DOI: 10.2478/s11686-014-0211-9 © W. Stefański Institute of Parasitology, PAS Acta Parasitologica, 2014, 59(1), 85–90; ISSN 1230-2821

Targeting tams-1 gene results in underestimation of Theileria annulata infection in diseased cattle in Egypt Ahmed M. Ghoneim* and Asmaa O. El-Fayomy Department of Zoology, Faculty of Science, Damietta University, New Damietta, Damietta, Egypt

Abstract Tropical theileriosis is considered one of the most economically important tick borne diseases that cause significant mortality and morbidity to livestock. In the context of epidemiological studies on livestock in Egypt, this report investigated the spread of Theileria annulata among diseased farm cows (Bos indicus) over one year. Blood samples collected from 130 cows were investigated by routine staining and 64 samples were investigated by PCR assay using two different probes targeting tams-1 gene. Microscopy revealed the infection of 33.8% of animals with Theileria while PCR detected infection in 51% of animals with one primer pair and the other primer pair detected the infection in 31% of animals. Combined PCR results indicated the infection of 68.8% of animals with T. annulata. Seasonal fluctuation of parasite infection was evident with the highest infection percentage and prevalence recorded during summer based on both microscopy and PCR data. For the first time, the current study reports the presence of two T. annulata isolates based on tams-1 gene partial sequence in Egypt. Targeting polymorphic genes for parasite detection may result in underestimation of infection and target gene diversity has to be considered. The high infection with these pathogens in the clinically ill cows necessitates implementing serious programs to minimize their economic burden on the Egyptian farming industry.

Keywords Tams-1 gene, PCR, epidemiology, Theileria, seasonal, Bos indicus, livestock

Introduction Tick-borne diseases cause major problems to health and management of domestic cattle in tropical and subtropical regions of the world (Jongejan and Uilenberg 1994). Tropical theileriosisis is one of the most economically important tick borne diseases (Callow 1984). It is caused by the Apicomplexan protozoan parasite Theileria transmitted by Ixodidae ticks (Caeiro 1999, Preston 2001, Silva et al. 2010) and affects millions of cattle in different regions of Africa. T. annulata is considered as the cause of tropical theileriosis (Spooner et al. 1989). Infective sporozoites, injected during tick feeding, rapidly enter target cells, escape from the surrounding host-cell membrane and differentiate to schizonts that interact with different host-cell components (Dobbelaere and Rottenberg 2003). This interaction includes host cell signaling pathways that regulate proliferation and cell survival (Chaussepied and Langsley 2011) and thus cause blastogenesis and clonal expansion of predominantly T and B cells (Fawcett et al. 1982, Baldwin et al. 1988, Spooner et al. 1989). Merozoites released

from these schizonts subsequently infect red blood cells and become trophozoites. Lymphocytic stage of Theileria (schizont) is the cause of many of the severe disease manifestations like lymphadenopathy, pyrexia, thrombocytopenia, and panleukopenia (Homer et al. 2000). Although microscopy is considered as the “gold standard” for detecting acute infections with piroplasms (Nayel et al. 2012), it is not suitable for detecting low parasitemia (Friedhoff and Bose 1994, Bose et al. 1995). Serological and indirect fluorescent antibody tests can result in false data due to crossreactions or improper specific immune response (Billiouw et al. 2005). Recently, molecular tools have been developed for detection and quantification of many infectious pathogens and they proved to be highly accurate and sensitive. Reverse line blotting assay is currently considered the most sensitive test for detecting T. annulata (Gubbels et al. 1999, Georges et al. 2001, Bilgic et al. 2010), however, this technique is a relatively cumbersome assay and is not entirely suitable for the routine diagnosis of Theileria infections (Santos et al. 2013). The alternative and most common PCR assays used for detecting T. annulata

*Corresponding author: [email protected]

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(e.g. D’Oliveira et al. 1995) target tams-1 gene which encodes the major piroplasm merozoite surface antigen of T. annulata. Epidemiological studies about tick borne diseases in Egypt are few and most of them were done using conventional staining techniques. In the current report we used conventional and PCR assays targeting tams-1 gene to accurately estimate the occurrence of T. annulata over one year in the farm cows Bos indicus showing signs of clinical illness and to check the reliability of a single tool in detecting this parasite.

