in the epidemiology of parasitic diseases under the three aspects of zoonoses, animal health and transmission to other livestock. Whipworms are widespread soil-transmitted helminths that can be found in a wide range of hosts, including humans (Trichuris trichiura), non-human primates (NHP) (Trichuris spp.), pigs (Trichuris suis), dogs (Trichuris vulpis), goats, sheep, bovines and camelids (Trichuris ovis, Trichuris discolor and Trichuris skrjabini), among others (Cutillas et al., 2007, 2009; Liu et al., 2012; Callej´ on et al., 2017; Ahmad et al., 2019). These parasites are transmitted by the direct fecal-oral route and are among the most common intestinal parasites of humans and animals, causing significant disease and economic loss globally (Jex et al., 2014; Roepstorff et al., 2011). For many years, the systematics of the genus Trichuris Roeder, 1761 at the species level has been considered controversial. Morphological and biometric studies were performed initially to identify the different Trichuris species. However, these approaches are not always satisfactory since Trichuris species overlap in most morpho-biometric features, especially in the closest species where differentiation is challenging (Cutillas et al., 2009; Robles, 2011). rDNA markers have provided * Correspondence to: Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Prof. García Gonz´ alez 2, 41012 Seville, Spain. E-mail address: [email protected] (C. Cutillas). 1 ORCID: 0000-0002-3886-1231 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar https://doi.org/10.1016/j.vetpar.2023.109886 Received 28 September 2022; Received in revised form 17 January 2023; Accepted 18 January 2023 Veterinary Parasitology 315 (2023) 109886 2 genetic information that allows taxonomically characterizing different species of the genus and even establishing relationships between sister species (Gasser et al., 2001; Rivero et al., 2020). Subsequently, mitochondrial markers have been used due to their rapid rate of evolutionary change, less recombination, and moderately conserved genomic structures for population genetics, systematics and phylogenetic relationships, such as cytochrome oxidase 1 (cox1), cytochrome b (cob) or 16 S gene (Cavallero et al., 2015, 2019; Callej´ on et al., 2017; Rivero et al., 2020, 2021a). In addition, some studies on the complete analysis of the mitochondrial genome have been determined to obtain the greatest amount of information for the taxonomic and phylogenetic studies (Liu et al., 2012; Hawash et al., 2015; Wang et al., 2019; Rivero et al., 2021b). Some authors evaluated the utility of mitochondrial and ribosomal genes for the molecular systematics of Trichuris and parasitic nematodes and concluded that combined molecular analysis based on nuclear and mitochondrial genes allows for a more complete understanding of the taxonomy and evolution of Trichuris species (Chan et al., 2020; Rivero et al., 2020). In recent years, the designation of parasitic species within the Trichuris genus has been determined using an integrative approach based on the combination of morphological and molecular methodologies (Rivero et al., 2020, 2021a, 2022). Within the genus Trichuris, various authors have referenced synonymies (Oliveros et al., 2000), cryptic species (Callej´ on et al., 2012), different lineages (Rivero et al., 2020), and new species (Liu et al., 2013; Cutillas et al., 2014; Robles et al., 2014; Callej´ on et al., 2017; Wang et al., 2019). From ruminants, more than 23 Trichuris species have been previously described (Knight, 1974). Several studies have reported that whipworms from domestic ruminants (T. ovis Abildgaard, 1795, T. globulosa Von Linstow, 1901, Trichuris discolor Von Linstow, 1906 and T. skrjabini Baskakov, 1924) may parasitize different hosts since nematodes isolated from sheep or cattle have been found in camels. Hence, in addition to the species of Trichuris specific to camelids (Trichuris tenuis) (Rickard and Bishop, 1991; Cafrune et al., 1999), these can host more Trichuris species (e.g., T. ovis, T. globulosa, T. skrjabini and Trichuris spp.) (Tait et al., 2002; Callej´ on et al., 2015a; Montalbano Di Filippo et al., 2020). Therefore, a complex of Trichuris species can be found in camels. Detailed epidemiological studies on camel parasites strongly demand molecular diagnostic tools to correctly classify species and genotypes in order to improve existing diagnostic tools and provide more detailed insight into the epidemiology, transmission and risk factors of these parasites. These results will advance an economically important integrated control program against camel parasites and include parasites in camel health surveillance. For this reason, we carried out a molecular and phylogenetic study of Trichuris population from C. bactrianus obtained from a zoological garden. We analyzed the relationships among the different species of Trichuris that parasitize camels and other closed Trichuris species. 