Open Access

A pan-Theileria FRET-qPCR survey for Theileria spp. in ruminants from nine provinces of China

  • Yi Yang1,
  • Yongjiang Mao2,
  • Patrick Kelly3,
  • Zhangpin Yang2,
  • Lu Luan1,
  • Jilei Zhang1,
  • Jing Li1,
  • Heba S El-Mahallawy1, 4 and
  • Chengming Wang1Email author
Parasites & Vectors20147:413

https://doi.org/10.1186/1756-3305-7-413

Received: 3 July 2014

Accepted: 24 August 2014

Published: 31 August 2014

Abstract

Background

Theileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections.

Methods

Primers and probes for a genus-specific pan-Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China.

Results

The pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle (380/1,235). Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (104.88 vs. 103.00Theileria 18S rRNA gene copies/per ml whole blood).

Conclusions

The pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp.

Keywords

Theileria spp FRET-qPCR Prevalence Ruminants

Background

Theileria spp. are tick-transmitted, intracellular protozoan parasites infecting leukocytes and erythrocytes of wild and domestic large and small ruminants. Several Theileria spp., transmitted by ixodid ticks of the genera Rhipicephalus, Hyalomma, Amblyomma and Haemaphysalis, have been described in cattle, water buffaloes, sheep and goats in different geographical zones of the world [15]. Theileriosis is primarily limited to tropical and sub-tropical areas of the world, with infections mainly reported in Africa and the Middle East but also in southern Europe and northern Asia [611]. Infections by Theileria spp. can cause fever, anemia and hemoglobinuria and, in severe cases, death although many species are benign. Animals recovered from acute or primary infections usually remain persistently infected and may act as reservoirs of infecting ticks [12, 13].

While there have been numerous reports of theileriosis in various animal species in China since 1958 [1427], many have been reported in Chinese and some were based on microscopic detection of parasites which can be difficult with low parasitemia and does not allow ready differentiation of species. Serological studies, although sensitive and easy to perform, are not specific as there is cross reactivity between Theileria spp. Although molecular studies have been performed, these have been to detect Theileria of specific domestic animal species, for example sheep and goats [27]. There have been no highly sensitive and specific molecular methods described which enable studies on various animals from widely divergent areas of China where different Theileria spp. might occur. To address this problem, we developed and validated a highly sensitive genus-specific Theileria FRET-qPCR that detects the recognized Theileria spp. of domestic animals and investigated the molecular prevalence of Theileria in cattle, water buffaloes, goats and sheep from nine provinces in China.

Methods

Animals and blood collection

Between 2007 and 2013, whole blood samples (around 6 ml) were collected in EDTA from apparently healthy cattle (n = 1,235), water buffaloes (29), goats (270) and sheep (111) from 9 provinces/municipality of China (Table 1). The Bos primigenius (p.) taurus studied (n = 975) were Holsteins, Simmentals, Bohai blacks, Luxis and Wannans while the Bos. p. indicus (n = 260) were the Yunlings, Minnans, and Leiqiongs (Table 1). The water buffaloes, goats and sheep in the study were bred in China and were indigenous breeds. Gender information was available for cattle from Yunnan province. After collection, the blood samples were frozen at -20°C and shipped on ice (over 2 days) to Yangzhou University where they were frozen at -80°C until thawed at room temperature for DNA extraction as described below. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University and animal owners gave written permissions for blood collection.
Table 1

Molecular prevalence of Theileria spp. in cattle, water buffalo, goat and sheep

Animal species

Subspecies /breed

Province

City

Coordinate of city

Theileria positivity

  

positive /total

%

Cattle (n = 1235)

Bos p. taurus

Simmental

Inner Mongolia

Chifeng

42.17°N, 118.58°E

19/132

14.4%

Bohai black

Shandong

Binzhou

37.22°N, 118.02°E

4/66

6.1%

Luxi

Shandong

Jining

35.23°N, 116.33°E

40/40

100%

Holstein

Jiangsu

Yancheng

33.22°N, 120.08°E

72/321

22.4%

Holstein

Jiangsu

Yangzhou

32.23°N, 119.26°E

17/144

11.8%

Holstein

Shanghai

Shanghai

31.14°N, 121.29°E

9/255

3.5%

Wannan

Anhui

Wuhu

31.19°N, 118.22°E

17/17

100%

Bos p. indicus

Yunling

Yunnan

Kunming

25.04°N, 102.42°E

124/161

77.0%

Minnan

Fujian

Putian

24.26°N, 119.01°E

4/25

16.0%

Leiqiong

Hainan

Haikou

20.02°N, 110.20°E

74/74

100%

Water buffalo (n = 29)

