Genetic diversity and phylogenetic analysis of Tams1 of Theileria annulata isolates from three continents between 2000 and 2012.

Theileria annulata, which is part of the Theileria sergenti/Theileria buffeli/Theileria orientalis group, preferentially infects cattle and results in high mortality and morbidity in the Mediterranean, Middle East, and Central Asia. The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of T. annulata that could be used as a marker for epidemiological studies and phylogenetic analysis. In the present study, a total of 155 Tams1 sequences were investigated for genetic diversity and phylogenetic relationships through phylogenetic analysis. Results showed that the Tams1 sequences were divided into two major groups and that distribution for some isolates also exhibited geographic specificity. As targeting polymorphic genes for parasite detection may result in underestimation of infection, polymerase chain reaction (PCR) assay using two different probes targeting tams-1 genes of these two groups can be more credible. In addition, the direction of the spread of the disease was discovered to be from the Mediterranean or the tropical zone to the Eurasian peninsula, Middle East, Southern Asia, and Africa, particularly for Group 2. A similar occurrence was also found between the Ms1 gene of Theileria lestoquardi and the Tams1 gene of T. annulata, which explains cross-immunogenicity to a certain extent. However, no potential glycosylation site in the Tams1 of T. annulata was found in this study, which illustrated that instead of N-glycosylation, other modifications have more significant effects on the immunogenicity of the Tams1 protein.

Introduction

Various species of Theileria bovine parasites are widespread in Southern Europe, North Africa, and Southern Asia, thus presenting a significant threat to livestock productivity [1]. Among these, the tick-borne protozoan parasite Theileria annulata is the causative agent of lymph proliferative theileriosis, an important disease with high mortality and morbidity. Recently, in an epidemiological survey of bovine Babesia and Theileria parasites, T. annulata has been found to be the most common blood parasite in cattle, buffalo, and sheep populations, which were bred in different geographical locations in Egypt [2]. Theileria annulata parasitizes the reticuloendothelial system and the red blood cells of cattle. The intermediate host then exhibits symptoms such as high fever (40°C to 42°C), depression, cough, runny nose and watery eyes, anemia, and jaundice. Given that the main vectors of ring theileriosis are Hyalommadetritum, H. anatolicumanatolicum, H. anatolicumexcavatum, H. asiaticum, H. dromedarii, and H. marginatummarginatum [3], the disease typically begins in May, with outbreaks occurring in June and July, and then gradually subsides.

The polypeptide Tams1 elicits a protective response as an immunodominant major merozoite piroplasm surface antigen against the protozoan parasite T. annulata [4, 5], and is considered as a candidate for inclusion in a sub-unit recombinant vaccine [6]. The Tams1-encoding gene has been developed for PCR-based assays, which could use bovine blood samples to detect T. annulata infections [1]. Tams1 protein has also been reported as a candidate to develop a diagnostic enzyme-linked immunosorbent assay (ELISA) because it exhibits significant sequence diversity and no geographic specificity [4]. However, sequencing and phylogenetic analyses of the Egyptian T. annulata showed that theTams-1 sequences are relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries [2]. Moreover, Tams1 diversity has been reported as being generated by the random mutation of nucleotides during asexual reproduction as well as by the selection of changes that confer a biological advantage instead of the differential expression of the members of a gene family [7, 8].

Several attempts have been made to analyze the phylogenetic characteristics of the Tams1 gene of T. annulata [8]. However, no distinct classification for all reported Tams1 sequences obtained from three continents (Asia, Africa, and Europe) is yet available. In the present study, 155 complete Tams1 genes of T. annulata occurring over a wide geographical range were first sequenced. Given that the taxonomic status and epidemiology of T. annulata remain undefined [9], studying the phylogenetic variability and molecular genetic characterization of Tams1 will help provide an understanding of the relationship between the molecular evolutionary history of T. annulata and the emergence, breakout, and spread of new T. annulata epidemics.

In addition, attempts were made to analyze the phylogenetic diversity and distribution of T. annulata. A phylogenetic tree was constructed and analyzed. Comparisons and additional analyses were also performed, including predicting potential glycosylation sites as well as finding volatile regions and evolutionary regular patterns [10].

