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Introduction
Nematodes of the Trichuris genus can parasitise the cecum and colon of humans, cattle, sheep, pigs and other animals, causing trichuriasis (also known as whipworm disease), which is harmful to human and livestock health and livestock production (2, 15, 23, 28). In ruminants, Trichuris spp. infection can cause chronic cicatricial inflammation of the cecum and colon, resulting in wasting, anaemia and even death (7, 24). Currently, the disease is widespread and globally distributed, seriously threatening the sustainability and expansion potential of the farming industry (20).
The Trichuris genus includes several species such as T. trichiura, T. suis and T. vulpis, which can infect humans and various animals and are rightfully accorded medical and veterinary importance (9, 11, 14, 18). Since Roederer first named the genus in 1761, so far at least 24 Trichuris species have been documented in ruminants (26, 27). In 2012, 415 domesticated adult ruminants were investigated for evidence of gastrointestinal protozoan and helminthic infections in India and a 9.15% infection rate of T. trichiura was noted (3).
Xinjiang is one of the most important sheep farming regions in China, with a current stock of 36 million sheep. Digestive tract parasitic infections are very common in sheep and result in serious economic losses to farmers who graze their flocks in the pasture areas of Xinjiang. However, the infection status of the region’s sheep and the prevalent species of Trichuris in them are still unknown. Therefore, the main objective of this study was to investigate the prevalences of Trichuris spp. in sheep in the Tianshan pasture areas of Xinjiang and characterise Trichuris spp. using morphological and molecular biological protocols. The investigation’s findings are hoped to provide valuable epidemiological data for the prevention and control of trichuriasis.
Material and Methods
Overview of the surveyed areas
The five pastoral areas in the Xinjiang Tianshan Mountains investigated in this survey are Urumqi, Yili, Bole, Tacheng and Altay (Supplementary Fig. S1). The five pastoral areas are located between 44° and 48°N and 83° and 88°E. The region has a temperate continental climate with an annual precipitation of 500 to 600 mm and an average annual temperature of −4 to 9°C. The average temperatures in summer range from 20 to 25°C and in winter range from −15 to −25°C.
Animals surveyed
During 2019–2021, 1,216 sheep were investigated, of which approximately 70% were under one year old and which had all been slaughtered in five pastoral slaughterhouses. All of the sheep had been reared by grazing. All slaughtered sheep were sorted, registered and numbered. After slaughter and evisceration, the large intestine was collected and transported at low temperature to the Xinjiang Key Laboratory of Animal Disease Prevention and Control.
Methods of investigation
Briefly, the collected large intestine was placed in a basin pre-filled with saline, the intestine was dissected and the contents washed out into the liquid, and then the mucosa was examined carefully in a sequential manner. Meanwhile, the washed-out contents were repeatedly sedimented with saline until the liquid ran clear. The sediment was then taken and the worms carefully examined. Adult Trichuris nematodes were collected and placed in 75% alcohol supplemented with 5% glycerol (18, 29).
Morphological identification of Trichuris spp
The morphological identification of Trichuris spp. was based on the size of the adult male worm, the ratio of whip to body, the length of the copulatory spine, and the shape and size of the spiny sheath (6, 11, 20, 22, 26, 27). All individuals of Trichuris spp. were subjected to morphometrical identification using an Olympus BX53 microscope (Olympus, Tokyo, Japan).
Molecular and phylogenetic analysis of Trichuris spp
Primers were designed based on the conserved sequence of a mitochondrial marker, the cytochrome c oxidase I (cox1) gene. Their sequences were FP: 5′-ACYACATAGTAGGTRTCATG-3′ and RP: 5′-TGATTTTTTGGTCACCCTGAAGTTTA-3′. Total genomic DNA was isolated from an individual male specimen of different Trichuris species with a DNA extraction kit (TaKaRa, Shiga, Japan) according to the instructions. Then, a PCR was performed to amplify the cox1 gene. The PCR reaction system consisted of 21 μL of water, 1 μL of each FP1 and RP1 primer (0.2 μmol/L), 25 μL of 2× Premix Ex Taq and 2 μL of DNA template. The reaction was carried out for 35 cycles of 94°C for 40 s, 56°C for 40 s and 72°C for 35 s followed by extension at 72°C for l0 min. The PCR products were detected using 1.6 % agarose gel electrophoresis, purified using a PCR purification kit (TaKaRa), and sequenced at the Huada Biological Company (Shenzhen, China). Each product was sequenced three times. Sequences that were identical were used for comparisons. Subsequently, a phylogenetic tree based on the cox1 gene was constructed using the neighbour-joining method using MEGA software version 7.0 (12).
Statistical analysis of data
All data were statistically analysed using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA). Continuous and categorical variables were analysed using one-way analysis of variance and chi-squared tests, respectively. Differences were considered significant when P<0.05 and highly significant when P<0.01.