Haizi

Jiangsu

Yancheng

33.22°N, 120.08°E

0/29

0%

Goat (n = 270)

Xinjiang

Xinjiang

Urumqi

43.45°N, 87.36°E

4/98

4.1%

Yangtse River Delta White

Jiangsu

Yangzhou

32.23°N, 119.26°E

116/172

67.4%

Sheep (n = 111)

Wuranke

Inner Mongolia

Xilingol

43.57°N, 116.03°E

36/72

50.0%

Sishui Fur

Shandong

Jining

35.23°N, 116.33°E

23/39

59.0%

DNA extraction

DNA was extracted from whole blood samples using a standard phenol-chloroform method previously described [28]. Two ml whole blood was used to extract DNA which was resuspended into 200 μl 1 × T10E0.1 buffer. The concentration of the extracted DNA was established with a Microscale Ultraviolet Spectrophotometer. Negative controls consisting of sterile molecular grade water were used to detect cross- contamination during DNA extraction and processing. The HMBS-based FRET-PCR was performed to verify if the extracted DNAs from blood samples were appropriate for molecular detection of tick-borne pathogens [29, 30].

Theileria spp. FRET-qPCR

Primers and probes

The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 4 other closely related protozoan species were obtained from GenBank: T. orientalis (HM538222), T. buffeli (HQ840967), T. annulata (KF429799), T. sergenti (EU083804), T. luwenshuni (JX469527), T. velifera (AF097993), T. ovis (AY508458), T. parva (L02366), T. uilenbergi (JF719835), T. equi (AB515310), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. cervi (AY735119), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585); Babesia bovis (KF928529), B. divergens (LK935835), B. bigemia (LK391709), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105) and Toxoplasma gondii (L37415) (Figure 1). The Clustal Multiple Alignment Algorithm was used to identify a highly conserved region of the 18S rRNA gene common to all the above Theileria spp. but significantly different from the other protozoan species (Figure 1). The primers and probes we developed were situated within the conserved region and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The Theileria FRET-qPCR we established amplifies a 149-bp target with the positions of primers and probes shown in Figure 1: forward primer: 5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′; reverse primer: 5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′; anchor probe: 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′; reporter probe: 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-phosphate-3′.
Figure 1

Alignment of oligonucleotides for Theileria PCR used in this study. Primers and probes are shown at the top of the boxes. Dots indicate nucleotides identical to primers and probes, and dashes denote absence of the nucleotide. The upstream primer is used as the demonstrated sequences without gaps while the two probes and downstream primer are used as antisense oligonucleotides. The designed oligonucleotides show minimum mismatching with Theileria spp. (0 mismatch with 11 species, 1 mismatch with 3 species, 2 mismatches with 2 species and 4 mismatches with 1 species), but significant numbers of mismatches with Babesia bovis (25 mismatches), B. divergens (23 mismatches), B. bigemina (22 mismatches), Cytauxzoon felis (8 mismatches), Hepatozoon americanum (16 mismatches) and Toxoplasma gondii (15 mismatches). The 6-FAM label is directly attached to the 3-terminal nucleotide of the fluorescein probe, and the LCRed-640 fluorescein label is added via a linker to the 5′-end of the LCRed-640 probe.

Thermal cycling

The Theileria FRET-PCR was performed in a LightCycler 480® II real-time PCR platform with 20 μl volumes comprising 10 μl reaction master mix and 10 μl of sample. Thermal cycling consisted of a 2 min denaturation step at 95°C followed by 18 high-stringency step-down thermal cycles, 40 low-stringency fluorescence acquisition cycles, and melting curve determination between 38°C and 80°C. The parameters for qPCR were 6 × 12 sec at 64°C, 8 sec at 72°C, 0 sec at 95°C; 9 × 12 sec at 62°C, 8 sec at 72°C, 0 sec at 95°C; 3 × 12 sec at 60°C, 8 sec at 72°C, 0 sec at 95°C; 40 × 8 sec at 54°C and fluorescence acquisition, 8 sec at 72°C, 0 sec at 95°C.