Material and methods

Theileria sampling, DNA extraction, polymerase chain reaction amplification, and sequencing

A total of 81 adult ticks were collected from 61 cattle in a randomly selected dairy farm in Xinjiang, a northwestern city in China, in 2012. DNA extraction, as well as polymerase chain reaction (PCR) detection and amplification, were performed by Meng et al. [3]. In brief, a previously reported primer set (Forward 5-GTAACCTTTAAAAAC-GT-3, Reverse 5-CAGTTACGAACATGGGTTT-3) was used to detect Tams1 DNA specifically [1, 11]. The recombinant plasmid pMD18-T vector (Takara Bio Inc., Japan) was transformed into Escherichia coli TOP10 competent cells after purified DNA fragments were cloned and inserted. Then, the E. coli that was cultured overnight were purified and sent to a private company [Sangon Biotech (Shanghai) Co., Ltd., China] for sequencing. The results were identified by comparing the obtained sequences with the registered sequences in GenBank through BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). From the clinical isolate, an identical sequence with the Tams1 gene of T. annulata was found (GenBank accession no. JX475044).

Sequence alignment and phylogenetic analysis

Tams1 gene sequences published in GenBank were also included. Blasting was performed at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The amplified sequences and Tams1 were both blasted to ensure that all known Tams1 genes of T. annulata were brought forward. Consequently, a total of 155 isolates were discovered (Table 1) including RefSeq (Turkey, 1), Spain (14), Portugal (5), Italy (5), Tunisia (46), Iran (4), Bahrain (9), Turkey (18), India (7), Mauritania (22), Iraq (7), China (2), Sudan (5), Sri Lanka (3), unknown origin (4), and isolates of T. lestoquardi (4) [11].

