# Molecular Detection ofTheileria spp. in Livestock on Five Caribbean Islands

Authors: Jilei  Zhang; Patrick   Kelly; Jing  Li; Chuanling  Xu; Chengming  Wang
Journal: BioMed Research International
 (2015)

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## Abstract

Theileria spp. are tick-transmitted, intracellular apicomplexan protozoan parasites infecting a wide range of animals. As there is very limited information on the prevalence ofTheileria spp. in the Caribbean we used the recently described genus-specific pan-Theileria FRET-qPCR to identify infected animals in the region and a standard 18S rRNA gene PCR and sequencing to determine the species involved. We foundTheileria spp. in 9% of the convenience samples of animals (n=752) studied from five Caribbean islands. Donkeys (20.0%: 5/25) were most commonly infected, followed by sheep (17.4%, 25/144), cattle (6.8%; 22/325), goats (5.0%; 12/238), and horses (5.0%; 1/20). Six species ofTheileria were identified:T.equi (donkeys, cattle, goats, and sheep),Theileria sp. OT3 (sheep and goats),Theileriasp. NG-2013a (cattle),Theileria sp. YW-2014 (donkeys),Theileria sp. B15a (goats), andBabesia vulpes or a closely related organism (sheep and goats). OnlyT. equi has been previously reported in the Caribbean. Our findings expand the known host ranges ofTheileria spp. and the known distribution of the organisms around the world.