2. Materials and methods 2.1. Ethics statement This study does not require approval by an ethics committee. Trichuris specimens were collected from stool samples from C. bactrianus that were housed in a zoological garden in Spain and handled in strict accordance with good animal practices. 2.2. Collection samples Whipworms of Trichuris spp. were collected from the fecal samples after anthelmintic treatment of C. bactrianus. These camels were housed in “Parque de la Naturaleza de Cab´ arceno” in Cantabria province, Spain. Four adult worms were recollected and washed extensively in 0.9% saline solution and then, stored at −20º C until further studies. Adults of Trichuris sp. from C. bactrianus were identified at the genus level exclusively based on the external characteristics of the adults (Cutillas et al., 1995; Callej´ on et al., 2015a) due to adult worms were deteriorated due to the treatment received to obtain the fecal samples from the camels, and morphological studies could not be completed. 2.3. Molecular and phylogenetic analysis The total genomic DNA from whipworms were extracted according to the manufacturer’s protocol using DNeasy Blood and Tissue Kit (Qiagen). 0.8% agarose gel electrophoresis with SYBR® Safe DNA gel stain was assessed for the quality of extractions. The molecular markers sequenced (cox1, cob and rrnL mtDNA, and ITS1 and ITS2 rDNA) were amplified by the polymerase chain reaction (PCR) using a thermal cycler (Eppendorf AG; Hamburg, Germany). PCR primers, PCR conditions and PCR mix were summarized in Supplementary Table S1. The PCR products were verified on SYBR® Safe stained 2% Tris-Borate-EDTA (TBE) agarose gels. PCR products were purified using QWizard SV Gel and PCR Clean-Up System Kit (Promega, Madison, WI, U.S.A.). Then, the purified products were concentrated and sequenced by Stab Vida (Lisbon, Portugal). For all markers analyzed, the identity of the of Trichuris sequences from this study were compared to other Trichuris spp. sequences obtained from the National Centre for Biotechnology Information (NCBI) GenBank database. The number of nucleotide differences per sequence was determined using Compute Pairwise Distances based on the number of differences method of MEGA X v10.1.8 (Kumar et al., 2018). To understand and analyze the relationships among the Trichuris species and Trichuris sp. from C. bactrianus (obtained in this study), additional sequences from the NCBI GenBank™ database were incorporated into alignments (Table 1). All phylogenetic trees were rooted by including Trichinella spp. as an outgroup. In addition, nucleotide sequence data obtained in this work are available in the GenBank™ database with the accession number reported in Table 1 and Supplementary Table S2. MUSCLE alignment method in MEGA X was used to obtain the nucleotide sequence alignment files (Kumar et al., 2018). Two methods based on nucleotide data were inferred to produce the phylogenetic trees, based on either Maximum Likelihood (ML) and Bayesian Inference (BI). ML was generated using PhyML 3.0 (Guindon et al., 2010), and MrBayes v3.2.6 to produce BI (Ronquist and Huelsenbeck, 2003). To determine the best-fit substitution model for the parasite data, jModelTest was utilized (Posada, 2008). According to Akaike information criterion, evolution models were chosen for subsequent analysis (Huelsenbeck and Rannala, 1997; Posada and Buckley, 2004). To Table 1 List of Trichuris samples obtained in the present work based on the different markers (cox1, cob, rrnL, ITS1 and ITS2) including GenBank accession numbers, G +C content and length of sequences. ID sample Accession number Marker Length (bp) G +C content (%) TCH1 OM920890 cox1 369 39.60 TCH2 OM920891 369 39.60 TCH5 OM920892 369 39.60 TCM1 OM920893 369 39.60 TCH1 OM912468 cob 519 31.60 TCH2 OM912469 519 31.98 TCH5 OM912470 519 31.98 TCM1 OM912471 519 31.60 TCH1 OM920886 rrnL 362 27.60 TCH2 OM920887 362 27.60 TCH5 OM920888 362 27.90 TCM1 OM920889 362 27.60 TCH1 OM920875 ITS1 521 59.90 TCH2 OM920876 521 59.30 TCH5 OM920877 521 59.50 TCM1 OM920878 521 59.30 TCH1 OM920881 ITS2 522 58.60 TCH2 OM920882 522 58.30 TCH5 OM920883 522 58.50 TCM1 OM920884 522 58.30 J. Rivero et al. Veterinary Parasitology 315 (2023) 109886 3 investigate the phylogenetic relationships, the partitioned datasets were analyzed by gene and models for individual genes (two rDNA (ITS1 and ITS2) and three mtDNA (cox1, cob and rrnL)), and the concatenation of two mitochondrial markers (cox1 and cob), two ribosomal markers (ITS1 and ITS2), and the concatenation