Specificity

PCR products were verified using electrophoresis (1.5% MetaPhor agarose gels), followed by purification with a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and genomic sequencing (GenScript, Nanjing, Jiangsu, China). The sequencing data from randomly selected positive Theileria samples (n = 37) were compared with the existing Theileria sequences in the GenBank using BLAST. The specificity of the PCR was further verified with the amplification of T. orientalis rRNA-containing pIDTSMART cloning Vector (Integrated DNA Technologies, Coralville, IA, USA) and 100 DNA copies of the rRNA gene of B. canis, H. americanum, C. felis and T. gondii (kindly provided by the parasitological laboratory of Yangzhou University College of Veterinary Medicine).

Sensitivity

For use as quantitative standards, the PCR products of DNAs of 5 Theileria species (T. orientalis, T. sergenti, T. buffeli, T. luwenshuni, T. ovis) were gel purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). After using the estimated molecular mass of the rRNA gene and the Quanti-iT TM PicoGreen ® dsDNA Assay Kit (Invitrogen Corporation, Carlsbad, CA, USA) to calculate the molarity of the solution, dilutions were made to give solutions containing 10,000, 1,000, 100, 10, 1 gene copies per PCR reaction system. These dilutions, and further dilutions providing 2, 4, 6 and 8 gene copies per PCR reaction, were used to determine the minimal detection limit. The 10-fold dilutions were used as quantitative standards in the FRET-PCR surveys to enable standard curves to be developed for the calculation of the gene copy numbers in positive samples.

Identification of Theileria spp. by PCR and sequencing

The amplicon of the pan-Theileria FRET-qPCR we established has a sequence which is highly conserved among the different Theileria species. To differentiate Theileria spp. in a positive reaction, we used a standard PCR to amplify a highly polymorphic region of the 18S rRNA gene (591–594 nucleotides for different Theileria spp.) and sequenced the products (GenScript, Nanjing, Jiangsu, China). For the PCR we designed a forward primer (5′-CCTGAGAAACGGCTACCACATCT-3′) that amplified all Theileria species and used a previously described reverse primer (5′-GGACTACGACGGTATCTGATCG-3′) that also amplified all species [31].

Statistical analysis

Differences in positivity of Theileria spp. were analyzed by Chi-squared Test while numbers of copies of the Theileria 18S rRNA gene determined in the Theileria FRET-qPCR were log10-transformed and analyzed using the Student’s T-test. Differences of P < 0.05 were considered statistically significant.

Results

Development of the pan-Theileria FRET-PCR

Comparison of the sequences in the highly conserved region of the Theileria spp. we used showed the region is highly conserved, but is substantially different from those in closely related protozoan species (Figure 1). The two primers and two probes we chose for the pan-Theileria FRET-qPCR had 0–4 nucleotide mismatches with the Theileria spp. in GenBank, but had 25, 23, 22, 8, 16 and 15 mismatches with B. bovis, B. divergens, B. bigemina, C. felis, H. americanum and T. gondii, respectively (Figure 1). The specificity of the pan-Theileria FRET-PCR was further confirmed when it gave positive reactions with the T. orientalis control, but gave negative reactions with DNAs of B. canis, C. felis, H. americanum and T. gondii. The pan-Theileria FRET-qPCR had a specific melting curve (Tm 57.5°C) with Theileria spp. DNA. Using the gel-purified PCR products as quantitative standards, we determined the detection limit of the pan-Theileria FRET-qPCR was 2 copies of the Theileria 18S rRNA gene per reaction for T. orientalis, T. sergenti and T. luwenshuni, T. buffeli and T. ovis.

Prevalence of Theileria spp. DNA in ruminants

Animals positive for Theileria were found in each of the nine provinces sampled with several animals of each species being positive at each location, except in the case of water buffaloes which were all negative in the one site they were studied. The overall prevalences of Theileria spp. DNA in sheep (53.2%; 59/111) and goats (44.4%; 120/270) were significantly higher than in cattle (30.8%; 380/1,235) (two-tailed Chi-squared Test, P < 10-4). The pan-Theileria FRET-PCR showed that sheep had an average of 102.4 copies of Theileria 18S rRNA/ml whole blood which was significantly lower than the 104.3 copies in cattle and 105.8 copies in goats (Student’s t Test, P < 10-4). While the prevalence of Theileria spp. DNA varied greatly from 3.5% (9/255) in Holsteins from Shanghai to 100% in Luxi cattle from Shandong (40/40) and Leiqiong cattle from Hainan (74/74), the prevalence did not differ significantly in sheep from Inner Mongolia and Shandong (Table 1, Figure 2).
Figure 2

Sites in China where samples ruminants were tested for Theileria spp. DNAs. Dots of different colors represent sites where samples obtained from cattle, water buffalo, goat and sheep of nine provinces were tested by pan-Theileria FRET-qPCR in this study.

