Epidemiological and Molecular Characteristics of <i>Piroplasmids</i> and <i>Anaplasma</i> spp. in Tan Sheep, Ningxia, Northwest China

Zhou, Jiali; Li, Zhixin; Zhou, Zicheng; Ma, Yue; Hu, Junhao; Dan, Xingang; Zhao, Hongxi

doi:https://doi.org/10.1155/2024/2529855

Transboundary and Emerging Diseases

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Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Ethical Approval Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2024 | Article ID 2529855 | https://doi.org/10.1155/2024/2529855

Epidemiological and Molecular Characteristics of Piroplasmids and Anaplasma spp. in Tan Sheep, Ningxia, Northwest China

Jiali Zhou,¹Zhixin Li,²Zicheng Zhou,¹Yue Ma,¹Junhao Hu,¹Xingang Dan,¹and Hongxi Zhao¹

Academic Editor: Horacio Bach

Received10 Jan 2024

Revised14 Apr 2024

Accepted18 Apr 2024

Published29 May 2024

Abstract

Piroplasmosis and anaplasmosis are important zoonotic diseases of animal origin, which can be transmitted by ticks to infect animals. However, there is limited information on the infection of piroplasmosis and anaplasmosis in Tan sheep in Ningxia, China. In order to understand the prevalence of piroplasmosis and anaplasmosis in Tan sheep in Ningxia, 150 blood samples of Tan sheep from farms in five urban areas of Ningxia were detected by PCR, and some positive samples were sequenced to establish a phylogenetic tree. PCR revealed that the prevalence of Anaplasma spp. in Tan sheep in Ningxia was 28.0%. The overall prevalence of Piroplasmids was 33.3%, of which Theileria spp. and Babesia spp. were 20.7% and 12.7%, respectively. Among the samples of different ages, the highest detection rates of Piroplasmids and Anaplasma spp. were found in Tan sheep aged 20–30 months, and the detection rate of Theileria spp., Babesia spp., and Anaplasma spp. were 25.4%, 23.6%, and 36.3%, respectively. In this study, one Theileria species was identified as Theileria uilenbergi, two Babesia species were identified as Babesia molasi and Babesia ovis, and two Anaplasma species were identified as Anaplasma ovis and Anaplasma phagocytophilum, and the dominant species were A. ovis and T. uilenbergi. To the best of our knowledge, this is the first report detailing the infection rate and genotype of Piroplasmids and Anaplasma spp. in Tan sheep in Ningxia, China. The results of this study provide valuable data for understanding the epidemiology of tick-borne disease in Tan sheep in Ningxia, China, and lay a theoretical foundation for the prevention and control of piroplasmosis and anaplasmosis in Tan sheep in Ningxia, northwest China.

1. Introduction

As an obligate blood-sucking ectoparasite, ticks are the second largest insect vector after mosquitoes [1], which can act as a vector for the transmission of variety of pathogens including bacteria, fungi, viruses, and protozoa [2]. These organisms can cause many important emerging or re-emerging infectious diseases, such as severe fever with thrombocytopenia syndrome [3], human granulocytic anaplasmosis [4], and Crimean–Congo hemorrhagic fever [5], causing great harm to human and animal health worldwide [6, 7, 8]. However, piroplasmosis and anaplasmosis are common tick-borne diseases in cattle and sheep. Piroplasmosis is a tick-borne protozoan disease caused by Babesia and Theileria in ruminants [9]. Among them, babesiosis of sheep is a blood parasitic disease characterized by anemia, emaciation, and jaundice caused by different species of Babesia species parasitic in mammalian erythrocytes [10]. It is reported that eight species of Babesia spp. have been found in horses, cattle, sheep, and dogs in China. Five species of Babesia spp. have been reported to infect sheep, B. motasi, B. ovis, Babesia crassa, Babesia taylori, and Babesia foliata [11]. Among them, B. ovis infection is the most serious blood parasitic disease, with clinical manifestations including fever, hemoglobinuria, hemolytic anemia, and jaundice, resulting in the death of 30%–50% of infected sheep worldwide [12, 13]. Theileriosis is a blood protozoal disease that is transmitted to sheep by the protozoa of the genus Theileria through tick-infected red blood cells, macrophages and lymphocytes. The disease is characterized by high fever, superficial lymphadenopathy, anemia, anorexia, lethargy, respiratory distress, and death, and Giemsa-stained blood smears showing polymorphic erythrocytes parasitized by Theileria [14, 15]. In recent years, Theileria luwenshuni, Theileria uilenbergi, and Theileria ovis have been reported to infect sheep in China [16, 17, 18]. Anaplasma belongs to Rickettsiales Anaplasmataceae, which is an obligate and intracellular parasitic Gram-negative bacteria. It is mainly transmitted by ticks and can infect a variety of intracellular parasitic pathogens in animals and humans [19]. At present, the identified species include Anaplasma bovis, A. phagocytophilum, and A. ovis [20]. The clinical symptoms of sheep infected with the above pathogens are similar, and all of them can be transmitted by ticks. With the rapid development of the sheep industry in China, exchanges of sheep and lamb products in different regions have become more frequent, and the epidemic areas of sheep piroplasmosis and anaplasmosis have gradually increased in China [21, 22].