of two mitochondrial and two ribosomal markers (cox1, cob, ITS1 and ITS2). The rrnL marker was not used for concatenated trees due to the lack of sequences. For ML inference, the best-fit nucleotide substitution model included a transition model with gamma-distributed rate variation and a proportion of invariable sites, TIM2 +I +G for the cox1 marker and TIM1 +I +G for rrnL marker; a transversion model with gamma-distributed rate variation and a proportion of invariable sites, TVM +I +G for cob marker; a general time reversible with gamma-distributed rate variation and a proportion of invariable sites, GTR +I+G for ITS1; and a general time reversible with gamma-distributed rate variation among sites for ITS2. Bootstrapping was utilized to examine the support for the topology (heuristic option) (Felsenstein, 1985) using over 1000 replications to evaluate the relative reliability of clades. For MrBayes, the command used was nst=6 with gamma rates (ITS2) and nst=6 with invgamma rates (ITS1, cox1, cob and rrnL). For BI, the standard deviation of split frequencies was used to determine whether the number of generations completed was sufficient. The chain was sampled every 500 generations and each dataset was run for 10 million generations. Based on an assessment of convergence, trees from the first million generations were discarded. Burn-in was resolved empirically by examination of the log-likelihood values of chains. The Bayesian Posterior Probabilities (BPP) include the significance scores for the nodes. 3. Results 3.1. Molecular Results Four individuals of Trichuris sp. from C. bactrianus were extracted and all the markers (ITS1, ITS2, cox1, cob and rrnL) were amplified and sequenced of each specimen. The length of the sequences, the G+C content and the GenBank accession numbers is summarized in Table 1. The multiple alignment of 45 cox1 nucleotide sequences, including outgroups, yielded a dataset of 291 characters. For cob, the multiple alignment was among 44 nucleotide sequences and a dataset of 384 characters. For rrnL, the multiple alignment of 17 nucleotide sequences yielded a dataset of 392 characters. Finally, for ITS1 and ITS2, the multiple alignment of 35 nucleotide sequences yielded a dataset of 1114 characters and the multiple alignment of 40 nucleotide sequences yielded a dataset of 910 characters, respectively. For cox1, cob, rrnL, ITS1 and ITS2 sequences the percentages of interspecific identity between our sequences and the other species of Trichuris from Camelus spp. are shown in Supplementary Tables S3, S4, S5, S6 and S7, respectively. Phylogenetic trees inferred by ML and BI methods of each marker showed similar results among Trichuris sequences assessed in this study. Fig. 1. Phylogenetic tree of Trichuris species based on analysis of ITS2 ribosomal DNA inferred using Bayesian Inference. Maximum Likelihood bootstrap values of clades are listed first, followed by Bayesian Posterior Probabilities, respectively, for clade frequencies exceeding 60%. J. Rivero et al. Veterinary Parasitology 315 (2023) 109886 4 In all trees, the four sequences from Trichuris sp. from C. bactrianus from Spain were clustered together in the same clade with high bootstrap values and BPP (Figs. 1–5). Due to the lack of Trichuris sequences in some markers, not all the trees could be compared with the same sequences and non-representative trees were obtained for each marker separately. Hence, the rrnL marker could not be concatenated with other markers and instead, the ITS2 dataset was analyzed independently due to many sequences of Trichuris sequenced for this marker. The phylogenetic analysis of the ITS2 dataset yielded a tree with no well-supported values to infer the phylogenetic relationship between Trichuris species (Fig. 1). However, it revealed a moderated support to delimit species. Thus, Trichuris sp. from C. bactrianus from Spain and the Czech Republic were grouped with strongly supported nodes related to T. leporis, but not associated with the T. globulosa sequences from C. dromedarius. The concatenated dataset of partial ITS1 and ITS2 rDNA sequences included 2024 aligned sites and 30 taxa (including the outgroup). The phylogeny revealed the existence of four different clades: clade 1 including Trichuris spp. from humans, NHP and suid; clade 2, including Trichuris spp. from canids and rodents; clade 3, including Trichuris spp. from camel, sheep, goat, and hare; and clade 4 including cows, goats, sheep, and the greater cane rat. Clades were well supported based on ML and BI methods except that clade 4 with a bootstrap value of less than 65% based on ML methods. Phylogenetic inferences of Trichuris sp. From C. bactrianus with respect to other Trichuris spp. revealed a sister