Sequencing of 87 randomly selected amplicons (52 from cattle, 14 from goats and 21 from sheep) from Theileria DNA positive samples showed that T. orientalis/ T. sergenti/T. buffeli group [29] were present in cattle while T. luwenshuni was found in goats in Jiangsu province and T. ovis and T. luwenshuni in sheep from Inner Mongolia and Jiangsu province, respectively.

Factors associated with the occurrence of theileriosis in cattle

When we analyzed factors that might be associated with the prevalence of Theileria spp. DNA in cattle we found that Bos p. indicus animals had significantly higher positivity (77.7% vs. 18.3%; P < 10-4) and copy number of the Theileria 18S rRNA gene (104.81 vs. 103.73 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. Similarly, Bos p. indicus cattle were more likely to be positive (65.2% vs. 13.7%; P < 10-4) and have higher copy numbers of the Theileria 18S rRNA gene (104.88 vs. 103.00 copies/per ml whole blood; P < 10-4) than Bos p. taurus animals. The cattle from southern China had significantly higher Theileria 18S rRNA gene copy numbers (104.39 vs. 103.87 copies/per ml whole blood; P = 0.02) than those from northern China but this difference in the prevalence was not significant (31.8% vs. 26.5%). In cattle from Yunnan province where gender information was available, female animals were more commonly positive (76.7% vs. 41.3%; P < 10-4) and copy numbers (105.02 vs. 102.93Theileria/per ml whole blood; P < 10-4) than males.

Gene accession numbers

The Theileria rRNA nucleotide sequences obtained in this study that were not identical to existing entries in GenBank were deposited with the following gene accession numbers: KJ850933 and KJ850938 (T. sergenti); KJ850936 and KJ850940 (T. buffeli); KJ850934, KJ850937, KJ850943, KJ850939 and KJ850941 (T. orientalis); KJ850942 (T. ovis); KJ850935 and KM016463 (T. luwenshuni). The sequences obtained were very similar (0–4 nucleotide mismatches) to Theileria spp. sequences deposited by other laboratories in China, USA, France, Australia and Iran (Table 2).
Table 2

Comparison of isolates identified in this study and similar sequences in GenBank by BLASTN

Isolates identified in this study

Highly similar sequences in GenBank

Theileria spp.

Gene accession #

Source/origin

Gene accession #

Source/origin

Mismatches

T. orientalis

KJ850934

Simmental cattle, Inner Mongolia

AP011948

Cattle, Shintoku of Japan

0/547

KJ850939

Luxi cattle, Shandong

HM538220

Cattle, Suizhou of China

0/547

KJ850937

Yunling cattle, Yunnan

AB520956

Cattle, New South Wales of Australia

2/491

KJ850941

Yunling cattle, Yunnan

AB520955

Cattle, Raymond of Australia

0/509

KJ850943

Holstein cattle, Jiangsu

AB520956

Cattle, New South Wales of Australia

0/549

T. sergenti

KJ850933

Australian Holstein cattle, Jiangsu

JQ723015

Cattle, Hunan of China

0/541

KJ850938

Yunling cattle, Yunnan

JQ723015

Cattle, Hunan of China

0/492

T. buffeli

KJ850936

Leiqiong cattle, Hainan

HM538196

Cattle, Hubei of China;

1/508

KJ850940

Wannan cattle, Anhui

AY661513

Cattle, USA

0/545

T. ovis

KJ850942

Wuranke sheep, Inner Mongolia

FJ603460

Sheep, Xinjiang of China;

0/529

T. luwenshuni

KJ850935

from Sishui-Fur sheep, Shandong

KC769996, JX469518, JF719831

Sheep, China

0/549

KM016463

Yangtse River Delta White goat, Jiangsu

KC769997

Goat, Beijing of China

0/571

Discussion

By systematically aligning the 18S rRNA sequences of representative Theileria spp. and other related protozoa, we identified a highly conserved region to place primers and probes for a pan-Theileria FRET-qPCR. The primers and probes we designed enabled us to amplify all our reference Theileria species with a detection limit of at least 2 Theileria 18S rRNA gene copies per PCR system. None of the other protozoan species tested gave reaction products. To the best of our knowledge, this is the first FRET-qPCR which specifically detects all Theileria species.