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## Body

## 1. Background

Theileria spp. are tick-transmitted, intracellular apicomplexan protozoan parasites infecting leukocytes and erythrocytes of a wide range of animals [1, 2]. The organisms have been described in all livestock species and can cause significant economic losses to farmers. They are transmitted by a variety of ixodid ticks of the generaRhipicephalus,Hyalomma,Amblyomma, andHaemaphysalis [3]. Infections with someTheileria spp. can result in fever, anemia, hemoglobinuria, and death in severe cases, but many species are benign and cause minor or no signs. Animals that recover from acute or primary infections usually remain persistently infected and may act as reservoirs for tick vectors [4, 5]. Infected animals are found particularly in tropical and subtropical regions in Africa, the Middle East, Southern Europe, and Asia [6–11].There is little information on infectious agents in livestock in the Caribbean although animal production is an important source of income for many people in the region. In the case ofTheileria spp., morphological and serological evidence has been presented thatT. mutans andT. velifera, both benign species transmitted byAmblyomma spp., occur in cattle on Guadeloupe [12]. Also, an organism with the morphology ofT. mutans was seen in a blood smear from a bovine on Martinique [13]. In Trinidad,T. equi (previouslyBabesia equi) has been demonstrated in horses with a specific nested 18S rRNA PCR [14, 15] and a serosurvey has provided supporting evidence for its presence [16].While there are many tests to detectTheileria spp. in animals, their specificity varies, as does their usefulness in finding the full spectrum of organisms present in an area. Microscopic detection of parasites can be difficult with low parasitemia and does not readily allow differentiation of species [2]. Serological studies, although sensitive and relatively easy to perform, are not specific as there is cross-reactivity betweenTheileria spp. [17]. Although molecular techniques have been described, many are for specific species which limits their usefulness in surveys. Reverse line blotting (RLB) assays enable the simultaneous identification of multiple species [18, 19], but they are cumbersome and time demanding to perform and identifying stringent species-specific oligonucleotide sequences can be challenging [20]. Recently, a sensitive genus-specific pan-Theileria FRET-qPCR has been described that detects the recognizedTheileria spp. of domestic animals in a single reaction (Table 1) [21]. To provide further data onTheileria spp. in the Caribbean, we used the pan-Theileria FRET-qPCR to screen livestock from five islands for evidence of infection. Further, we used a standard 18S rRNA PCR and gene sequencing on positive reactors to identify theTheileria spp. involved. The results of this survey are described below.Table 1
Alignment of the nucleotides used in the primers and probes of the pan-Theileria FRET-qPCR used in this study.
Forward primer
LCRed-640
6-FAM
Reverse primer
T
AGTGACAAGAAATAACAATACGGGGC- -TT
GTCTTGTAATTGGAATGATGGGAATT
AAACCTCTTCCAGAGTATCAATTGG
AGTTAAAAAGCTCGTAGTTGAATTTCTGCTG
T. orientalis
...........................- -..
..........................
.........................
...............................
T. buffeli
...........................- -.C
..........................
.........................
...............................
T. annulata
...........................
- -..
..........................
.........................
...............................
T. sergenti
...........................
- -..
..........................
.........................
...............................
T. luwenshuni
...........................
- -..
..........................
.........................
...............................
T. velifera
...........................
- -..
.C........................
.........................
..............................A
T. ovis
...........................
- -..
..........................
.........................
...............................
T. parva
...........................
- -..
..........................
.........................
...............................
T. uilenbergi
...........................
- -..
..........................
.........................
.............................C.
T. lestoquardi
...........................
- -..
..........................
.........................
...............................
T. equi
......................A....
- -..
..........................
.....C...................
...............................
T. separata
...........................
- -..
..........................
.........................
......................C........
T. capreoli
...........................
- -..
..........................
.........................
...............................
T. bicornis
...........................
- -..
.....C....................
.........................
...............................
T. taurotragi
...........................
- -..
..........................
.........................
...............................
T. mutans
...........................
- -.C
.C.......................C
.....C...................
...............................
Theileria sp. OT3
...........................
- -..
..........................
.........................
...............................
Theileria sp. NG
...........................
- -..
..........................
.........................
...............................
Theileria sp. YW
...........................
- -..
..........................
.........................
...............................
Theileria sp. B15
...........................
- -.C
.C......................CC
.....C...................
.............................C.
B. vulpes
......................A....
- -..
.........................C
.....CT.C................
......G......................CT
B. hongkongensis
......................A....
- -AA
.....................TG.C.
.....CTCA........G.......
....................T....T...GT
B. divergens
......................A....
- -AA
.....................TG.CC
.....CTCA........A.......
.........................T...GT
B. bovis
...............C...........
- -.A
.CTC.........C...GG..CG.CC
TC...CTCG..C.....C.C.....
........................C.A-.GT
B. bigemina
......................A....
- -..
.....................TG..G
.C.A.CTCA........C.......
....G...............T.....A..CT
B. gibsoni
......................A....
- -AA
.....................TG.CG
...ATCTCA........A.......
.A...........................GT
B. microti
......................A....
- -..
.........................C
.....CT.C................
......G......................CT
B. felis
......................A....
- -..
......................G.CC
.....CT.C................
......G......................CT
B. canis
......................A....
- -.A
.....................TG.C.
.....CTCA........G.......
.........................TA..GT
C. felis
.......................A...
- -..
..................C..A....
..G.TCT....G.............
...............................
H. americanum
......................AA...AA..
.CT................A.A....
....ACT..TT.A............
............................T.A
T. gondii
...................C..T..AAAT..
T...A..G...........A.....C
.....C...T.......A.......
....................G..........
Primers and probes are shown at the head of the table. 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 withTheileria spp. 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. The 18S rRNA sequences for the available recognized Theileria spp. on GenBank and 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 (AY534882), T. lestoquardi (JQ917458), T. separata (AY260175), T. capreoli (AY726011), T. bicornis (AF499604), T. taurotragi (L19082), T. mutans (FJ213585), Babesia vulpes(JX454779), Theileria sp. OT3 (KF470868), Theileria sp. NG-2013a (KF597076), Theileria sp. YW-2014 (AB981984), Theileria sp. B15a (JN572700); B. hongkongensis (JQ867356), B. divergens (AJ439713), B. bovis (JQ723013), B. bigemia (JQ723014), B. gibsoni (EU583386), B. microti (AB219802), B. felis (AF244912), B. canis (HM590440), Hepatozoon americanum (AF176836), Cytauxzoon felis (AY679105), and Toxoplasma gondii (L37415).