group that includes T. skrjabini, Trichuris sp. from Ovis spp. and T. leporis with high support values (Fig. 2). The partial rrnL mtDNA dataset included 392 aligned sites and 17 taxa (including outgroups). Phylogenetic inferences for the genus based on rrnL were not resolved because some were in polytomy (Fig. 3). Even so, Trichuris sp. from C. bactrianus formed a solid group related to T. skrjabini as a sister group with high bootstrap values and BPP. The concatenated dataset of partial cox1 and cob mtDNA sequences included 675 aligned sites and 35 taxa (including outgroups). In congruence with concatenated dataset rDNA (ITS1 and ITS2), the four main clades cited were revealed with high branches support. An additional clade (clade 5), including Trichuris sp. from Hystrix cristata, was considered for mitochondrial datasets. Trichuris spp. from camels were separated into different clades. In this way, the population of Trichuris sp. from C. bactrianus from Spain was related to T. skrjabini as a sister group with high bootstrap values and BPP within clade 3, and on the other way, Trichuris sp. from C. bactrianus from Italy (rDNA sequences not available) appeared within clade 4 clustered with T. ovis and more related to Trichuris sp. from Addax nasomaculatus from Italy and T. globulosa from C. dromedarius from Iran. In any case, of all the clades in the genus Trichuris, both clades (which included Trichuris spp. parasitizing herbivorous host species) were the most phylogenetically related, being more separated from clades 1, 4 and 5 (Fig. 4). Finally, the concatenated dataset of partial ribosomal (ITS1 and ITS2 rDNA) and mitochondrial (cox1 and cob mtDNA) sequences included 2728 aligned sites and 15 taxa (including outgroup). Due to missing sequences in most markers, many Trichuris species sequences could not be added to the analysis. The four main clades were obtained in congruence with phylogenetic inferences based on mitochondrial and ribosomal datasets. Trichuris sequences obtained in this study clustered together, and this population appeared as a sister group to T. skrjabini (clade 3) with high BPP and bootstrap values separated from all other Trichuris clades (Fig. 5). Fig. 2. Phylogenetic tree of Trichuris species based on combined analysis of ribosomal DNA (ITS1 and ITS2) inferred using Bayesian Inference. Maximum Likelihood bootstrap values of clades are listed first, followed by Bayesian Posterior Probabilities, respectively, for clade frequencies exceeding 60%. J. Rivero et al. Veterinary Parasitology 315 (2023) 109886 5 4. Discussion The present paper reflects the current knowledge of the phylogenetic analysis of the Trichuris lineages of camels from C. bactrianus from Spain and other camel populations from different geographical origins. Molecular techniques have been carried out to clarify the Trichuris systematics in camelids. No biometrical and morphological data were obtained for Trichuris from C. bactrianus in this work and, therefore, comparative morpho-biometrical studies could not be performed. To assess the utility of all the different markers (cox1, cob and rrnL mtDNA, and ITS1 and ITS2 rDNA) used in this study, intra and inter-specific identity and phylogenetic tree studies have been performed. In all markers analyzed, the identity percentage obtained among the Trichuris sequences obtained in this study from C. bactrianus showed values corresponding to intra-specific identity according to the range cited by several authors in different studies for the genus Trichuris (Blouin et al., 2002; Callej´ on et al., 2015b; Chan et al., 2020). Comparative analysis of Trichuris sequences obtained in this study with other Trichuris sequences from camels revealed that only two sequences of Trichuris from C. bactrianus from the Czech Republic, one sequenced for the cox1 mtDNA and one for ITS2 rDNA markers, showed the same percentage of identity (98–100%) with our sequences, corresponding with the same species. Furthermore, Trichuris sp. sequences from C. bactrianus from Italy and T. globulosa showed values within the range of inter-specific identity (Blouin et al., 2002). Thus, the identity percentage observed between T. globulosa and Trichuris sp. sequences from C. bactrianus from Italy were higher than the values of T. globulosa with Trichuris sp. from C. bactrianus from Spain. Hence, the species compared above, having a lower inter-specific identity, also appeared to be in the range of different species. Moreover, the greatest inter-specific identity among our sequences and other Trichuris species was with T. skrjabini. These values were higher in rDNA markers (approximately 95%) than in mtDNA markers. In addition, the interspecific identity was usually higher in rDNA markers (ITS1 and ITS2) than in mtDNA markers (cox1, cob). However, the rrnL mitochondrial marker presented intermediate values. This fact agrees with several authors (Callej´ on et al., 2015b; Chan et al., 2020; Rivero et al., 2021a), who support the hypothesis that ITS regions show a higher nucleotide saturation than mitochondrial markers due to this region accumulating substitutions more slowly. For this reason, ITS markers were not sufficient to infer phylogenetic relationships among the different related populations of Trichuris, but they were effective in differentiating among other species that were more evolutionarily separated. With respect to mitochondrial markers, the rrnL partial gene has been suggested as the Fig. 3. Phylogenetic tree of Trichuris species based on analysis of rrnL mitochondrial DNA inferred using Bayesian Inference. Maximum Likelihood bootstrap values of clades are listed first, followed by Bayesian Posterior Probabilities, respectively, for clade frequencies exceeding 60%. J. Rivero et al. Veterinary Parasitology 315 (2023) 109886 6 best marker to differentiate among close genetic lineages since nucleotide saturation was lower than ITS markers but higher than cox1 and cob genes. This means that the rrnL gene provided a higher resolution for phylogenetic studies than the other mitochondrial markers due to the rrnL gene presenting a lower intraspecific variability (Rivero et al., 2021a). Nevertheless, because of the lack of sequences for the rrnL partial gene in Trichuris species, only a few sequences have been able to be studied. For this reason, a concatenated dataset with rrnL could not be done. Consequently, a concatenated dataset of mitochondrial (cox1 and cob genes) and ribosomal (ITS1 and ITS2) markers supplied higher resolution for phylogenetic studies (Rivero et al., 2020, 2021a), providing in this study strongly supported nodes, differentiating among the different clades. Attending to the identity percentage observed for the other markers, we suggest that the population of Trichuris sp. from C. bactrianus from Spain corresponds to a different species from those cited for C. bactrianus and C. dromedarius and other herbivores. Therefore further studies based on parallel morpho-biometric and molecular data are necessary for its description. The results obtained in the identity study corresponded with the phylogenetic results. The sequences obtained in this study were grouped in the same clade as the Trichuris sequence of C. bactrianus from the Czech Republic (clade 3). However, other populations of Trichuris parasitizing camels (C. bactrianus from Italy and C. dromedarius from Iran) were shown separated into a different clade (clade 4) (Fig. 4). In all phylogenetic trees, both in the individual marker data sets and in the concatenated data sets, our sequences showed a close relationship with T. skrjabini as a sister group with both populations forming a wellsupported clade separate from all other clades. The identity results were consistent with the phylogenetic results. Hence, our sequences obtained the highest identity values compared to T. skrjabini and lower compared to T. globulosa and Trichuris sp. from C. bactrianus from Italy, appearing these last populations within clade 4, suggesting that these populations correspond to different species according to interspecific and phylogenetic results but closely related genetically, being separated from the population of Trichuris sp. from C. bactrianus from Spain. This result may be due to the geographical difference among them, the type of food, and the inter-specific transmission route among related host species, or it may be related to the passive transport of eggs through zookeepers or freely circulating animals (Kvapil et al., 2017; Montalbano Di Filippo et al., 2020). On the other hand, the evolutionary relationship between the domestic and wild Bactrian camel (two-- humped camels) and the actual origin of the domestic Bactrian is not clarified. Ji et al. (2009) revealed that the extant wild two-humped camel might not share a common ancestor with the domestic Bactrian camel and they are not the same subspecies except in their maternal origins. Complete mitochondrial genome sequences indicated that the wild camel is a separate lineage but not the direct progenitor of the domestic Bactrian camel. Later, phylogenetic analyses suggested that the wild Bactrian camel appeared monophyletic in evolutionary origin and that the domestic Bactrian camel could come from a single wild population. Our results showed two different genetic lineages in the populations of Trichuris parasites of C. bactrianus, one of them (from Spain and the Czech Republic) genetically related to T. skrjabini and another Fig. 4. Phylogenetic tree of Trichuris species based on combined analysis of mitochondrial DNA (cox1, cob) inferred using Bayesian Inference. Maximum Likelihood bootstrap values of clades are listed first, followed by Bayesian Posterior Probabilities, respectively, for clade frequencies exceeding 60%. J. Rivero et al. Veterinary Parasitology 315 (2023) 109886 7 population (from Italy) associated with T. globulosa of C. dromedarius. Future studies should focus on whether a coevolution process corresponds to the wild or domestic character of C. bactrianus and C. dromedarius. 