Ahmed M. Ghoneim and Asmaa O. El-Fayomy

95°C for initial enzyme activation followed by 40 cycles of 30 sec at 95°C for denaturation, 30 sec at the optimized temperature for annealing and 1–2 min at 72°C for extension, and a final extension step for 10 min at 72°C. PCR products were loaded together with Simply Load 100 bp DNA ladder (#50327, Lonza) on 1.5% agarose gels and run for 40 min. Gels were stained for 20 min with Ethidium Bromide (Bioshop, Canada) photographed in a gel documentation system (Photo Doc-IT Imaging system, USA). DNA sequencing

Materials and Methods Sample collection Blood samples were collected by a veterinarian from the jugular vein of 130 clinically ill Bos indicus cows that needed urgent treatment in EDTA-rinsed vacuotainer tubes. All samples were collected from 16 small scale farms at Port Said Governorate in the Delta region of Egypt. Blood smears were prepared immediately, dried and stored for later Giemsa staining. The remaining blood was frozen until DNA extraction for molecular investigation. Conventional parasite detection The prepared thin smears were fixed in methanol, stained with Giemsa, and microscopically examined at 1000× magnification for Theileria detection. Smears were recorded as negative for infection if no parasites were detected in 50 oil-immersion fields (Moretti et al. 2010).

PCR products were run on agarose gels and DNA bands were excised for DNA extraction with EZ-10 Spin Column Gel extraction kit (#BS353, Bio Basic, Canada) according to the manufacturer’s instructions. Purified products were sequenced with the primer Th5_2 using Standard Sanger method combined with new technology on ABI 3730XL DNA Sequencer at GATC Biotech (Germany). Chromatograms were checked and corrected using ABI sequence viewer software. Statistical analysis Differences between the percentage (or prevalence) of infection recorded by different detection methods (or during different seasons) were tested by Chi-square test and the differences were considered significant if the significance level was 0.05 or less.

Results

Molecular detection of T. annulata

Detection of Theileria parasites by microscopy

DNA was extracted from 500 µl of whole blood using GeneJET genomic DNA purification kit (K#0721, Fermentas, Germany) or G-spin total DNA extraction kit (17045, Intron Biotechnology, South Korea) according to the manufacturer’s instructions. Two pairs of oligonucleotides specific for tams1 gene of T. annulata; Th5_1 (Forward 5’-GTAACCTTTAAAAACGT-3’, reverse 5’-GTTACGAACATGGGTTT-3’ (d’Oliveira et al. 1995)) and Th5_2 (Forward 5’-CAAATTCGAGACCTACTACGATG-3’, reverse 5’-CCACTTRTCGTC CTTAAGCTCG-3’ (Santos et al. 2013)) were synthesized in Bioneer Corporation (South Korea) and used to detect the parasite DNA. PCRs were performed on 64 DNA samples using Maxima Hot Start PCR master mix kit (#K1051, Fermentas, Germany) or i-Taq DNA polymerase master mix (#25027, Intron Biotechnology, South Korea) according to the manufacturer’s instructions. In both types of PCR, 25 μl of Master mix (containing Taq DNA polymerase, dNTPs and Mg2+) were mixed with 4 pmoles of the oligonucleotides, 2 μl of genomic DNA and the reaction was brought to a final 50 μl with nuclease-free water. Reactions were loaded to a thermal cycler (Multigene, Labnet, USA) programmed as follows: 4 min at