Table 1

Origin of Tams1 isolates used in the present study

No.	Country	Year	Accession no.
1	Spain	2000	AF214813.1
2	Spain	2000	AF214816.1
3	Spain	2000	AF214814.1
4	Spain	2000	AF214815.1
5	Spain	2000	AF214827.1
6	Italy	2000	AF214862.1
7	Italy	2000	AF214860.1
8	Spain	2000	AF214809.1
9	Iran	2006	EF092915.1
10	Tunisia	2000	AF214879.1
11	Tunisia	2000	AF214864.1
12	Tunisia	2000	AF214903.1
13	Tunisia	2000	AF214882.1
14	Tunisia	2000	AF214865.1
15	Spain	2000	AF214812.1
16	Spain	2012	JX683683.1
17	Spain	2006	Z48739.1
18	Spain	1995	U22888.1
19	Bahrain	2000	AF214802.1
20	Bahrain	2000	AF214795.1
21	Bahrain	2000	AF214799.1
22	Turkey	2000	AF214835.1
23	Mauritania	2000	AF214851.1
24	Mauritania	2000	AF214856.1
25	Mauritania	2000	AF214853.1
26	Mauritania	2000	AF214850.1
27	Portugal	2000	AF218426.1
28	Portugal	2000	AF218425.1
29	Portugal	2000	AF218429.1
30	Portugal	2000	AF218428.1
31	Tunisia	2000	AF214919.1
32	Tunisia	2000	AF214895.1
33	Tunisia	2000	AF214893.1
34	Tunisia	2000	AF214894.1
35	Tunisia	2000	AF214880.1
36	Bahrain	2000	AF214794.1
37	Bahrain	2000	AF214801.1
38	Bahrain	2000	AF214798.1
39	Turkey	2000	AF214836.1
40	Turkey	2000	AF214837.1
41	Bahrain	2000	AF214800.1
42	India	2000	AF214844.1
43	Tunisia	2000	AF214868.1
44	Tunisia	2000	AF214866.1
45	Mauritania	2000	AF214817.1
46	Mauritania	2000	AF214822.1
47	Iraq	2010	Gu130194.1
48	Iraq	2010	Gu130193.1
49	Tunisia	2000	AF214869.1
50	Tunisia	2000	AF214871.1
51	Tunisia	2000	AF214870.1
52	Bahrain	2000	AF214796.1
53	Bahrain	2007	EF618728.1
54	India	2007	EF618726.1
55	Tunisia	2000	AF214878.1
56	Tunisia	2000	AF214877.1
57	Tunisia	2000	AF214887.1
58	Tunisia	2000	AF214867.1
59	Tunisia	2000	AF214900.1
60	Tunisia	2000	AF214889.1
61	Tunisia	2000	AF214888.1
62	Turkey	2000	AF214909.1
63	Turkey	2000	AF214915.1
64	Turkey	2010	AJ276654.1
65	Turkey	2000	AF214918.1
66	Turkey	2008	XM948626.1
67	Turkey	2000	AF214916.1
68	Mauritania	2000	AF214858.1
69	Mauritania	2000	AF214846.1
70	Tunisia	2000	AF214898.1
71	Tunisia	2000	AF214897.1
72	Mauritania	2000	AF214854.1
73	Mauritania	2000	AF214821.1
74	Mauritania	2000	AF214824.1
75	Mauritania	2000	AF214823.1
76	Mauritania	2000	AF214848.1
77	Mauritania	2000	AF214845.1
78	Mauritania	2000	AF214819.1
79	Tunisia	2000	AF214883.1
80	Tunisia	2000	AF214881.1
81	Tunisia	2000	AF214901.1
82	Tunisia	2000	AF214920.1
83	Tunisia	2000	AF214885.1
84	Iran	2004	AY672541.1
85	China	2012	JX475044.1
86	Turkey	2000	AF214917.1
87	Turkey	2000	AF214914.1
88	Turkey	2000	AF214838.1
89	Turkey	2000	AF214839.1
90	Mauritania	2000	AF2w14857.1
91	Mauritania	2000	AF214820.1
92	China	2008	EU593912.1
93	India	2000	AF214842.1
94	Mauritania	2000	AF214818.1
95	Mauritania	2000	AF214849.1
96	Mauritania	2000	AF214855.1
97	Sudan	2000	AF214834.1
98	Sudan	2000	AF214831.1
99	Sudan	2000	AF214832.1
100	Sudan	2000	AF214833.1
101	Sudan	2000	AF214830.1
102	India	2000	AF214843.1
103	South India	2012	JX648210.1
104	India	2000	AF214841.1
105	India	2000	AF214840.1
106	Mauritania	2000	AF214847.1
107	Tunisia	2000	AF214899.1
108	Tunisia	2000	AF214872.1
109	Turkey	2000	AF214911.1
110	Tunisia	2000	AF214873.1
111	Tunisia	2000	AF214886.1
112	Tunisia	2000	AF214906.1
113	Tunisia	2000	AF214907.1
114	Tunisia	2000	AF214905.1
115	Tunisia	2000	AF214884.1
116	Iran	2006	EF092919.1
117	Iran	2006	EF092918.1
118	Iraq	2010	GU130192.1
119	Turkey	2000	AF214910.1
120	Turkey	2000	AF214912.1
121	Turkey	2000	AF214913.1
122	Iraq	2010	GU130191.1
123	Iraq	2010	GU130190.1
124	Italy	2000	AF214859.1
125	Italy	2000	AF214863.1
126	Italy	2000	AF214861.1
127	Bahrain	2000	AF214797.1
128	Spain	2000	AF214808.1
129	Spain	2000	AF214807.1
130	Spain	2000	AF214805.1
131	Spain	2000	AF214804.1
132	Spain	2000	AF214803.1
133	Spain	2000	AF214810.1
134	Spain	2000	AF214811.1
135	Spain	2000	AF214806.1
136	Sri Lanka	2012	AB690865.1
137	Sri Lanka	2012	AB690864.1
138	Sri Lanka	2012	AB690863.1
139	Iran	2006	EF092916.1
140	Iran	2000	AF004775.2
141	Iran	1999	AJ006447.1
142	Iran	1999	AJ006448.1
143	Iraq	2010	FJ159695.1
144	Tunisia	2000	AF214876.1
145	Tunisia	2000	AF214874.1
146	Tunisia	2000	AF214875.1
147	Tunisia	2000	AF214896.1
148	Tunisia	2000	AF214892.1
149	Tunisia	2000	AF214890.1
150	Tunisia	2000	AF214891.1
151	Turkey	2000	AF214908.1
152	Mauritania	2000	AF214852.1
153	Tunisia	2000	AF214904.1
154	Tunisia	2000	AF214902.1
155	Iraq	2010	GU130189.1

Multiple sequence alignments were performed using the ClustalW algorithm [12]. The phylogenetic tree was constructed through the neighbor-joining method [13] and a bootstrap value of 1000 replicates, using the MEGA 5.1 software [14].