5. Conclusions Camels play a crucial role in the economy of many marginal and desert areas of the world, where they survive under harsh conditions. Nevertheless, there is insufficient knowledge regarding camel’s parasite fauna which can reduce their milk and meat production. This work provides data on Trichuris species parasitizing C. bactrianus based on ribosomal and mitochondrial markers. The percentage of identity observed for the different markers revealed that the population of Trichuris sp. from C. bactrianus from Spain corresponds to a different species from those cited for camels and other herbivores showing a sister relationship with respect to T. skrjabini. These results could allow the scientific community and organizations responsible for formulating camel health policies to clarify the role of these animals in the epidemiology of zoonotic parasitic diseases and view a possible increase in their prevalence. Hence, further studies based on parallel morpho-biometric and molecular data are necessary to clarify the knowledge of Trichuris species that may be responsible for trichuriasis in camels. Funding Grant CGL2017–83057-P funded by MCIN/AEI/ 10.13039/ 501100011033 and, as appropriate, by “ERDF A way of making Europe”. The Junta de Andalucía (BIO-338), and a grant from the V and VI Plan Propio de Investigaci´ on of the University of Seville, Spain. CRediT authorship contribution statement Julia Rivero: Methodology, Validation, Investigation, Formal analysis, Software, Data curation, Writing – original draft, Visualization. Cristina Cutillas: Conceptualization, Formal analysis, Writing – review & editing, Funding acquisition, Resources, Project administration, Supervision. Rocío Callej´ on: Methodology, Formal analysis, Software, Data curation, Writing – original draft, Visualization, Writing – review & editing, Supervision. Declaration of Competing Interest The authors declare no competing interests. Acknowledgements Special thanks to “Parque de la Naturaleza de Cab´ arceno” (Cantabria, Spain) for providing faeces samples of Camelus bactrianus with adults of Trichuris sp. We are in debt with Dr. Steven Doyle for the critical correction of the manuscript. Ethics approval and consent to participate Not applicable. This study does not require approval by an ethics committee. C. bactrianus, from which Trichuris specimens were collected from their feces post-treatment with anthelminthic. The specimen was handled and housed in a zoo in strictly following good animal practices. Fig. 5. Phylogenetic tree of Trichuris species based on combined analysis of mitochondrial DNA (cox1, cob) and ribosomal DNA (ITS1 and ITS2) inferred using Bayesian Inference. Maximum Likelihood bootstrap values of clades are listed first, followed by Bayesian Posterior Probabilities, respectively, for clade frequencies exceeding 60%. J. Rivero et al. Veterinary Parasitology 315 (2023) 109886 8 Consent for publication Not applicable. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.vetpar.2023.109886. References Ahmad, A.A., Shabbir, M.A.B., Xin, Y., Ikram, M., Hafeez, M.A., Wang, C., Zhang, T., Zhou, C., Yan, X., Hassan, M., Hu, M., 2019. Characterization of the Complete Mitochondrial Genome of a Whipworm Trichuris skrjabini (Nematoda: Trichuridae). Genes 10 (6), 438. https://doi.org/10.3390/genes10060438. Cafrune, M.M., Aguirre, D.H., Rickard, L.G., 1999. Recovery of Trichuris tenuis Chandler, 1930, from camelids (Lama glama and Vicugna vicugna) in Argentina. J. Parasitol. 85, 961–962. Callej´ on, R., Cutillas, C., Nadler, S.A., 2015b. Nuclear and mitochondrial genes for inferring Trichuris phylogeny. Parasitol. Res. 114 (12), 4591–4599. https://doi.org/ 10.1007/s00436-015-4705-7. Callej´ on, R., Guti´ errez-Avil´ es, L., Halajian, A., Zurita, A., de Rojas, M., Cutillas, C., 2015a. Taxonomy and phylogeny of Trichuris globulosa Von Linstow, 1901 from camels. A review of Trichuris species parasitizing herbivorous. Infect. Genet. Evol. 34, 61–74. https://doi.org/10.1016/j.meegid.2015.06.011. Callej´ on, R., Halajian, A., Cutillas, C., 2017. Description of a new species, Trichuris ursinus n. sp. (Nematoda: Trichuridae) from Papio ursinus Keer, 1792 from South Africa. Infect. Genet. Evol. 51, 182–193. https://doi.org/10.1016/j. meegid.2017.04.002. Callej´ on, R., Halajian, A., de Rojas, M., Marrugal, A., Guevara, D., Cutillas, C., 2012. 