Theileriosis, babesiosis, and anaplasmosis are all transmitted by ticks in sheep, and mixed infection is easy to occur. The morphology of the three pathogens is similar in red blood cells, and it is not easy to distinguish them by blood smear, but PCR can be used for differential diagnosis. Tan sheep, one of the sheep breeds in Ningxia, mainly distributed in the central region of Ningxia, is the main pillar industry of animal husbandry in some places. Because of its tender meat and delicious taste, it has become an important means for local farmers to increase their income. Piroplasmosis and anaplasmosis have seriously affected the healthy development of the Tan sheep industry, but there are no related data on Piroplasmids and Anaplasma spp. in Ningxia Tan sheep. In this study, 150 blood samples of Tan sheep from farmers in five regions of Ningxia Hui Autonomous Region were collected, and Theileria, Babesia, and Anaplasma were detected by PCR. This study aimed to investigate the prevalence and genetic diversity of Piroplasmids and Anaplasma spp. infections in Tan sheep in Ningxia Hui Autonomous Region and to lay a theoretical foundation for the prevention and control of Piroplasmids and Anaplasma spp. infections in Tan sheep in Ningxia.

2. Materials and Methods

2.1. Sample Collection

From June 3 to 9, 2023, blood samples were randomly collected from 150 Tan sheep with similar body weight and clinical manifestations of fever (the body temperature exceeds 40°C), emaciation, and jaundice in Yanchi County, Haiyuan County, Tongxin County, Lingwu City, and Hongsibu District of Ningxia Hui Autonomous Region. 30 samples from each region (Figure 1). Blood samples were collected aseptically from the jugular vein of Tan sheep. These samples were immediately placed into EDTA anticoagulated tubes. Concurrently, detailed information such as the time and location of collection, as well as the age of the sheep, was recorded. Samples were stored in ice boxes after collection and stored at 4°C in the laboratory until screening.

2.2. Extraction of Genomic DNA

Following the instructions form the whole blood genomic DNA extraction kit by Tiagen Biochemical Technology (Beijing) Co.Ltd., sheep blood samples were extracted, and then, the DNA was stored at −20°C. It was used for PCR amplification of target genes.

2.3. PCR Detection of Piroplasmids and Anaplasma spp.

2.3.1. PCR detection of Theileria and Babesia in Tan Sheep

According to the 18S rRNA gene sequence of Theileria spp., specific primers for T. luwenshuni, T. uilenbergi, and T. ovis were synthesized, respectively. According to the 18S rRNA gene sequence of Babesia spp., specific primers for B. ovis and B. motasi were designed, respectively. All primers were synthesized by BioEngineering (Shanghai) Co.Ltd. Theileria and Babesia were amplified from the blood samples of Tan sheep by PCR, and the PCR products were electrophoresed in 1% (w/v) agarose gel, and the results were observed under a gel imager.