Predicting N-glycosylation sites

Glycosylation sites have an important role in determining the properties of the concerned protein, such as antigenic properties, among others [15, 16]. Considering that the glycosylation sites of the protein were formed after processing, only potential glycosylation sites were predicted at the amino acid level. In this investigation, referring to the phylogenetic tree, 39 isolates (including XM948626.1, AF214906.1, AF214872.1, AF214920.1, AF214904.1, AF214819.1, AF214840.1, JX648210.1, AF214832.1, AF214852.1, AF214849.1, AF214818.1, AF214898.1, EU563912.1, JX475044, AJ276654.1, EF618726.1, AF214869.1, AF214900.1, GU130193.1, AF214866.1, AF214856.1, AF214835.1, U22888.1, AF214800.1, AF214801.1, AF214812.1, AF214879.1, EF092915.1, AF214825.1, AF214815.1, AB690864.1, AF214805.1, AF214797.1, AF214863.1, AF214875.1, GU130190.1, EF092918.1, FJ159695.1) on behalf of 155 isolates were selected, followed by the submission of the amino acid sequence of the protein to NetNGlyc Server (http://www.cbs.dtu.dk/services/NetNGlyc/) [17], and the prediction of the N-glycosylation sites in the amino acid sequence of the protein.

Results

Based on the multiple sequence alignments and the neighbor-joining method, the sequence obtained from the clinical isolate and the 154 sequences (including four Ms1 genes from T. lestoquardi) in GenBank of Tams1 isolates derived from the three continents (Asia, Africa and Europe) were assigned to two groups (Fig. 1). Sequences from Spain, Italy, Tunisia, Iran, Bahrain, Turkey and Iraq were found in both groups, while in Portugal, India, Mauritania, China, Sudan and Sri Lanka, sequences were only found in Group 1. In the phylogenetic tree, isolates from the same continents (Africa and Europe) got-together in Group 1 (Fig. 2). The N-glycosylation sites’ prediction by online software showed that no potential glycosylation sites were found.

Fig. 1

Phylogenetic tree for the Tams1 gene of the 155 Theileria annulata isolates

Fig. 2

The geographical distribution of Theileria annulata

Discussion

The phylogenetic tree constructed with 155 Tams1 gene sequences(including four Ms1 genes from T. lestoquardi) shows that T. annulata isolates were divided into two major groups, which were called Groups 1 and 2. This discovery was very important and significant, as almost every research about Tams1 would bring up some similar guesses [18]. Habibi [19] classified 17 Tams1 gene sequences into 2 clusters. However, classifying all reported Tams1 sequences obtained from three continents (Asia, Africa, and Europe) has not yet been performed.

Several studies found that the Tams1 gene is highly polymorphic, raising questions concerning the suitability of Tams1 gene-targeted primers to detect all T. annulata isolates [1]. And especially in clinical practice, as targeting polymorphic genes for parasite detection may result in underestimation of infection, PCR assay using two different probes targeting tams-1 genes of these two groups, for example the primers (Forward 5-GTAACCTTTAAAAAC-GT-3, Reverse 5-CAGTTACGAACATGGGTTT-3) [3] and primers (Forward 5-ATGTGTCCAGGACCACCC-3, Reverse 5-GGGTTTTAAAGGAAGTAAAGG-3) [4], can be more credible than just relying on one primer pair [18]. The sequences from Spain, Italy, Tunisia, Iran, Bahrain, Turkey, and Iraq were found in both groups, whereas the sequences from Portugal, India, Mauritania, China, Sudan, and Sri Lanka were only found in Group 1. In this group, isolates from the same continents (Africa and Europe) were obviously clustered in the phylogenetic tree (Fig. 1).

Previous studies have reported that no geographic specificity was observed and nearly identical sequences occurred in different geographic areas. Moreover, a panmictic (from panmixia, that is, the ability of individuals in a population to move freely within their habitat, and thus breed with other members of the population) population structure was suggested by the results of several studies [4]. In the present study, however, the comparison of the Tams1 gene sequences obtained from three continents revealed that particular sequence types belong to definitive regions, such as Sudan, Sri Lanka, and so on (marked in dark green and purple in Fig. 1). Nearly no identical Tams1 sequence was found in widely separated regions.