Investigation of 130 blood smears by routine Giemsa staining indicated the infection of 44 animals (33.8%) with Theileria parasites (Table I). Infections were detected both in erythrocytes and in leucocytes (Fig. 1, A) and a high number of lymphocytes was frequently noticed (Fig. 1, B). Detection of Theileria annulata by PCR For specific detection of Theileria annulata, 64 samples were chosen randomly and their extracted DNA was investigated by PCR using two primer pairs targeting different regions of Tams1 gene. As shown in Fig. 2, panel A and C, the first primer pair (Th5_1) successfully amplified ~721 bp fragment of tams-1 gene as expected in 33 samples (51%). Two samples (62 and 63), however, resulted in a PCR product with a size higher than expected. The second primer pair (Th5_2) amplified ~319 bp fragment as expected in 20 samples (31%) (Fig. 2, panels B and D). Samples 1, 5, 13 and 14, however, resulted in a PCR product with a size higher than expected. Statistically, the results obtained with the first PCR (with Th5_1) were significantly different from those obtained by the second PCR (with Th5_2) or Microscopy.

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Targeting tams-1 gene underestimates Theileria annulata infection

Table I. Results of microscopy and the two PCR methods used for parasite detection in all samples investigated in this study Seial No. Microscopy PCR (Th5_1) PCR (Th5_2) Seial No. Microscopy PCR (Th5_1) PCR (Th5_2) Seial No. Microscopy PCR (Th5_1) PCR (Th5_2) Seial No. Microscopy PCR (Th5_1) PCR (Th5_2) Seial No. Microscopy PCR (Th5_1) PCR (Th5_2)

1 – n n 27 – – – 53 – – + 79 + n n 105 + – –

2 – – – 28 – n n 54 – n n 80 – n n 106 – – –

3 – n n 29 + n n 55 – n n 81 – n n 107 + + –

4 – n n 30 – + + 56 – n n 82 + n n 108 + + –

5 – – – 31 – n n 57 – n n 83 – n n 109 – + +

6 – – – 32 – + – 58 – n n 84 – n n 110 + – +

7 – – – 33 – n n 59 – – – 85 + + – 111 + n n

8 + + – 34 + + – 60 + + + 86 – + – 112 + n n

9 – + – 35 – + – 61 – n n 87 – + – 113 – n n

10 + + – 36 + – – 62 – n n 88 + + – 114 – – +

11 + – – 37 + + – 63 – – – 89 – + + 115 – n n

12 – – + 38 – n n 64 – – – 90 + + – 116 + – +

13 – n n 39 – n n 65 – – – 91 + + – 117 + n n

14 – + + 40 – n n 66 – n n 92 – + – 118 + n n

15 – + – 41 – n n 67 – n n 93 + + – 119 – n n

16 – – – 42 – – – 68 + n n 94 – + – 120 + – +

17 + + + 43 – – – 69 + n n 95 + + – 121 + – +

18 – n n 44 + – – 70 + + – 96 + – – 122 – n n

19 – n n 45 + – – 71 – + – 97 + n n 123 – n n

20 – + + 46 + – + 72 + + – 98 – n n 124 – n n

21 – n n 47 + n n 73 – + + 99 + n n 125 – n n

22 – n n 48 – n n 74 – – – 100 + n n 126 + n n

23 – n n 49 – – – 75 – n n 101 – n n 127 + n n

24 – – + 50 – n n 76 – n n 102 – n n 128 – n n

25 – – + 51 – n n 77 – n n 103 + + + 129 – n n

26 – n n 52 – n n 78 – n n 104 – + + 130 + n n

–, negative; +, positive; n, not determined

However, there was no significant difference between the results obtained with the second PCR and Microscopy. In total, 44 samples (68.8%) were positive with either Th5_1 or Th5_2 with PCR products of the expected sizes. Results of both microscopy and the two PCR methods used for investigating all samples in this study are shown in Table I. Seasonal variation of T. annulata occurrence Based on microscopy data, the highest percentage of infection with Thelieria was recorded during summer season (71.1%) compared to spring (15.8%), winter (10.5%) and fall (2.6%) (Table II). A very close pattern was obtained with PCR where

the highest percentage of T. annulata infection was recorded during summer (77.3%) followed by spring and winter (9.1%) and fall (4.5%). Parasite prevalence was highest in summer (36.5% and 79.1%) according to microscopic and PCR data respectively (Table II). Thus, parasitic infection during summer was the highest as indicated by both methods and the difference in the parasite prevalence was confirmed to be significantly different at 0.01 level. Partial sequencing of tams-1 gene of T. annulata Sequencing of the 319 bp PCR products amplified with the primer Th5_2 revealed the presence of two different tams-1