2.3.2. PCR Detection of Anaplasma spp.

According to the 16S rRNA gene sequence of A. phagocytophilum and A. bovis, specific primers for A. phagocytophilum and A. bovis were synthesized, respectively, and specific primers for A. ovis msp4 gene were used for PCR amplification. The primers were synthesized by BioEngineering (Shanghai) Co.Ltd. PCR products were electrophoresed in a 1% (w/v) agarose gel, and the results were visualized under a gel imager. The sequences of each primer are shown in Table 1.

2.4. Phylogenetic Analysis

Positive PCR products were sent to BioEngineering (Shanghai) Co.Ltd. for sequencing. After shearing the sequencing results, blast alignment in NCBI revealed sequences with high similarity to the target fragment. Phylogenetic tree analysis was performed using MEGA 7.0 software.

3. Results

3.1. Detection Rates of Piroplasmids and Anaplasma spp. in Tan Sheep in Different Regions

A total of 150 blood samples were detected by PCR from five regions in Ningxia (Table 2). The highest infection rate of Theileria spp. was found in Yanchi County at 30.0% (10/30), followed by Hongsibu County with 26.7% (8/30), Lingwu City with 23.3% (7/30), Haiyuan County with 13.3% (4/30), and Tongxin County with the lowest rate at 6.0% (2/30). The infection rates of Babesia spp. were 16.7% (5/30), 16.7% (5/30), and 30% (9/30) in Yanchi County, Lingwu City, and Hongsibu County, respectively, and none was detected in Haiyuan County and Tongxin County, and Hongsibu County had the highest infection rate of Babesia. The infection rate of Anaplasma spp. was 46.7% (14/30) in Yanchi County, 26.7% (8/30) in Haiyuan County, 20% (6/30) in Tongxin County, 3.3% (1/30) in Lingwu City, and 43.3% (13/30) in Hongsibu County, respectively. The highest infection rate of Anaplasma spp. was in Yanchi County, followed by Hongsibao County.

The detection of different species of Piroplasmids and Anaplasma spp. in five areas of Ningxia showed that among the infected Theileria spp., the dominant species of infection was T. uilenbergi, but T. luwenshuni and T. ovis were not detected (Table 3). Among infected Babesia spp., the detection rates of B. ovis and B. molasi were similar. Among the infected Anaplasma spp., the highest detection rate was A. ovis, while the detection rate of A. phagocytophilum was relatively low. Totally, 150 blood samples were collected from Tan sheep, and Theileria spp. were detected from 31 blood samples (20.7%); Babesia spp. were detected from 19 blood samples (12.7%); Anaplasma spp. were detected from 42 blood samples (28%); and 7 blood samples (4.7%) were mixed infection.

3.2. Age Distribution of Piroplasmids and Anaplasma spp. in Tan Sheep

According to Table 4, statistics on the detection of Theileria spp.in Tan sheep of different ages showed that the highest detection rate was 25.4% in sheep aged 20–30 months, while the lowest detection rate was 12.2% in sheep aged 30–40 months. A significant difference in the infection rate of Theileria spp. was among sheep of different age groups (). The highest detection rate of Babesia spp. was 23.6% in sheep aged 20–30 months, which was significantly different from the rates in those aged 30–40 months and 40–50 months (). The highest detection rate of Anaplasma spp. was 36.3% in the 20–30 months old group, while the lowest was 18.3% in the 30–40 months old group, indicating a significant difference in detection rates across age groups ().

3.3. Sequence Analysis and Establishment of Phylogenetic Trees

The 18S rRNA gene sequences of five T. uilenbergi isolates identified by sequencing were submitted to GenBank, and the sequence accession numbers were OR739069–OR739073. From the constructed phylogenetic tree (Figure 2), the five isolates of T. uilenbergi from Ningxia, China, were divided into two groups. T. uilenbergi Ningxia (OR739069–OR739071) belonged to the same clade with T. uilenbergi isolates from Lintan (AY262116), Qinghai (JF719835), and Egypt (KF922215). T. uilenbergi Ningxia (OR739072–OR739073) and T. uilenbergi (KU554728) isolated from Zhangye, China, were in the same branch, and the similarity reached 99%. In addition, the five T. uilenbergi isolates were not closely related to T. luwenshuni and T. ovis prevalent in China, and the dominant T. uilenbergi strain was found in Tan sheep in some areas of Ningxia.