Our data indicate infections with Theileria spp. are very widespread and common in cattle in China. We found positive animals in each province where we tested and an overall average of 30.77% of animals being positive. This relatively high level of positivity is similar to that obtained in the only other molecular survey for Theileria in China which showed 13.46% being positive in the northeast [32]. Sequencing data showed cattle are infected with the T. orientalis/T. sergenti/T. buffeli group which are generally recognized to be benign species [33] although some strains might cause economic losses [34].

Although we sequenced relatively few positive amplicons, we found no evidence of the more pathogenic strains, T. annulata and T. parva, which is not unexpected in the case of T. parva which has only been reported from Africa [8]. T. annulata, however, has been reported in H. asiaticum in northwestern China [35]. Our failure to demonstrate the organism in our study might be because T. annulata has an uneven distribution in China.

Although we found no evidence of Theileria spp. in the 29 water buffaloes we sampled, He [36] reported 58/304 (19.1%) positive for T. buffeli in Hubei province, south China. More extensive studies are necessary to determine the epidemiology of theileriosis in water buffaloes.

We found both goats and sheep were infected with T. luwenshuni (Table 2) using the pan-Theileria FRET-PCR and sequencing. This is a highly pathogenic organism that is known to occur in China where it is transmitted by Haemaphysalis qinghaiensis and may cause significant economic losses. While PCR assays have been described which detect and differentiate T. luwenshuni from T. uilenbergi[37, 38], they do not enable detection of all Theirleria spp. in all species and are thus not as versatile as our pan-Theileria FRET-PCR. Yin et al.[37] found the prevalence of T. luwenshuni varied from 0% to 85% in 4 provinces and we also found a wide range of positive values (4.1% to 67.4%). Such variability is probably due to tick prevalence rates, geoclimatic factors and livestock management systems. Further research using sensitive detection methods will be important in determining the best mechanisms to control infections.

T. ovis was first identified in China in 2011 by PCR and sequencing and an infection rate of 78% was found in Xinjiang but no positive animals were found in twelve other provinces [26, 27, 39]. Our study confirms the presence of the organism in China although T. ovis is considered benign and economically unimportant as it might only cause signs in some animals which are stressed [40].

It is well recognized that exotic breeds are more susceptible to disease following infection with Theileria spp. than local stocks. Recent studies, however, have indicated that different breeds of animals might have similar susceptibilities to infections with Theileria spp. although some breeds are more capable of controlling the pathogenic effects of the organism [41].

Generally, B. p. taurus breeds are more susceptible to theileriosis than B. p. indicus breeds which might be associated with innate or immune mechanisms and/or general resistance to ticks. These factors have been mainly investigated with T. parva infections and thus it is interesting that our pan-Theileria FRET-PCR testing showed that in China it is the B. p. indicus breeds that are more likely to be positive than the B. p. taurus breeds and that the former generally had higher copy numbers indicating heavier infections. While our sample sizes were small and we cannot exclude the possibility of sample bias, our findings might be due to host genetic factors relating to infections with less pathogenic Theileria spp. or other factors such as differences in tick control and husbandry practices on different farms. Further studies taking these factors into account are needed to more precisely investigate the relationships between infections with benign Theileria spp. and the genetic background of the host.

Conclusion

In summary, our study has described the development and testing of a FRET-PCR which can detect recognized Theileria spp. The pan-Theileria FRET-PCR should be a useful diagnostic tool as it will enable diagnostic laboratories to detect infections in all domestic species with a single test.

