Veterinary Parasitology 229 (2016) 31–36

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Veterinary Parasitology

jou rnal homepage: www.elsevier.com/locate/vetpar

Research paper

Detection of Theileria orientalis in mosquito blood meals in the United Kingdom

a b,c a a

M. Fernández de Marco , V.A. Brugman , L.M. Hernández-Triana , L. Thorne ,

a d a,e a,f,∗

L.P. Phipps , N.I. Nikolova , A.R. Fooks , N. Johnson

Animal and Plant Health Agency, Woodham Lane, Addlestone, Surrey KT15 3NB, United Kingdom

Department of Disease Control, The London School of Tropical Medicine and Hygiene, London WC1E 7HT, United Kingdom

Entomology Group, The Pirbright Institute, Ash Road, Woking GU24 0NF, United Kingdom

Biodiversity Institute of Ontario, University of Guelph, 50 Stone East Road, Ontario N1G 2W1, Canada

Department of Clinical Infection, Microbiology and Immunology, Institute for Infection and Global Health, University of Liverpool, L69 7BE, United Kingdom

Faculty of Health and Medicine, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom

r t i c l e i n f o a b s t r a c t

Article history: Theileria spp. are tick-borne protozoan parasites that infect a wide range of wild and domestic animals.

Received 14 June 2016

In this study, the utility of xenosurveillance of blood-fed specimens of Culiseta annulata for detecting

Received in revised form

the presence of piroplasms in livestock was investigated. Blood-fed mosquitoes were collected at Elmley

14 September 2016

National Nature Reserve, Kent, United Kingdom. All specimens were morphologically identiﬁed, and

Accepted 17 September 2016

DNA barcoding was used to conﬁrm the morphological identiﬁcation. Both the vertebrate host species

and Theileria genome was detected within the bloodmeal by real-time PCR. Sequencing was used to

Keywords:

conﬁrm the identity of all amplicons. In total, 105 blood-fed mosquitoes morphologically identiﬁed as

Mosquitoes

Cs. annulata were collected. DNA barcoding revealed that 102 specimens were Cs. annulata (99%), while a

Blood meal

single specimen was identiﬁed as Anopheles messeae. Two specimens could not be identiﬁed molecularly

Culiseta annulata

Theileria orientalis due to PCR ampliﬁcation failure. Blood meal analysis revealed that Cs. annulata fed almost exclusively on

Polymerase chain reaction cattle at the collection site (n = 100). The application of a pan-piroplasm PCR detected 16 positive samples

Xenosurveillance (15.2%) and sequence analysis of the amplicons demonstrated that the piroplasms present in the blood

meal belonged to the Theileria orientalis group. This study demonstrates how xenosurveillance can be

applied to detecting pathogens in livestock and conﬁrms the presence of Theileria species in livestock

from the United Kingdom.

1. Introduction mental importance, mosquitoes are a target group in surveillance

programmes for disease transmission and control across the globe.

The family Culicidae (Diptera) includes 112 genera and at least The analysis of host selection and host preferences of

3547 described species (Web Reference 1). In some species, the mosquitoes and other haematophagous arthropods via the iden-

female requires a blood meal for egg maturation, and it is this tiﬁcation of their blood meals has become a focus of studies on

requirement that makes members of this family important as bit- the dynamics of vector-host-pathogen interactions (Kent, 2009;

ing pests and vectors of pathogens of humans and livestock (Becker Kent and Norris, 2005). The understanding of these complex inter-

et al., 2010). Mosquitoes transmit numerous protozoa, viruses and actions could facilitate the implementation of control methods

nematodes to humans, livestock, as well as both domestic and wild of vector-borne disease outbreaks (Alcaide et al., 2009; Chaves

birds that are responsible for disease outbreaks all over the world et al., 2010; Brugman et al., 2015). This non-invasive approach

(Forattini, 1998; Becker et al., 2010; Medlock et al., 2012; Schaffner has recently been termed xenosurveillance (Grubaugh et al., 2015),

et al., 2013). As a result of their medical, veterinary and environ- and several studies have demonstrated that blood-fed mosquitoes

could be used to detect inﬂuenza virus (Barbazan et al., 2008),

Epstein-Barr virus and canine distemper virus (Ng et al., 2011).

Current molecular techniques for blood meal identiﬁcation and the

∗

increasing volume of open access databases for host species identi-

Corresponding author at: Animal and Plant Health Agency, Woodham Lane,

Addlestone, Surrey KT15 3NB, United Kingdom. ﬁcation such as GenBank (Web Reference 2) and The Barcode of Life

E-mail address: [email protected] (N. Johnson).

http://dx.doi.org/10.1016/j.vetpar.2016.09.012

32 M. Fernández de Marco et al. / Veterinary Parasitology 229 (2016) 31–36

Databases (Web Reference 3) have facilitated this area of research ing. Each DNA extract was used for three purposes as demonstrated

(Ratnasingham and Hebert, 2007; Brugman et al., 2015). previously (Brugman et al., 2015): Firstly, to conﬁrm the species