The 18S rRNA gene sequences of six Babesia spp. isolates identified by sequencing were submitted to GenBank, and the sequence accession numbers were OR735152–OR735157. From the constructed phylogenetic tree (Figure 3), the six Chinese isolates of Babesia. spp. Ningxia were divided into three groups. The B. ovis isolates from Iran (AY362829, AY349159) and Spain (AY150058) shared 100% similarity with B. ovis Ningxia (OR735157). The Babesia sp. isolates from Lintan (AY260181) and Tianzhu (DQ159072) shared 100% similarity with B. sp. Ningxia (OR735152). And B. motasi Ningxia (OR735153-OR735156) shared 99% similarity with B. motasi isolates from the Netherlands (AY260179, AY260180).

The msp4 gene sequences of eight A. ovis strains identified by sequencing were submitted to GenBank, and the sequence accession numbers were OR766697–OR766704. From the constructed phylogenetic tree (Figure 4), the A. ovis isolates from Italy (AY702924) and Xinjiang (OP503167, OP169079) had 100% similarity with A. ovis Ningxia (OR766697–OR766704). The other two groups were A. centrale and A. phagocytophilum, which were not in the same branch as A. ovis isolated from Tan sheep in Ningxia and other A. ovis isolated from domestic and foreign areas, which also indicated that all A. ovis strains isolated in this study were A. ovis.

The 16S rRNA gene sequences of five A. phagocytophilum strains identified by sequencing were submitted to GenBank, and the sequence accession numbers were OR735468–OR735472. According to the phylogenetic tree constructed (Figure 5), the isolated A. phagocytophilum pathogens had complex and variable genotypes, including unique local strains in Ningxia and strains with similar genotypes with domestic and foreign strains. Five isolates were closely related to Jilin (DQ449948) and Gansu (MW397035) in China. The isolates from Turkey (KP745629) and Japan (AY969010) shared high similarity. The other two groups were A. centrale and A. ovis, which were not in the same branch as A. phagocytophilum isolated from Tan sheep in Ningxia and A. phagocytophilum isolated from other countries at home and abroad, indicating that all A. phagocytophilum strains isolated in this study were A. phagocytophilum.

4. Discussion

In recent years, many articles have reported the prevalence of sheep infected with Piroplasmids and Anaplasma spp. in China, ranging from 15% to 50% [2, 22, 26, 27, 28]. Li et al. [29] nested polymerase chain reaction (nPCR) assays and gene sequencing were used to identify tick-borne Babesia spp., Theileria spp., and Anaplasma spp. infections in border regions, northwestern China. Out of 323 samples tested in this study, 225 (69.7%) sheep were infected with Babesia spp., Theileria spp., and Anaplasma spp. Two hundred six (63.8%), 60 (18.6%), 54 (16.7%), 51 (15.8%), 32 (9.9%), 19 (5.9%), and 16 (5.0%) were positive for A. ovis, B. motasi-like, A. bovis, T. uilenbergi, A. phagocytophilum, T. luwenshuni, and B. motasi-like Xinjiang, respectively. Molecular detection of tick-borne pathogens harbored by ticks collected from livestock in the Xinjiang Uygur Autonomous Region, China, founding the most common pathogen was Rickettsia raoultii (36.80%), followed by Brucella sp. (26.2%), A. ovis (22.4%), Babesia caballi (14.8%), Theileria equi (8.7%), and T. ovis (8.5%) [30]. In addition, El-Alfy et al. [31] conducted a systematic review and meta-analysis of the molecular epidemiology and species diversity of tick-borne pathogens in animals in Egypt and found that the infection rates of tick-borne pathogens (TBPs), Babesia spp., and Theileria spp. in sheep and goats were 3.8% and 11.0%, respectively. Meanwhile, diverse faunas of TBPs were identified in sheep, including various species of the genus Babesia (B. bovis, B. bigemina, and B. ovis), the genus Theileria (T. annulata, T. ovis, and Theileria lestoquardi), and the genus Anaplasma (A. marginale, A. ovis, A. phagocytophilum, A. platys, and A. platys-like). In this study, the detection rate of Theileria spp. was 20.7%, which was lower than the detection rate reported in most previous studies in China, such as 60.8% in Gansu [32], 50.0% in Hubei [33] and 31.3% in Qinghai [34]. These differences may be related to regions, feeding patterns, and breeds of sheep. The detection rate of Babesia spp. was 12.7%, which was slightly higher than that reported in previous major studies in China, such as 3.9% in Xinjiang, 2.0% in Shaanxi, and 1.5% in Gansu [26]. The detection rate of Anaplasma spp. was 28%, which is higher than that in some areas of China, such as Henan (4.4%) and Guizhou (2.6%) [22]). This may be related to the increased awareness of disease prevention among local farmers in recent years, and the reduction of grazing times and vector tick distribution during the grazing season. Previous studies have shown that the impact of parasite on young sheep is greater than that on adult sheep and the detection rate is higher. In this study, because the Tan sheep whose blood was collected were all adult sheep, the age difference is small, but the detection rate of Babesia in 20–30 months old sheep is higher than older sheep in other months, which may be due to the difference in feeding management and tick control in the area where the samples were collected at this age.