Declarations

Acknowledgment

This project was supported by grants from the National Natural Science Foundation of China (NO 31272575, 32472225), and by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Authors’ Affiliations

(1)
Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine
(2)
Yangzhou University College of Animal Science and Technology
(3)
Ross University School of Veterinary Medicine
(4)
Faculty of Veterinary Medicine, Suez Canal University

References

  1. Aktas M, Altay K, Dumanli N: PCR-based detection of Theileria ovis in Rhipicephalus bursa adult ticks. Vet Parasitol. 2006, 140 (3–4): 259-263.View ArticlePubMedGoogle Scholar
  2. Morrison WI: Progress towards understanding the immunobiology of Theileria parasites. Parasitology. 2009, 136 (12): 1415-1426. 10.1017/S0031182009990916.View ArticlePubMedGoogle Scholar
  3. Muleya W, Namangala B, Simuunza M, Nakao R, Inoue N, Kimura T, Ito K, Sugimoto C, Sawa H: Population genetic analysis and sub-structuring of Theileria parva in the northern and eastern parts of Zambia. Parasit Vectors. 2012, 5: 255-10.1186/1756-3305-5-255.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Gachohi J, Skilton R, Hansen F, Ngumi P, Kitala P: Epidemiology of East Coast fever (Theileria parva infection) in Kenya: past, present and the future. Parasit Vectors. 2012, 5: 194-10.1186/1756-3305-5-194.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Otranto D, Dantas-Torres F, Giannelli A, Latrofa MS, Cascio A, Cazzin S, Ravagnan S, Montarsi F, Zanzani SA, Manfredi MT, Capelli G: Ticks infesting humans in Italy and associated pathogens. Parasit Vectors. 2014, 7: 328-10.1186/1756-3305-7-328. doi: 10.1186/1756-3305-7-328PubMed CentralView ArticlePubMedGoogle Scholar
  6. Balkaya I, Utuk AE, Piskin FC: Prevalance of Theileria equi and Babesia caballi in donkeys from Eastern Turkey in winter season. Pakistan Vet J. 2010, 30 (4): 245-246.Google Scholar
  7. Hussain MH, Saqib M, Raza F, Muhammad G, Asi MN, Mansoor MK, Saleem M, Jabbar A: Seroprevalence of Babesia caballi and Theileria equi in five draught equine populated metropolises of Punjab, Pakistan. Vet Parasitol. 2014, 202 (3–4): 248-256.View ArticlePubMedGoogle Scholar
  8. Muhanguzi D, Picozzi K, Hatendorf J, Thrusfield M, Welburn SC, Kabasa JD, Waiswa C: Prevalence and spatial distribution of Theileria parva in cattle under crop-livestock farming systems in Tororo District, Eastern Uganda. Parasit Vectors. 2014, 7: 91-10.1186/1756-3305-7-91.PubMed CentralView ArticlePubMedGoogle Scholar
  9. Razmi GR, Eshrati H, Rashtibaf M: Prevalence of Theileria spp. infection in sheep in South Khorasan province, Iran. Vet Parasitol. 2006, 140 (3–4): 239-243.View ArticlePubMedGoogle Scholar
  10. Ros-García A, Nicolás A, García-Pérez AL, Juste RA, Hurtado A: Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle. Parasit Vectors. 2012, 5: 171-10.1186/1756-3305-5-171.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Gomes J, Soares R, Santos M, Santos-Gomes G, Botelho A, Amaro A, Inácio J: Detection of Theileria and Babesia infections amongst asymptomatic cattle in Portugal. Ticks Tick Borne Dis. 2013, 4 (1–2): 148-151.View ArticlePubMedGoogle Scholar
  12. Tian Z, Liu G, Yin H, Xie J, Wang S, Yuan X, Wang F, Luo J: First report on the occurrence of Theileria sp. OT3 in China. Parasitol Int. 2014, 63 (2): 403-407. 10.1016/j.parint.2013.12.014.View ArticlePubMedGoogle Scholar