Results
Of the 1,216 sheep examined, 1,047 were infected by Trichuris spp., which equated to an infection rate of 86.1%. Of the five pasture areas, Altai had the sheep with the highest infection rate (91.4%), while Urumqi had the animals with the lowest (80.7%) (Table 1). The infection rates were as high as 99.8% for lambs less than one year old and were accompanied in these youngest animals by an average infection intensity of 17.8 Trichuris spp. (All feces in the large intestine). However, the infection in sheep aged one to four years was less prevalent and weaker, with an 80.7% infection rate and an average intensity of infection of 9.7. Sheep over four years old had a prevalence of 38.7% and an average intensity of 6.7 Trichuris spp. The survey showed that there were no significant differences between infection rates in different grazing areas (P>0.05), while both infection rates and intensity varied significantly (P<0.05) between lambs less than one year old and adult sheep over four years old (Table 2).
Survey on the prevalences of Trichuris spp. infection in sheep from the Tianshan Mountains pastoral areas in China
Year
Total
2019
2020
2021
Pastoral area
Number of sheep surveyed
Number of infected sheep (%)
Number of sheep surveyed
Number of infected sheep (%)
Number of sheep surveyed
Number of infected sheep (%)
Number of sheep surveyed
Number of infected sheep (%)
Urumqi
86
69 (80.2)
84
66 (78.6)
79
66 (83.5)
249
201 (80.7)
Yili
78
70 (89.7)
85
73 (85.9)
82
70 (85.4)
245
213 (86.9)
Bole
85
74 (87.0)
88
74 (84.1)
75
65 (86.7)
248
213 (85.9)
Tacheng
75
65 (86.7)
78
68 (87.2)
88
74 (84.1)
241
207 (85.9)
Altai
72
66 (91.7)
77
71 (92.2)
84
76 (90.5)
233
213 (91.4)
Total
396
344 (86.9)
412
352 (85.4)
408
351 (86.0)
1,216
1,047（86.1)
Infection of Trichuris spp. in sheep at different ages
Sheep age (years)
<1
1–4
≥4
Number of sheep surveyed
862
119
235
Number of infected sheep (%)
860 (99.8) a
96 (80.7) a
91 (38.7) b
NotesDifferent superscript letters (a, b) in the same row indicate significant difference (P<0.05)
A total of 16,849 Trichuris spp. (7,548 males and 9,301 females) from sheep were collected in this survey. Six documented species and one undefined species of Trichuris, namely T. gazellae, T. lani, T. ovina, T. longispiculus, T. concolor, T. discolor and Trichuris sp. (Fig. 1), were morphologically identified based on key characteristics such as the ratio of whip to body, the length of the copulatory spine and the shape and size of male spiny sheaths (Table 3). It was indicated that the undefined species of Trichuris obtained from sheep in the pastoral areas of the Tianshan in Xinjiang was different in its morphological characteristics from previously reported Trichuris species. The spicule sheath of Trichuris sp. was covered with small spines and shaped such that the beginning of the sheath flared and then gradually became thinner, with no spines at the end. Trichuris gazellae and T. lani were the dominant species, accounting for 34.5% and 31.0% of all Trichuris, respectively. The other species of Trichuris, which were T. ovina, T. longispiculus, T. concolor, T. discolor and Trichuris sp., accounted for 15.8%, 6.9%, 5.2%, 3.9% and 2.7%, respectively.
Fig.1
Morphological characteristics of the spine and spicule sheath of male specimens of Trichuris spp. from sheep from the Tianshan Mountains pastoral areas in China. A – gross morphology of Trichuris spp.; B – T. lani; C – T. gazellae; D – T. ovina; E – T. discolor; F – T. longispiculus; G – T. concolor; H – Trichuris sp.
Key characteristics used in the identification of Trichuris spp. in sheep from the Tianshan Mountains pastoral areas in China
Species of Trichuris of which a male was examined
Length of body (mm)
Whip length/body length
Length of spicule (mm)
Length of the spicule sheath (mm)
Morphology of the spicule sheath
T. lani
46.5 ± 10.3
1.8/1
3.2–4.5
0.18–0.2
Tubular; the spicule sheath surface densely covered with small spines, the outer shape resembling a baseball bat
T. gazellae
52.1 ± 6.6
2.2/1
1.5–2.2
0.2–1.2
Proximal 3/4 of the sheath densely covered with an expanded small spines; part as spiny it approaches part forming the unarmed part; distal 1/4 thinner, unarmed
T. ovina
47.4 ± 8.7
2.5/1
0.8–1.2
0.18–0.32
Proximal small spines; 1/2 of distal sheath 1/2 densely unarmed covered with
T. discolor
45.2 ± 1.8
1.9/1
1.2–1.5
0.18–0.20
Surface sheath end densely dilated covered and spherical with small spines,
T. longispiculus
45.6 ± 10.4
2.0/1
1.3–2.5
0.21–0.66
Surface sheath ends densely flared covered and enlarged with small spines,
T. concolor
36.8 ± 7.4
2.2/1
1.1–1.5
0.25–0.3
Rod-shaped, densely covered with small spines
Trichuris sp.