## 2. Materials and Methods

### 2.1. Samples Collection

Jugular venipuncture was used to collect blood in EDTA from convenience samples of apparently healthy livestock (cattle, goats, sheep, donkeys, and horses) on five Caribbean islands [22]. This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Ross University School of Veterinary Medicine (RUSVM), St. Kitts. Owners of animals gave permission for the blood samples to be collected.


### 2.2. DNA Extraction

The DNA was extracted from aliquots (200μL) of the whole blood samples with the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. The DNAs were eluted into 200 μL Buffer AE and couriered to Yangzhou University College of Veterinary Medicine, China, at room temperature where they were frozen at −80°C until PCRs were performed.


### 2.3. PCRs forTheileria Detection and Species Determination

All the PCRs were performed on a Roche Light-Cycler 480-II platform with the HMBS gene as an endogenous control [23]. Samples found positive in the pan-Theileria FRET-qPCR were tested in a conventional PCR with primers targeting a highly polymorphic 584–610-nucleotide region of the 18S rRNA gene ofTheileria spp. (Table 2, Figure 1) [21]. The amplicons from positive conventional PCRs were sequenced directly with forward and reverse primers to determine theTheileria spp. present (BGI, Shanghai, China) [21] as has been done with 18S rRNA sequences in a number of previous studies [11, 14, 17, 21, 24].Table 2
Primers and probes used in this study.
PCR
Primer/probe
Nucleotides sequence
Amplicon
FRET-qPCR
ForwardReverse6-FAMLCRed-640
5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′ 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′ 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-Phosphate-3′
178 bp
Conventional PCR
UpstreamDownstream
5′-CCTGAGAAACGGCTACCACATCT-3′5′-GGACTACGACGGTATCTGATCG-3′
T. orientalis 593 bp; T. buffeli 591 bp; T. annulata 591 bp; T. sergenti 591 bp; T. luwenshuni 594 bp; T. velifera 592 bp; T. ovis 595 bp; T. parva 592 bp; T. uilenbergi 592 bp; T. equi 596 bp; T. cervi 595 bp; T. lestoquardi 591 bp; T. separata 593 bp; T. capreoli 599 bp; T. bicornis 610 bp; T. taurotragi 587 bp; T. mutans 585 bp;  B. vulpes609 bp; Theileria sp. OT3 600 bp; Theileria sp. NG 597 bp; Theileria sp. YW 593 bp; Theileria sp. B15 584 bpFigure 1
Alignment of the sequences in the polymorphic region of the 18S RNA gene ofTheileria spp. and related organisms that was targeted in the standard PCR used in the study. Nucleotides that are identical for all species are highlighted in blue while those that vary between species and can be used for differentiation are not highlighted.


## 2.1. Samples Collection

Jugular venipuncture was used to collect blood in EDTA from convenience samples of apparently healthy livestock (cattle, goats, sheep, donkeys, and horses) on five Caribbean islands [22]. This study was reviewed and approved by the Institutional Animal Care and Use Committee of the Ross University School of Veterinary Medicine (RUSVM), St. Kitts. Owners of animals gave permission for the blood samples to be collected.


## 2.2. DNA Extraction

The DNA was extracted from aliquots (200μL) of the whole blood samples with the QIAamp DNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. The DNAs were eluted into 200 μL Buffer AE and couriered to Yangzhou University College of Veterinary Medicine, China, at room temperature where they were frozen at −80°C until PCRs were performed.