  13. McKeever DJ: Bovine immunity - a driver for diversity in Theileria parasites?. Trends Parasitol. 2009, 25 (6): 269-276. 10.1016/j.pt.2009.03.005.View ArticlePubMedGoogle Scholar
  14. Chen Z, Liu Q, Liu JQ, Xu BL, Lv S, Xia S, Zhou XN: Tick-borne pathogens and associated co-infections in ticks collected from domestic animals in central China. Parasit Vectors. 2014, 7: 237-10.1186/1756-3305-7-237. doi: 10.1186/1756-3305-7-237PubMed CentralView ArticlePubMedGoogle Scholar
  15. Yang FG, Feng ZG, Yu GH, Liu JX, Wei ZQ, He XC: The report of sheep theileriosis in Ganning agriculture and animal husbandry station, Garze Tibetan Autonomous Prefecture. Chinese J Vet Med. 1958, 2: 33-37. (In Chinese)Google Scholar
  16. Wang FX, Li O, Li W, Wang WX, Hua D, Wang LZ, Guo RM, Shi WZ, Li ZM: Investigation of theileriosis in counties of Huangyuan, Menyuan and Jianzha. Chinese Qinghai J Animal Vet Sci. 1980, 3: 29-32. (In Chinese)Google Scholar
  17. Li ZM, Hao YK, Chen TH, Yu XQ, Xi HL, Zuo YR, Chen YQ, Liu J, Ding YT, Meng ZX: The report of theileriosis of sheep in Dianan Commune, Longde county. Gansu Animal Vet Sci. 1984, 1: 13-14. (In Chinese)Google Scholar
  18. Luo J, Yin H: Theileriosis of sheep and goats in China. Trop Anim Health Prod. 1997, 29 (4 suppl): S 8-S 10.View ArticleGoogle Scholar
  19. Guo S, Yuan Z, Wu G, Wang W, Ma D, Du H: Epidemiology of ovine theileriosis in Ganan region, Gansu Province, China. Parasitol Res. 2002, 88 (13 Suppl): S36-S37.View ArticlePubMedGoogle Scholar
  20. Guo SZ, Ma DL, Mou YJ, Yang SM, Wang WB, Ge GH, Ga DJ, Fang BQ: Epidemiological investigation of theileriosis in goats and sheep in northern China. Chinese J Vet Parasitol. 2005, 4: 15-17. (In Chinese)Google Scholar
  21. Yuan YQ, Li YQ, Liu ZJ, Yang JF, Chen Z, Guan GQ, Luo JX, Yin H: Epidemiological investigation of theileriosis in goats and sheep in southern China. Chinese Vet Sci. 2013, 43: 551-556. (In Chinese)Google Scholar
  22. Hu SY, Zhang SF, Qian NC, Jia LJ, Xue SJ, Sun KN, Lin WJ: Molecular epidemiological survey of Theileria sergenti infection in cattle in part areas of Jilin province. Chinese J Prev Vet Med. 2013, 35: 539-541. (In Chinese)Google Scholar
  23. Jian ZJ, Ma SZ, Sun QZ, Shen JY, Lv W, Miao ZQ: Epidemiologic survey for detection of Theileria annulata-infected cattle in Xinjiang by an improved indirect tams1 ELISA assay. Xinjiang Agric Sci. 2011, 48: 1509-1513. (In Chinese)Google Scholar
  24. Kahar S, Cao WL, Zhang Y, Wang BJ, Bayinchahan : Epidemiological investigation of infection of Theileria annulata for cattle in some epidemic areas of Turpan. Xinjiang Agric Sci. 2013, 50: 1161-1164. (In Chinese)Google Scholar
  25. Li Y, Chen Z, Liu Z, Liu J, Yang J, Li Q, Li Y, Cen S, Guan G, Ren Q, Luo J, Yin H: Molecular identification of Theileria parasites of Northwestern Chinese Cervidae. Parasit Vectors. 2014, 7 (1): 225-10.1186/1756-3305-7-225.PubMed CentralView ArticlePubMedGoogle Scholar
  26. Ge Y, Pan W, Yin H: Prevalence of Theileria infections in goats and sheep in Southeastern China. Vet Parasitol. 2012, 186 (3–4): 466-469.View ArticlePubMedGoogle Scholar
  27. Li Y, Zhang X, Liu Z, Chen Z, Yang J, He H, Guan G, Liu A, Ren Q, Niu Q, Liu J, Luo J, Yin H: An epidemiological survey of Theileria infections in small ruminants in central China. Vet Parasitol. 2014, 200 (1–2): 198-202.View ArticlePubMedGoogle Scholar
  28. Sambrook J: Molecular Cloning: A Laboratory Manual. 1989, New York: Cold Spring Harbor LaboratoryGoogle Scholar