Six species of Theileria spp. infesting sheep have been reported and named, including T. luwenshuni, T. uilenbergi, T. ovis, T. lestoquardi, Theileria recondita, and Theileria separata, among which T. lestoquardi, T. luwenshuni, and T. uilenbergi are highly pathogenic and are known as malignant Theileria spp., and T. ovis, T. recondita, and T. separata are known as mild-type Theileria spp. because of their weak or no obvious pathogenicity [35]. The molecular evidence, genetic characteristics, and phylogenetic analysis of Theileria spp. in sheep of Egypt have been reported abroad. The results showed that the detection rate of T. ovis in sheep was 4.37%, and the detection rate of T. lestoquardi was 0.87% [36]. In addition, Al-Fahdi et al. [37] conducted a molecular survey of livestock Theileria spp. genus in four provinces of Oman, and the results showed that the prevalence of Theileria spp. was 72.3%, 36.7%, and 2.7% among cattle, sheep, and goats, respectively. Among the reported sheep Theileria spp. in China, T. luwenshuni is the dominant one. [38]. However, in this study, the 18S rRNA gene sequences of Theileria spp. isolated from sheep were examined in Ningxia, China. The results revealed that only T. uilenbergi was detected; T. ovis and T. luwenshuni were not detected. Some studies have shown that most of T. luwenshuni and T. uilenbergi were mixed infection in northwest China [39]. However, only T. luwenshuni exists in central, southeast, and southwest China [33, 40], and some scholars have reported that T. luwenshuni can infect goats, sheep, yellow sheep, cattle, yak, roe deer, sika deer, red deer, Mongolian gazelle, and hedgehog [41], which is different from our results. There are many reasons for this, which may be related to the species of ticks in Ningxia. Different Piroplasmids are transmitted by different vectors of ticks. In China, the transmission vectors of T. luwenshuni and T. uilenbergi are Haemaphysalis qinghaiensis and Haemaphysalis longicornis [39]. It may also be due to the rapid development of sheep breeding and frequent cross-border transport in China and transmission in other regions or abroad. In addition, Ningxia is located in the northwest of China, which has a typical continental climate, long sunshine time, large temperature difference between day and night, and high temperature in summer, which is suitable for the growth and reproduction of ticks. After the bites of ticks on sheep, animals are in a state of insect-carrying immunity for a long time, and it is difficult to be eradicated. The cured animals with Theileria spp. disease show growth retardation and decreased milk and meat production, which has a very negative impact on the development of the breeding industry. The high detection rate of Theileria spp. infection in this study may be due to the significant seasonal and regional transmission and prevalence of Theileria spp. infection in sheep, which is closely related to the distribution of ticks in the local vector, and the time when we collected samples is a high incidence period of Theileria spp. infection. Meanwhile, the reason for the high detection rate of Theileria spp. is not only due to the wide variety and seasonal prevalence of Theileria spp., which can be infected by the bite of a variety of ticks, but also due to the fact that among the blood samples collected in this study, Tan sheep in Tongxin County were vaccinated 2 months ago, while the Tan sheep in other sample collection areas were not vaccinated. This may also be one of the reasons for the high local detection rate of Theileria spp. In conclusion, the incidence of Theileria spp. in sheep is gradually increasing, and mild or inapparent infection is common in natural cases, which increases the difficulty of prevention and control of this disease. In Ningxia, especially in the areas with a high detection rate of Theileria spp., it is necessary to do a good job of prevention and early diagnosis. Tick control should be carried out on the sheep’s surface and pens in spring to autumn, and blood samples should be collected to screen positive cases through early diagnosis and treat them timely, so as to minimize the economic loss of herdsmen.