40.5 ± 5.6
2.0/1
1.1–1.5
0.25–0.3
Surface covered with small spines; the beginning gradually of becomes the sheath thinner, flares with and no then spines at the end
After amplification (Fig. 2) and sequencing of the cox1 gene of the six known species, it was found to share 99.1–99.8% identities with other Trichuris spp. After the same steps, the undefined species’ cox1 gene transpired to share only 93.21% identity with other Trichuris spp. The cox1 genes of the investigated species of Trichuris were submitted to GenBank and given these accession numbers: T. gazellae, ON190112, ON190105 and ON190107; T. lani, ON190106 and ON213215; T. ovina, ON190109; T. longispiculus, ON190110; T. concolor, ON190108; T. discolor, ON184014; and Trichuris sp., ON189049, ON189053 and ON189054. Phylogenetic analysis revealed that Trichuris spp. divided into two genetic clades, namely clade I and clade II. The six known species and the undefined species investigated in this study were all classified into clade I. However, these species shared a higher genetic relatedness with T. muris and T. pampeana, with the exception of T. ovina, which displayed a closer genetic relationship with T. navonae.
Fig. 2
Amplification of the mitochondrial cox1 gene of Trichuris spp. M – standard DNA marker DL-2000 (2,000, 1,000, 750, 500, 250 and 100 bp); 1–7 – positive samples detected by RT-PCR; 8 – positive control
Moreover, the phylogenetic tree based on cox1 gene also indicated that those Trichuris spp. derived from sheep had closer genetic relationships with T. trichiura, while they were genetically far from T. suis and T. vulpis, which supported the morphological classification in terms of molecular taxonomy.
Fig. 3
Phylogenetic analysis of different species of the Trichuris genus based on the cox1 gene using MEGA 7.0 software (neighbour-joining method, bootstrap values 1,000)
Discussion
Trichuris spp. are soil-transmitted helminths, which can naturally infect a wide range of hosts including rodents, cats, dogs, pigs, ruminants, humans, and non-human primates and cause trichinosis (5, 25, 28). These helminths have strong resistance to degrading factors in the external environment because of the thick shells of their eggs, thus they can spread widely in domestic animals grazing on pasture (5, 7). Since most sheep in Xinjiang are mainly grazed, the sheep are very heavily infected with digestive parasites. However, the infection status of sheep and prevalences of Trichuris spp. in them are still unknown.
The prevalences of Trichuris species and their infection intensities in sheep were investigated in the northern Xinjiang region. Existing studies have confirmed that there are approximately 24 species of Trichuris which parasitise ruminants (11, 26, 27). In this epidemiological survey, 6 documented species and 1 undefined species of Trichuris were found in sheep in Xinjiang and characterised. It is assumed that this breadth of species variety may be related to the frequency of trade in sheep and the geography. Among the nematodes detected, T. gazellae and T. lani were the dominant species, which accounted for 34.5% and 31.0% of the total number of parasites detected, respectively. The overall infection rate in sheep in the five grazing areas was 86.1% and the highest infection rate of 91.4% was in Altai. The prevalences and infection intensity of Trichuris spp. were significantly higher in lambs than those in adult sheep, which may be related to the incomplete development of the immune system in lambs or the immunity to the parasites of adult individuals.
Accurate identification of Trichuris spp. is essential for epidemiological investigations (15, 21). Generally speaking, the features of Trichuris spp. in females are indistinguishable for various species (17). In the present study, distinguishing Trichuris females corresponding to each species of male was difficult because of co-infection with several species. Therefore, females were not identified in this study, and the identification of Trichuris species was based on the key characteristics of the reproductive organs of adult males as is common practice (6, 11, 15, 20). Studies have revealed that the features such as the body length, the whip length to body length proportion, the length of the spicule and its sheath, and the morphometric characteristics of the spines of the spicule sheath are of high discriminatory value in differentiating Trichuris species (6, 11). The morphological characteristics of males of six known species and one undefined species of Trichuris were detailed through this survey, which enriched the body of taxonomic information of different species of Trichuris in ruminants. However, key characteristics are not present in some Trichuris species in the typical form, so it is necessary to combine morphological and molecular protocols for definitive and comprehensive identification (1, 8, 10, 12, 19, 21).
Phylogenetic analysis based on the cox1 gene revealed that Trichuris species can be divided into two genetic clades. Clade I was where the six known species and the single undefined species investigated in this study were classified. However, obvious inter- and intra-species genetic diversity was displayed by Trichuris. Considering that the single cox1 gene provides very limited genetic information, a new molecular typing method based on multiple genes, such as MLST, should be developed for the identification of species and elucidation of the genetic diversity of Trichuris spp. in the future (14, 29).
In summary, this study revealed for the first time that six documented species and one undefined species of Trichuris were prevalent in sheep in the Tianshan pasture areas of Xinjiang, China. These observations provided valuable morphological and molecular data for the control of trichuriasis in sheep.
* These authors contributed equally to this study and should be considered co-first authors.