  29. Wei L, Kelly P, Zhang J, Yang Y, Zheng X, Tao J, Zhang Z, Wang C: Use of a universal hydroxymethylbilane synthase (HMBS)-based PCR as an endogenous internal control and to enable typing of mammalian DNAs. Appl Microbiol Biotechnol. 2014, 98: 5579-5587. 10.1007/s00253-014-5659-x. doi: 10.1007/s00253-014-5659-xView ArticlePubMedGoogle Scholar
  30. Wang C, Mount J, Butler J, Gao D, Jung E, Blagburn BL, Kaltenboeck B: Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs. Parasit Vectors. 2012, 5: 4-10.1186/1756-3305-5-4. doi: 10.1186/1756-3305-5-4PubMed CentralView ArticlePubMedGoogle Scholar
  31. Jefferies R, Ryan UM, Irwin PJ: PCR-RFLP for the detection and differentiation of the canine piroplasm species and its use with filter paper-based technologies. Vet Parasitol. 2007, 144 (1–2): 20-27.View ArticlePubMedGoogle Scholar
  32. Yu L, Zhang S, Liang W, Jin C, Jia L, Luo Y, Li Y, Cao S, Yamagishi J, Nishikawa Y, Kawano S, Fujisaki K, Xuan X: Epidemiological survey of Theileria parasite infection of cattle in northeast China byallele-specific PCR. J Vet Med Sci. 2011, 73 (11): 1509-1512. 10.1292/jvms.10-0507.View ArticlePubMedGoogle Scholar
  33. Kamau J, de Vos AJ, Playford M, Salim B, Kinyanjui P, Suqimoto C: Emergence of new types of Theileria orientalis in Australian cattle and possible cause of theileriosis outbreaks. Parasit Vectors. 2011, 4: 22-10.1186/1756-3305-4-22.PubMed CentralView ArticlePubMedGoogle Scholar
  34. McFadden AM, Rawdon TG, Meyer J, Makin J, Morley CM, Clough RR, Tham K, Mullner P, Geysen D: An outbreak of haemolytic anaemia associated with infection of Theileria orientalis in naive cattle. N Z Vet J. 2011, 59 (2): 79-85. 10.1080/00480169.2011.552857.View ArticlePubMedGoogle Scholar
  35. Meng K, Li Z, Wang Y, Jing Z, Zhao X, Liu J, Cai D, Zhang L, Yang D, Wang S: PCR-based detection of Theileria annulata in Hyalomma asiaticum ticks in northwestern China. Ticks Tick Borne Dis. 2014, 5 (2): 105-106. 10.1016/j.ttbdis.2013.09.006.View ArticlePubMedGoogle Scholar
  36. He L, Feng HH, Zhang WJ, Zhang QL, Fang R, Wang LX, Tu P, Zhou YQ, Zhao JL, Oosthuizen MC: Occurrence of Theileria and Babesia species in water buffalo (Bubalus babalis, Linnaeus, 1758) in the Hubei province, South China. Vet Parasitol. 2012, 186 (3–4): 490-496.View ArticlePubMedGoogle Scholar
  37. Yin H, Liu Z, Guan G, Liu A, Ma M, Ren Q, Luo J: Detection and differentiation of Theileria luwenshuni and T. uilenbergi infection in small ruminants by PCR. Transbound Emerg Dis. 2008, 55 (5–6): 233-237.View ArticlePubMedGoogle Scholar
  38. Zhang X, Liu Z, Yang J, Chen Z, Guan G, Ren Q, Liu A, Luo J, Yin H, Li Y: Multiplex PCR for diagnosis of Theileria uilenbergi, Theileria luwenshuni, and Theileria ovis in small ruminants. Parasitol Res. 2014, 113 (2): 527-531. 10.1007/s00436-013-3684-9.View ArticlePubMedGoogle Scholar
  39. Li Y, Guan G, Ma M, Liu J, Ren Q, Luo J, Yin H: Theileria ovis discovered in China. Exp Parasitol. 2011, 127 (1): 304-307. 10.1016/j.exppara.2010.07.002.View ArticlePubMedGoogle Scholar
  40. Ahmed J, Yin H, Bakheit M, Liu Z, Mehlhorn H, Seitzer U: Small ruminant theileriosis. Parasitol Res Monograph. 2011, 2: 135-153.Google Scholar
  41. Ndungu SG, Brown CG, Dolan TT: In vivo comparison of susceptibility between Bos indicus and Bos taurus cattle types to Theileria parva infection. Onderstepoort J Vet Res. 2005, 72 (1): 13-22.PubMedGoogle Scholar

Copyright

© Yang et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Advertisement