Babesiosis is an emerging parasitic disease, which is endemic in the world, including Europe, Asia, Africa, North and South America, and Australia. The United States has the largest number of babesiosis cases. Babesiosis is mainly distributed in northeast China, followed by southwest China [42, 43]. According to the current study, of the five Babesia spp. known to infect sheep and goats, B. motasi and B. crassa can infect large animals, and B. ovis, B. taylori, and B. folie can infect small animals [44, 45]. In this study, the 18S rRNA gene sequence of a B. ovis isolate from the northern part of sheep was detected in Ningxia, China. Meanwhile, B. ovis and B. motasi were detected with the infection rates of 6.7% and 6.0%, respectively. Relevant studies have shown that B. ovis is mainly distributed in Europe, Africa, Asia, and the Far East. In recent years, studies have shown the prevalence of Babesia spp. infection among sheep in different countries and regions, with rates ranging from 5.85% to 86.4% [46, 47]. B. motasi originated from southern Europe, and the infection rate of B. motasi is 16.7% in the northwest border area of China [29]. Compared with previous studies, the infection rate of Babesia spp. was lower in Ningxia, which might be related to the effective prevention and control of Babesia spp. in region in recent years. The genotype of Babesia spp. isolated in Ningxia had 100% similarity with the isolates of Babesia Lintan and Babesia Tianzhu in China. The genotypes of B. ovis and B. motasi shared 100% similarity with other countries. There are many reasons for this situation, and we need to further explore. The research of babesiosis in sheep is still a long way to go. China is a big country of sheep raising, and we must pay attention to the prevention and control of babesiosis in sheep in the process of sheep breeding, so as to provide a guarantee for the healthy development of the sheep industry and human health.

Anaplasma is obligate intracellular bacteria of cells of hematopoietic origin and is etiological agents of tick-borne diseases of both veterinary and medical interest common in both tropical and temperate regions. The pathogen has a wide range of hosts and can infect humans, domestic animals, and a variety of wild animals, among which infection of various wild animals is an important reservoir host to maintain its long-term existence in nature [48]. There are six kinds of pathogens A. phagocytophilum, A. Marginale, A. bovis, A. centrale, A. ovis, and A. capra [49]. At present, the disease of Anaplasma spp. has been found all over the world. For instance, a study by Wei et al. [50] investigated the prevalence of Anaplasma spp. infection in goats in Beijing, China. They found that 44.6% (41/92) of goats were infected with A. capra, while the 22.8% (21/92) were infected with Anaplasma. In addition, in China, the prevalence rates were 19.4%, 19.3%, 10%, 8.8%, 6.8%, 1.8%, and 0% in goats from Guizhou Province, Henan Province, Inner Mongolia Autonomous Region, Shanxi Province, Xinjiang Uygur Autonomous Region, Yunnan Province, and Gansu Province, respectively [51]. However, in this study, A. phagocytophilum and A. bovis 16S rRNA gene sequences and A. ovis msp4 gene sequences were studied in Tan sheep in Ningxia, China, and A. phagocytophilum and A. ovis could be detected, but A. bovis was not detected. In addition, the msp4 gene of A. ovis was detected to be similar to the “sheepXJ2” and “sheepXJ9” strains isolated from sheep in southern Xinjiang [52], indicating that they belong to the same clade as the sheep isolated from southern Xinjiang. A. phagocytophilum showed high homology with Jilin, Gansu, Turkey, and Japan isolates. There are many reasons for this situation, which may be a pathogen import phenomenon caused by the cross-border migration of vector ticks or live animals or animals, and the cross-regional transport of livestock products. Most importantly, humans and all kinds of animals are generally susceptible to anaplasmosis. Since the disease was first identified in the United States in 1994, A. phagocytophilum has also been detected in many countries. Ticks play an irreplaceable role in the transmission of this disease. Eliminating ticks and reducing contact with ticks play a key role in the prevention and treatment of this disease. Ningxia is located in northwest China, with a diverse ecological environment and a wide range of bacteria carrying animals and ticks. Herdsmen, veterinary workers, and mountaineers must prevent tick bites to reduce the risk of infection when they are out and about.