Fig. 1. Representative images showing the infection of cow leucocytes with Theileria (A) and the lymphoproliferation disorder (B). Arrow heads refer to the parasite within the leucocyte in (A)

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Ahmed M. Ghoneim and Asmaa O. El-Fayomy

A

M

1

2

3

4

5

6

7

8

9

10

11

12

M

33

34

35

36

37

38

39

40

41

42

43

44

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

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31

32

47

48

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51

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60

61

62

63

64

1000 700 500 300 100

B

1000 700 500 300 100

C

45

46

1000 700 500 300 100

1000 700

D 500 300 100

Fig. 2. PCR assay on 64 DNA randomly selected samples for detection of T. annulata with the primer pair Th5_1 (A, C) and primer pair Th5_2 (B, D)

Table II. Parasitic infection during different seasons based on microscopic examination and PC assay Season

Winter

Method

Microscopy PCR

No. of examined animals No. of infected animals Percentage of infection Prevalence of infection

17 4 10.5 23.5

Spring

8 4 9.1 50.0

Summer

Fall

Total

Microscopy

PCR

Microscopy

PCR

Microscopy

PCR

26 6 15.8 23.1

9 4 9.1 44.4

74 31 71.1 36.5

43 34 77.3 79.1

13 1 2.6 7.7

4 2 4.5 50.0

sequences in Egyptian isolates of T. annulata. These sequences were deposited in the gene bank under the accession numbers KF765518 and KF765519. When blasted to gene bank, one sequence (KF765518) was found to be 99% identical to T. annulata isolates with the accession numbers AF214829, AF214825, AF214816, AF214815, AF214814, AF214813, AF214809, EU563912.1, AF214898, AF214897, AF214862, AF214858, AF214857, XM_948626.1, AF214918, AF214917, AF214915, AF214914 and AF214909. The other sequence (KF765519), however, was 99% identical to T. annulata isolate JX088598. In general, the two tams-1 partial sequences differed in 5 positions 457 (T/G), 494 (T/A), 531 (C/T), 532 (A/C) and 533 (C/T). Translation of these 2 sequences revealed their difference in 3 amino acid residues; 153 (S/A), 165 (M/K) and 178 (T/L).

Discussion The current study has been undertaken in the context of profiling the spread of tick borne diseases in domestic animals in Egypt. Microscopy revealed the infection of 33.8% of animals with Theileria while PCR detected infection in 68.8% of animals with T. annulata in the randomly selected samples. The

Microscopy 130 42 32.3

PCR 64 44 68.8

highest infection percentage and parasite prevalence was recorded during summer based on both microscopy and PCR data. Our data were close to some previous reports undertaken in different regions of Egypt on symptomatic and non-symptomatic animals while differed considerably from others. For example, Adel (2007) found that the incidence peaks of T. annulata seropositive native breed cattle using IFAT were 27.8 and 22% in spring and summer, respectively in Gharbia governorate. Gamal EI-Dien (1993) reported 65.4% prevalence of T. annulata in EI-Behera governorate using stained blood films. This discrepancy is most probably due to variation in the susceptibility of the animal species and/or the variance in animal locale. One more reason could be the mis-identification of T. annulata by microscopy. In fact, several species of Theileria are known to infect cattle and it is difficult to differentiate Theileria species solely on the basis of the morphology of the piroplasm and schizont stages, and confusion may arise if mixed infections occur (d’Oliveira et al. 1995). With IFA, cross-reactions have been observed among T. annulata, T. parva, T. mutans, and T. taurotragi (Burridge et al. 1974, Grootenhuis et al. 1979, Kiltz et al. 1986). According to d’Oliveira et al. (1995), amplification of parasite DNA is by far more sensitive than parasite detection by light microscopy or IFA and PCR assay detects T. annulata parasites at low