## 2.3. PCRs forTheileria Detection and Species Determination

All the PCRs were performed on a Roche Light-Cycler 480-II platform with the HMBS gene as an endogenous control [23]. Samples found positive in the pan-Theileria FRET-qPCR were tested in a conventional PCR with primers targeting a highly polymorphic 584–610-nucleotide region of the 18S rRNA gene ofTheileria spp. (Table 2, Figure 1) [21]. The amplicons from positive conventional PCRs were sequenced directly with forward and reverse primers to determine theTheileria spp. present (BGI, Shanghai, China) [21] as has been done with 18S rRNA sequences in a number of previous studies [11, 14, 17, 21, 24].Table 2
Primers and probes used in this study.
PCR
Primer/probe
Nucleotides sequence
Amplicon
FRET-qPCR
ForwardReverse6-FAMLCRed-640
5′-TAGTGACAAGAAATAACAATACGGGGCTT-3′5′-CAGCAGAAATTCAACTACGAGCTTTTTAACT-3′ 5′-CCAATTGATACTCTGGAAGAGGTTT-(6-FAM)-3′ 5′-(LCRed640)-AATTCCCATCATTCCAATTACAAGAC-Phosphate-3′
178 bp
Conventional PCR
UpstreamDownstream
5′-CCTGAGAAACGGCTACCACATCT-3′5′-GGACTACGACGGTATCTGATCG-3′
T. orientalis 593 bp; T. buffeli 591 bp; T. annulata 591 bp; T. sergenti 591 bp; T. luwenshuni 594 bp; T. velifera 592 bp; T. ovis 595 bp; T. parva 592 bp; T. uilenbergi 592 bp; T. equi 596 bp; T. cervi 595 bp; T. lestoquardi 591 bp; T. separata 593 bp; T. capreoli 599 bp; T. bicornis 610 bp; T. taurotragi 587 bp; T. mutans 585 bp;  B. vulpes609 bp; Theileria sp. OT3 600 bp; Theileria sp. NG 597 bp; Theileria sp. YW 593 bp; Theileria sp. B15 584 bpFigure 1
Alignment of the sequences in the polymorphic region of the 18S RNA gene ofTheileria spp. and related organisms that was targeted in the standard PCR used in the study. Nucleotides that are identical for all species are highlighted in blue while those that vary between species and can be used for differentiation are not highlighted.