From the results of the present study, Anaplasma spp. appear to be more harmful than Theileria and Babesia, not only because it has the highest infection rate, but also because it is highly infectious to humans. It can cause fever, emaciation, leukopenia and thrombocytopenia, and multiorgan failure in the host. Health policy makers, veterinarians, and farmers can consider this information for scientific management of Tan sheep in Ningxia. At the same time, in view of the fact that the number of pure Tan sheep breeding scale is relatively small, breeding requirements are high, and cross with other breeds of sheep will not occur, so the collected Tan sheep samples are very pure, so the detection rate of Piroplasmids and Anaplasma spp. is relatively accurate in Ningxia. In addition, the limitation of this study is that the risk factors associated with this infection were not analyzed at the animal or farm level, which may be more helpful in developing optimal preventive measures by identifying the potential risk factors associated with tick-borne diseases, and therefore, further studies by a large number of researchers are needed.

5. Conclusions

This study is the first to reveal the incidence of T. uilenbergi, B. ovis, B. motasi, A. ovis, and A. phagocytophilum and the total prevalence of Piroplasmids and Anaplasma spp. in sheep in Ningxia, China. The infection rates of Theileria, Babesia,and Anaplasma were 20.7%, 12.7%, and 28.0%, respectively. The dominant parasite species were T. uilenbergi and A. ovis. The genotypes of the four detected parasites were similar to those found both domestically and internationally, with some genotypes unique to Ningxia and others similar to those observed elsewhere. Our results provide basic data for understanding the molecular epidemiology of Piroplasmids and Anaplasma spp. in Tan sheep in Ningxia and lay a theoretical foundation for the prevention and control of Piroplasmids and Anaplasma spp. in Tan sheep in Ningxia.

Data Availability

The nucleotide sequences obtained in this study were deposited in the GenBank database. The accession numbers were T. uilenbergi (OR739069–OR739073); Babesia spp. (OR735152–OR735157); A. phagocytophilum (OR735468–OR735472); and OR766697–OR766704 of A. ovis msp4 gene in sheep.

Ethical Approval

This study was approved by the Animal Ethics Committee of Ningxia University, NXU (No. NXU 22-73). The use of these Tan sheep samples was approved by the Animal Ethics Procedures and Guideline of China.

Conflicts of Interest

The authors declare no conflicts of interest.

Authors’ Contributions

Jia-li Zhou was responsible for investigation, formal analysis, data curation, writing of the original draft, writing, and reviewing and editing. Zhixin Li was responsible for resources, methodology, and investigation. Zicheng Zhou, Yue Ma, and Junhao Hu were responsible for resources and investigation. Xingang Dan was responsible for conceptualization, formal analysis, writing, reviewing and editing, and supervision. Hongxi Zhao was responsible for resources, conceptualization, formal analysis, writing, reviewing and editing, supervision, and funding acquisition.

Acknowledgments

We thank the farmers on five sheep farms in Ningxia for providing blood samples from Tan sheep. This work was supported by the Ningxia Natural Science Foundation (No. 2023AAC02017). We also thank the School of Animal Science and Technology, Ningxia University, for providing space to complete this study.

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