By referring to Fig. 2 and Table 2, the direction of the spread of the disease, particularly for Group 2, can be speculated. Based on the time (as the recording time reflects the isolates’ outbreak time in some degree) and distribution, the following conclusions can be drawn. Isolates from both groups were found in the European peninsula in 2000, in Iran in 2006, and in Iraq in 2010. However, only Group 1 isolates were found in China, India, and Sri Lanka. Group 2 isolates eventually spread to Iraq. The overall spread direction was from the Mediterranean or the tropical zone to the Eurasian peninsula, Middle East, Southern Asia, and Africa.

Table 2

Numbers of Theileria annulata isolates in groups 1 and 2 in each country

Country	Group 1	Year	Group 2	Year
Spain	6	2000	8	2000
Portugal	5	2000	0
Italy	2	2000	3	2000
Tunisia	39	2000	7	2000
Iran	1	2004	2	2006
	1	2006
Bahrain	8	2000	1	2000
Turkey	11	2000	4	2000
	1	2010
India	5	2000	0
	2	2007
	1	2012
Mauritania	22	2000	0
Iraq	2	2010	5	2010
China	1	2001	0
	1	2012
Sudan	5	2000	0
Sri Lanka	3	2012	0

Theileria annulata has the ability to transform the leukocytes of host animals and been called transforming Theileria. The evolution of the transforming Theileria has been accompanied by drastic changes in its genetic makeup, such as acquisition or expansion of gene families, which are thought to play critical roles in the transformation and the immune escapement of host cells [20]. Immune escape in Theileria is facilitated by genetic diversity in its antigenic determinants, which potentially results in a loss of T cell receptor recognition in its host. And in the case of Asn-Xaa-Ser/Thrsequons (incl. Asn-Pro-Ser/Thr), no potential glycosylation site was discovered. This positive verified the suspect of Katzer in 2002 that there were no N-glycosylation sites on Tams1 genes [21]. Consequently, Tams1 protein easily combined with the antibody as a stable protein antigen. These results indicate that compared with N-glycosylation, other modifications have more significant effects on the immunogenicity of the Tams1 protein.

Cows in the aforementioned three continents have been reported to have been infected with mixed populations of geographically variable Theileria parasites [9, 22]. Cross-immunity studies conducted by Leeman [23] suggested that partial cross-immunity from T. annulata to T. lestoquardi, and vice versa, developed in sheep, thus indicating a close relationship among parasite species. As the tree may not be a true representation of the evolutionary relationship of these sequences/species, however, Tams1 sequences for T. annulata would be more likely to cluster together than with sequences from a distinct species. Co-incidentally, four T. lestoquardi Ms1 gene isolates (Query cover: 86% to 95%, ident: 85% to 86%) were obtained by blasting the Tams1 gene of T. annulata and were included in Group 2 through phylogenetic assay. This finding provides evidence of a significant similarity between the Ms1 gene of T. lestoquardi and the Tams1 gene of T. annulata, thus explaining the immunogenicity of the Tams1 protein to a certain extent.

Given that it enables evasion from host immune response, diversity is generally believed to be a positively selected result [24]. In Fig. 3, several variable areas were marked. These areas represent the generation of Tams1 diversity that enabled T. annulata to escape from host immunity. This finding provides valuable information on the antigenic structure of T. annulata and may be helpful in designing vaccines.

Fig. 3

Analysis and comparison of amino acid mutations in Tams1

Conclusions

Tams1, as an immunodominant surface antigen, has been used to investigate genetic diversity and vaccine purposes for antigenicity [4]. In the present study, all 155 Tams1 isolates were classified into two major groups in the phylogenetic tree, which explained why PCR assay using one primer pair targeting tams-1 gene failed to detect infection of some animals. Geographic specificity was observed when comparing Tams1 gene sequences. In addition, based on the geographic distribution and recording time of T. annulata, the spread direction of the disease, particularly for Group 2, was hypothesized to be from the Mediterranean or the tropical zone to the Eurasian peninsula, Middle East, Southern Asia, and Africa. A similarity on immunodominant major surface antigen gene was also found between the Ms1 gene of T. lestoquardi and the Tams1 gene of T. annulata, which explains cross-immunogenicity to a certain extent. However, no potential glycosylation site was found in this study, thus instead of N-glycosylation, other modifications have been hypothesized to have more significant effects on the immunogenicity of the Tams1 protein.