## 3. Results and Discussion

The sensitive and specific pan-Theileria FRET-qPCR [21] we used in our study demonstrated that a substantial proportion of livestock (8.6%; 65/752) on the five Caribbean islands we studied were infected withTheileria (Table 3). Each island had positive animals and each livestock species we studied was found to be infected withTheileria spp. Sequencing of representative samples of positive 18S rRNA PCR amplicons we obtained (n=43) showed that there was one recognizedTheileria spp. (T. equi) present in the Caribbean, along with five less well characterizedTheileria sp. (Tables 3 and 4, Figure 2). The average copy number of 18S rRNA per mL whole blood was relatively low at 116.6±440.8, indicating that the animals we studied were chronically infected.Table 3
Prevalence ofTheileria spp. in livestock from five Caribbean islands.
Bovine
Goat
Sheep
Donkey
Horse
Total
Theileria spp.
Dominica
3/77(3.9%)
0/70(0.0%)
1/15(6.7%)
N/A∗
N/A
4/162 (2.5%)
T. equi, Theileria sp. NG-2013a, B. vulpes,or closely related organism
Grenada
N/A
2/31(6.5%)
N/A
N/A
N/A
2/31 (6.5%)
Theileria sp. B15a
Montserrat
0/12(0.0%)
8/19(42.1%)
24/62(38.7%)
N/A
N/A
32/93 (34.4%)
Theileria sp. OT3, B. vulpes,or closely related organism
Nevis
19/43(44.2%)
2/114(1.8%)
0/41(0.0%)
N/A
N/A
21/198 (10.6%)
T. equi, Theileria sp. NG-2013a
St. Kitts
0/193(0.0%)
0/4(0.0%)
0/26(0.0%)
5/25(20.0%)
1/20(5.0%)
6/268 (2.2%)
T. equi, Theileria sp. YW-2014
Total
22/325(6.8%)
12/238(5.0%)
25/144(17.4%)
5/25(20.0%)
1/20(5.0%)
65/752(8.6%)
Theileria spp.
T. equi,Theileria sp.NG-2013a
T. equi,Theileria sp. OT3,Theileria sp. B15a, B. vulpes,or closely related organism
T. equi,Theileria sp. OT3,B. vulpes,or closely related organism
T. equi,Theileria sp. YW-2014
T. equi
∗No specimen was available.Table 4
TheTheileriaspp. identified in livestock from five Caribbean islands and their similarity with reported organisms on GenBank.
Sequences identified in this study
Highly similar sequences in GenBank
Mismatch
Theileria spp.
Number
Source
GenBank #
Source
T. equi
14
8 cattle, 1 goat from Nevis;1 cow from Dominica;3 donkeys, 1 sheep from St. Kitts
KF559357
Horse from China
0/550
Theileria sp. OT3
11
8 sheep, 3 goats from Montserrat
KF470868
Sheep from China
0/555
Theileria sp. NG-2013a
7
6 cattle from Nevis;1 cow from Dominica
KF597076
Waterbuck from Kenya
0/552
Theileria sp. YW-2014
1
1 donkey from St. Kitts
AB981984
Sika deer from Japan
0/549
Theileria sp. B15a
1
1 goat from Grenada
JN572700
Buffalo from South Africa
0/539
B. vulpesor closely related organism
9
4 sheep, 4 goats from Montserrat;1 goat from Dominica
JX454779
Dog from France
0/178Figure 2
Phylogenetic tree of sequences identified in this study and closely relatedTheileria spp. Sequences from GenBank have gene accession numbers in black font; those from this study are in red font.B. vulpes or closely related organism DM indicates the organism detected on Dominica and Montserrat;Theileria sp. Gre indicates theTheileria sp. from Grenada;T. equi DNS indicates the sequence ofT. equi from Dominica, Nevis, and St. Kitts;Theileria sp. Mon indicates theTheileria sp. detected from Montserrat; andTheileria sp. SK is the sequence ofTheileria sp. from St. Kitts.Theileria equi and theTheileria sp. YW-2014 were the species we identified in equids.Theileria sp. YW-2014 has been described in a Sika deer (Cervus nippon) from Japan but there is little sequence data on the organism with only a 552 bp sequence of the 18S rRNA gene reported in GenBank (AB981984). On the other hand,T. equi is a well-recognized cause of equine piroplasmosis [25], an important disease of horses which has been recognized and studied in the Caribbean [14–16]. The organism has also been found in dogs in Spain [26], South Africa [27], and Nigeria [28] with some having clinical signs that responded to appropriate treatment [29]. Our finding thatT. equi also occurs in domestic ruminants further expands the host range of the organism. The significance, extent, and consequences of infections withT. equi in domestic ruminants require further investigation.Numerous species ofAmblyomma,Dermacentor, Hyalomma,Ixodes, andRhipicephalus are confirmed or suspected vectors ofT. equi [29]. Of these, onlyA. cajennense [30],R. microplus [30],R. sanguineus [31], andR. turanicus (unpublished observation) occur in the Caribbean. There is conflicting data thatAmblyomma cajennense is a competent vector ofT. equi [32] but the tick is localized to Jamaica, Trinidad, and Cuba in the Caribbean [30] and it appears, then, not to be the vector ofT. equi we found on Nevis, St. Kitts, and Dominica.Dermacentor nitens, the tropical horse tick, is very common in the Caribbean and the tropical Americas [33]. Although there is no data on the competence ofD. nitens as a vector ofT. equi and there is contradictory epidemiological evidence [32], PCR positiveD. nitens have been found [32] and Asgarali et al. [16] have suggested that this tick is a vector in Trinidad. They also suggested thatR. microplus [16], which is very common on cattle throughout the Caribbean, might also be a vector. While there is some evidence thatR. microplus is a competent vector [34] and our PCR identifiedT. equi in cattle on two islands, it seems unlikely thatR. microplus is an important natural vector as it is a one host species and transovarial transmission has not been demonstrated [32]. AlthoughR. sanguineus