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Targeting tams-1 gene underestimates Theileria annulata infection

parasitemias in carrier cattle and it can discriminate T. annulata from nonpathogenic Theileria species and other hemoparasites that may occur simultaneously in the same carrier animal. One more report (Mohamed et al. 2012) about the prevalence of T. annulata among clinically suspected animals selected from different localities in ELFayoum, EL-Minia, Assuit, Sohage and EL-Wady EL-Gaded governorates in Upper Egypt (during the period of May to August 2008) detected T. annulata in 48 (75%) and 4 (33.3%) cases out of the 64 and 12 clinically suspected Bos indicus cattle and water buffaloes, respectively. Seasonal fluctuation in T. annulata parasitemia was evident in the current study with summer season harboring T. annulata outbreaks. This finding coincides with previous reports worldwide where environmental conditions (especially temperature and humidity) are well known to be major determinants in tick-borne disease outbreak. Humidity plays an important role in tick population density, particularly I. ricinus (Milne 1950, L’Hostis et al. 2002). An increase in temperature may allow vectors to migrate into new areas or to allow a significant development of parasites in areas where ambient temperature was not convenient (Coles et al. 2001). The discrepancy in PCR data between Th5_1 (d’Oliveira et al. 1995) which detected T. annulata infection in 51% of animals and Th5_2 (Santos et al. 2013) which detected parasite infection in 31% of animals is most probably due to the high polymorphism of tams-1 gene at the primer sites. The 319 bp region of tams-1 gene amplified by Th5_2 primer pair is thought to be a conserved region and for which this primer pair was recently designed by Santos et al. (2013), however, amplification with Th5_1 primer pair originally set by d’Oliveira et al. (1995) seems to be more sensitive in detecting Egyptian isolates of T. annulata. In fact, both isolates of the current study have a point mutation at the position 687 of tams-1 gene, which is spanned by the primer Th5_2 reverse. The different results obtained with these two primer pairs highlight the necessity to use more than one primer pair for reliable parasite detection by PCR especially when targeting polymorphic genes like tams-1. For the first time, the current study provides a partial sequence of tams-1 gene in Egypt. None of the isolates sequenced here was 100% identical to any of the sequences deposited in the gene bank. One isolate (KF765518) was 99% identical to 19 different isolates while the other (KF765519) was 99% identical to one isolate only. tams-1 gene which encodes the major piroplasm merozoite surface antigen is known to be highly polymorphic (Gubbels et al. 2000) and more than 140 sequences of this gene were released (Santos et al. 2013). The encoded protein is involved in recognition and attachment of merozoites to erythrocytes and the amino acid replacements in this protein may imply its use by merozoites for alternative interaction with erythrocytes and thus ensuring more virulence. Considering the nature of the samples studied here, where all of them were diseased animals and a veterinarian was called for urgent treatment, it can be concluded that T. annu-

lata represents a true threat for livestock in Port Said governorate and possibly other governorates of Egypt. Epidemiological research is essential in providing updated knowledge about livestock infection with tick-borne diseases; however, appropriate preventive measures have to be considered to protect animal production and management. Further studies to investigate the relation between genetic diversity and parasite virulence have to be considered in Egypt. Acknowledgements. Authors would like to thank A. Khidr and Ola Abu-Samak, professors of Parasitology in Zoology Department, Faculty of Science, Damietta University, for their advice and help. Thanks are due to Radwa El-Tahan, a graduate student in the same Department for assistance in lab work.

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Received: June 14, 2013 Revised: October 27, 2013 Accepted for publication: December 19, 2013

Ahmed M. Ghoneim and Asmaa O. El-Fayomy

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