16S partial gene mitochondrial DNA and internal transcribed spacers ribosomal DNA as differential markers of Trichuris discolor populations. Vet. Parasitol. 186 (3–4), 350–363. https://doi.org/10.1016/j.vetpar.2011.11.033. Cavallero, S., De Liberato, C., Friedrich, K.G., Di Cave, D., Masella, V., D’Amelio, S., Berrilli, F., 2015. Genetic heterogeneity and phylogeny of Trichuris spp. from captive non-human primates based on ribosomal DNA sequence data. Infect. Genet. Evol. 34, 450–456. https://doi.org/10.1016/j.meegid.2015.06.009. Cavallero, S., Nejsum, P., Cutillas, C., Callej´ on, R., Doleˇ zalov´ a, J., Modrý, D., D’Amelio, S., 2019. Insights into the molecular systematics of Trichuris infecting captive primates based on mitochondrial DNA analysis. Vet. Parasitol. 272, 23–30. https://doi.org/10.1016/j.vetpar.2019.06.019. Chan, A., Chaisiri, K., Morand, S., Saralamba, N., Thaenkham, U., 2020. Evaluation and utility of mitochondrial ribosomal genes for molecular systematics of parasitic nematodes. Parasit. Vectors 13 (1), 364. https://doi.org/10.1186/s13071-020- 04242-8. Cutillas, C., Callej´ on, R., De Rojas, M., Tewes, B., Úbeda, J.M., Ariza, C., Guevara, D.C., 2009. Trichuris suis and Trichuris trichiura are different nematode species. Acta Trop. 111, 299–307. https://doi.org/10.1016/j.actatropica.2009.05.011. Cutillas, C., de Rojas, M., Ariza, C., Ubeda, J.M., Guevara, D., 2007. Molecular identification of Trichuris vulpis and Trichuris suis isolated from different hosts. Parasitol. Res. 100 (2), 383–389. https://doi.org/10.1007/s00436-006-0275-z. Cutillas, C., de Rojas, M., Zurita, A., Oliveros, R., Callej´ on, R., 2014. Trichuris colobae n. sp. (Nematoda: Trichuridae), a new species of Trichuris from Colobus guereza kikuyensis. Parasitol. Res 113 (7), 2725–2732. https://doi.org/10.1007/s00436-014- 3933-6. Cutillas, C., German, P., Arias, P., Guevara, D., 1995. Trichuris ovis and Trichuris globulosa: morphological, biometrical, and genetic studies. Exp. Parasitol. 81 (4), 621–625. https://doi.org/10.1006/expr.1995.1159. Demelash, K., Alemu, F., Niguse, A., Feyera, T., 2014. Prevalence of gastrointestinal parasites and efficacy of anthelmintics against nematodes in camels in Yabello District. South. Ethiop. Acta Parasitol. Glob. 5 (3), 223–231. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x. Gasser, R.B., Zhu, X., Beveridge, I., Chilton, N., 2001. Mutation scanning analysis of sequence heterogeneity in the second internal transcribed spacer (rDNA) within some members of the Hypodontus macropi (Nematoda: Strongyloidea) complex. Electrophoresis 22 (6), 1076–1085, 10.1002/1522-2683(-)22:6<1076::AIDELPS1076>3.0.CO;2-8. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59 (3), 307–321. https://doi. org/10.1093/sysbio/syq010. Hawash, M.B., Andersen, L.O., Gasser, R.B., Stensvold, C.R., Nejsum, P., 2015. Mitochondrial genome analyses suggest multiple trichuris species in humans, baboons, and pigs from different geographical regions. PLoS Negl. Trop. Dis. 9 (9), e0004059 https://doi.org/10.1371/journal.pntd.0004059. Huelsenbeck, J.P., Rannala, B., 1997. Phylogenetic methods come of age: testing hypotheses in an evolutionary context. Science 276 (5310), 227–232. https://doi. org/10.1126/science.276.5310.227. Jex, A.R., Nejsum, P., Schwarz, E.M., Hu, L., Young, N.D., Hall, R.S., Korhonen, P.K., Liao, S., Thamsborg, S., Xia, J., Xu, P., Wang, S., Scheerlinck, J.P., Hofmann, A., Sternberg, P.W., Wang, J., Gasser, R.B., 2014. Genome and transcriptome of the porcine whipworm Trichuris suis. Nat. Genet. 46 (7), 701–706. https://doi.org/ 10.1038/ng.3012. Ji, R., Cui, P., Ding, F., Geng, J., Gao, H., Zhang, H., Yu, J., Hu, S., Meng, H., 2009. Monophyletic origin of domestic bactrian camel (Camelus bactrianus) and its evolutionary relationship with the extant wild camel (Camelus bactrianus ferus). Anim. Genet. 40 (4), 377–382. https://doi.org/10.1111/j.1365-2052.2008.01848.x. Kinne, J., Ali, M., Wernery, U., Dubey, J.P., 2002. Clinical large intestinal coccidiosis in camels (Camelus dromedarius) in the United Arab Emirates: description of lesions, endogenous stages, and redescription of Isospora orlovi, Tsygankov, 1950 oocysts. J. Parasitol. 88 (3), 548–552. https://doi.org/10.1645/0022-3395(2002)088[0548: CLICIC]2.0.CO;2. Knight, R.A., 1974. Trichuris oreamnos sp. n. from the mountain goat, Oreamnos americanus (Blainville), in British Columbia, Canada, and a key to trichurids in North American ruminants. J. Parasitol. 60 (2), 275–279. Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K., 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35 (6), 1547–1549. https://doi.org/10.1093/molbev/msy096. Kvapil, P., Kastelic, M., Dovc, A., Bartova, E., Cizek, P., Lima, N., Strus, S., 2017. An eight-year survey of the intestinal parasites of carnivores, hoofed mammals, primates, ratites and reptiles in the Ljubljana zoo in Slovenia, 2017.013 Folia Parasitol. (Praha) 64. https://doi.org/10.14411/fp.2017.013. Liu, G.H., Gasser, R.B., Nejsum, P., Wang, Y., Chen, Q., Song, H.Q., Zhu, X.Q., 2013. Mitochondrial and nuclear ribosomal DNA evidence supports the existence of a new Trichuris species in the endangered françois’ leaf-monkey. PLoS One 8 (6), e66249. https://doi.org/10.1371/journal.pone.0066249. Liu, G.H., Wang, Y., Xu, M.J., Zhou, D.H., Ye, Y.G., Li, J.Y., Song, H.Q., Lin, R.Q., Zhu, X. Q., 2012. Characterization of the complete mitochondrial genomes of two whipworms Trichuris ovis and Trichuris discolor (Nematoda: Trichuridae). Infect. Genet. Evol. 12 (8), 1635–1641. https://doi.org/10.1016/j.meegid.2012.08.005. Montalbano Di Filippo, M., Berrilli, F., De Liberato, C., Di Giovanni, V., D’Amelio, S., Friedrich, K.G., Cavallero, S., 2020. Molecular characterization of Trichuris spp. from captive animals based on mitochondrial markers. Parasitol. Int. 75, 102043 https:// doi.org/10.1016/j.parint.2019.102043. Oliveros, R., Cutillas, C., De Rojas, M., Arias, P., 2000. Characterization of four species of Trichuris (Nematoda: Enoplida) by their second internal transcribed spacer ribosomal DNA sequence. Parasitol. Res. 86 (12), 1008–1013. https://doi.org/10.1007/ pl00008519. Posada, D., 2008. jModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. https://doi.org/10.1093/molbev/msn083. Posada, D., Buckley, T.R., 2004. Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests. Syst. Biol. 53, 793–808. https://doi.org/10.1080/10635150490522304. Radfar, M.H., Aminzadeh, G.M., 2013. Common gastrointestinal parasites of indigenous camels (Camelus dromedarius) with traditional husbandry management (free-ranging system) in central deserts of Iran. J. Parasit. Dis. 37, 225–230. https://doi.org/ 10.1007/s12639-012-0170-8. Rickard, L.G., Bishop, J.K., 1991. Redescription of Trichuris tenuis Chandler, 1930, from llamas (Lama glama) in Oregon with a key to the species of Trichuris present in North American ruminants. J. Parasitol. 71, 70–75. Rivero, J., Callej´ on, R., Cutillas, C., 2021b. Complete Mitochondrial Genome of Trichuris trichiura from Macaca sylvanus and Papio papio. Life 11 (2), 126. https://doi.org/ 10.3390/life11020126. Rivero, J., Cutillas, C., Callej´ on, R., 2021a. Trichuris trichiura (Linnaeus, 1771) From Human and Non-human Primates: Morphology, Biometry, Host Specificity, Molecular Characterization, and Phylogeny. Front. Vet. Sci. 7, 626120 https://doi. org/10.3389/fvets.2020.626120. Rivero, J., García-S´ anchez, ´ A.M., Zurita, A., Cutillas, C., Callej´ on, R., 2020. Trichuris trichiura isolated from Macaca sylvanus: morphological, biometrical, and molecular study. In: BMC Vet. Res, 16, p. 445, 10.1186/s12917-020-02661-4. Erratum in: BMC Vet. Res. 2021. 17(1), 160. Robles, M.R., 2011. New species of Trichuris (Nematoda: Trichuridae) from Akodon montensis Thomas, 1913, of the Paranaense Forest in Argentina. In: J. Parasitol, 97, pp. 319–327. https://doi.org/10.1645/GE-2434.1. Robles, M.R., Cutillas, C., Panei, C.J., Callej´ on, R., 2014. Morphological and molecular characterization of a new Trichuris species (Nematoda- Trichuridae), and phylogenetic relationships of Trichuris species of Cricetid rodents from Argentina. PLoS One 9 (11), e112069. https://doi.org/10.1371/journal.pone.0112069. Roepstorff, A., Mejer, H., Nejsum, P., Thamsborg, S.M., 2011. Helminth parasites in pigs: new challenges in pig production and current research highlights. Vet. Parasitol. 180 (1–2), 72–81. https://doi.org/10.1016/j.vetpar.2011.05.029. Ronquist, F., Huelsenbeck, J.P., 2003. MrBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. https://doi.org/10.1093/ bioinformatics/btg180. Sazmand, A., Joachim, A., 2017. Parasitic diseases of camels in Iran (1931-2017) - a literature review. Parasite 24, 21. https://doi.org/10.1051/parasite/2017024. Tait, S.A., Kirwan, J.A., Fair, C.J., Coles, G.C., Stafford, K.A., 2002. Parasites and their control in South American camelids in the United Kingdom. Vet. Rec. 150 (20), 637–638. https://doi.org/10.1136/vr.150.20.637. Wang, H.B., Zhang, H.J., Song, L.L., Zhu, L., Chen, M., Ren, G.J., Liu, G.H., Zhao, G.H., 2019. Morphological and molecular confirmation of the validity of Trichuris rhinopiptheroxella in the endangered golden snub-nosed monkey (Rhinopithecus roxellana). J. Helminthol. 93 (5), 601–607. https://doi.org/10.1017/ S0022149X18000500. J. Rivero et al.