andR. turanicus have been implicated as vectors ofT. equi, more recent studies have failed to confirm their role [29]. Further studies are needed to establish the epidemiology ofT. equi and its vectors in the Caribbean and the neighboring Americas [32].Theileria sp. OT3 was first described in sheep, deer, and chamois in Spain [35–37] and later in sheep in Italy [38], China [39], and Turkey [40]. Its pathogenicity and vectors have yet to be determined. Ours is the first report of the organism in the Caribbean and also the first report of theTheileria sp. OT3 in goats which further demonstrates that the organism has a wide distribution and host range. Although we used only convenience samples which were not representative of the islands, it is of note thatTheileria sp. OT3 was the most prevalent species we detected in ruminants. Similar high prevalences of infections have also been reported in the other countries where the organism has been described. The sequences of ourTheileria sp. OT3 were identical to one another and to that of an isolate from China (KF470868) [39]. A phylogenetic relationship tree established for the Chinese isolate showed that theTheileria sp. OT3 forms a separated cluster and that the organism is closely related toT. uilenbergi,T. luwenshuni, andT. ovis. Although the organism has only been found in apparently healthy animals, studies on its pathogenicity seem indicated as, with its high prevalence and wide distribution, it might be causing substantial economic losses for livestock farmers.Theileria mutans andT. velifera are African species that have been reported on Guadeloupe [12] and appear most likely to have been imported with cattle from West Africa in the 18th Century. This might also have been the case with the other AfricanTheileria sp. we identified. Seven of theTheileria we found had an identical sequence to that ofTheileria sp. NG-2013a (KF597076) described from waterbuck (Kobus defassa) in Kenya [41]. This organism has been found to cluster withT. equi but might represent a novel taxon. Its vectors and pathogenicity are unknown. One goat we studied had aTheileria spp. with a sequence identical to that ofTheileria sp. B15a (JN572700) from an African buffalo (Syncerus caffer) in South Africa [42]. This organism is close toT. mutans which is widespread in Africa where it is transmitted byAmblyomma spp. and causes benign theileriosis. PreviouslyT. mutans has been reported on Guadeloupe based on serological test results [12] and, since we found no confirmatory molecular evidence for the presence ofT. mutans, it might be that the cross-reacting antibodies detected in Guadeloupe were against this closely relatedTheileria sp. B15.The only non-Theileria sp., the pan-Theileria FRET-qPCR identified in our study, was one that appeared to beBabesia vulpes [43] or a closely related species. This organism was previously known as “T. annae” or the “Babesia microti-like organism” but was reclassified based on 18S rRNA and tubulin-beta gene sequence data [43]. The sequences of the short amplicons we obtained in the pan-Theileria FRET-qPCR were identical to those ofB. vulpes (JX454779; KF773740) and had one mismatch withB. microti (AB219802, HQ629933, and LC005772). We were unable, however, to obtain longer amplicons with the standard PCR, probably because there were only very low copy numbers present in the positive animals. We were, then, unable to use this additional sequencing data to confirm that the organism we detected wasB. vulpes or determine if it was a closely related species or strain. The pan-Theileria FRET-qPCR we used in our study was designed to detect seventeen recognizedTheileria spp., which did not include “T. annae” orB. vulpes (Table 1) [21]. These seventeen recognizedTheileria spp. differed from one another by only a maximum of 4 nucleotides in the regions of the primers and the probes used in the pan-Theileria FRET-qPCR. It is of note, then, that these primers and probes enabled the multiplication and detection ofB. vulpes, or a closely related species, with 8 nucleotide differences and it therefore appears that the pan-Theileria FRET-qPCR might not be as genus specific as first thought. Further work is currently underway in our laboratory to more clearly characterize theB. vulpes or closely related organism found in the Caribbean.


## 4. Conclusions

Our study has confirmed the sensitivity of the pan-Theileria PCR in the rapid detection of a wide range ofTheileria spp. but has also shown it might detectB. vulpes or closely related organisms. We found livestock infected withTheileria spp. on each of the five islands we studied. While we could not confirm previous reports ofT. mutans andT. velifera in cattle, we found that one recognized species,T. equi, four poorly characterizedTheileria spp., andB. vulpes or a closely related organism are present in the region. Further studies are indicated to more precisely determine the phylogenetic relationships of these organisms in the Caribbean with closely related organisms from other parts of the world. Also, the prevalences of infections on the different islands should be determined as well as the impact these poorly characterized organisms might have on livestock production, both in the Caribbean and around the world where they are found.



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*Source: 624728-2015-12-09.xml*