In the United Kingdom (UK), molecular methods for host pref- identity of collected mosquitoes, secondly, to identify the verte-

erence studies in mosquitoes have only been carried by Danabalan brate source of the blood meal and, ﬁnally, for targeted pathogen

et al. (2014), and more recently by Brugman et al. (2015), targeting detection, in this case of piroplasms.

mosquito species inhabiting farmland and wetland areas. In addi-

tion, Brugman et al. (2015) detected myxoma virus in blood meals 2.3. Morphological and molecular identiﬁcation of mosquitoes

of two members of the Anopheles maculipennis complex (Anophe-

les atroparvus and Anopheles messeae) that had fed on the European Female mosquitoes were morphologically identiﬁed as Cs. annu-

rabbit (Oryctolagus cuniculus), and advocated the same xenosurvel- lata based on the key of Cranston et al. (1987). Due to the

liance methodology for the targeted detection of pathogens in morphological similarities between several species of mosquitoes

livestock and wildlife. known to occur in the area, we tested the utility of DNA bar-

Piroplasms including Babesia and Theileria are tick-borne pro- coding to support our morphological identiﬁcation. We employed

tozoan blood parasites that infect a wide range of both domestic the cytochrome c oxidase I (COI) DNA barcoding approach (Hebert

and wild animals worldwide, including cattle and sheep (Mehlhorn et al., 2003a,b) to conﬁrm morphological identiﬁcation, in accor-

and Schein, 1985). Piroplasms are transmitted by Ixodid ticks dance with the approach used in Brugman et al. (2015). PCR

(Mehlhorn and Schein, 1985). However, there is some evidence products ampliﬁed with primers LCO1490 and HCO2198 (Folmer

for mechanical transmission by other biting arthropods includ- et al., 1994) were visualized on a 1.5% agarose gel, and sam-

ing mosquitoes, tabanid ﬂies, and lice (Turell and Knudson, 1987; ples showing bands of the correct size of 658 base pairs (bp)

® ®

Fujisaki et al., 1993; Hammer et al., 2015). were sequenced in both directions using the ABI PRISM BigDye

The samples analysed in this study were collected as part of Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Life

a larger investigation of feeding preference of farm-associated Technologies Ltd, Paisley, UK). The details of specimen records and

mosquitoes. It was noted that Culiseta (Cs.) annulata fed predomi- sequence information (including trace ﬁles), are publically avail-

nantly on cattle suggesting a possible means of surveying livestock able on the Barcoding of Life Database (Web Reference 4) using

for blood-borne diseases using a non-invasive method. The aim of the search term “DS-CSAN2014”. All sequences have been submit-

the present study was to test the utility of a targeted xenosurveil- ted to GenBank (accession numbers: KU748414–KU748459, KU74

lance approach for detecting piroplasms in blood-fed specimens of 8461–KU748470, KU748472–KU748495, KU748497–KU748505,

Cs. annulata collected within a livestock farm setting in the UK. The KU748507–KU748514).

successful application of this targeted methodology could enhance Paired bi-directional sequence traces (sequenced with the

the detection and surveillance of these parasites. LCO1490 and HCO2198 primers) for the Cs. annulata specimens

were combined to produce a single consensus DNA barcode

sequence. To achieve this, individual forward and reverse traces

2. Methods

were oriented, edited, and aligned using the Sequencer (v.4.5;

Genes Codes Corporation, Ann Harbour, MI), GenDoc (v. 2.6.02) and

2.1. Collection of blood-fed mosquitoes

ClustalX sequence analysis programs. The full data set was ana-

lysed in MEGA v.6 (Tamura et al., 2013). The K2P distance metric

Blood-fed female mosquitoes were collected from Elmley

was used to determine the intra- and inter-speciﬁc genetic dis-

National Nature Reserve, Isle of Sheppey (51.377445, 0.784068),

tances, and a neighbour-joining (NJ) tree analysis was carried out

Kent, UK, over 25 visits between June and October 2014. Elmley

to represent the specimen’s clustering pattern. Bootstrap values

is a freshwater coastal marsh used to graze approximately 700

were calculated to test the robustness of the cluster, which was

head of cattle (Fig. 1). Mosquitoes were primarily collected using a

obtained by conducting 1000 pseudoreplicates.

Prokopak aspirator (John W Hock, Gainsville, Florida, USA) from

inside resting traps (plywood resting boxes 500 × 500 × 500 cm,

2.4. Identiﬁcation of vertebrate host in the blood meal

painted black on the outside and red on the inside) or from

the inside of man-made structures at the site using an aspirator

Vertebrate host species in the blood meal were identiﬁed

(Brugman et al., 2015).

using a vertebrate speciﬁc, M13-tailed, triple-primer cocktail

Collected mosquitoes were placed into a cooler containing dry

(VF1 t1 + VF1d t1 + VFli t1/VR1 tl + VR1d t1 + VD1i t1) targeting a

ice and transported to the laboratory. Blood-fed specimens were

685 bp sequence of the COI gene (Ivanova et al., 2007; Brugman

separated from non-blood-fed specimens on the same day as col-

◦ et al., 2015). Ampliﬁcation products were sequenced in both

−

lection and stored at 20 C until processed. ® ®

directions using the ABI PRISM BigDye Terminator v3.1 Cycle

Sequencing Kit (Applied Biosystems, Life Technologies Ltd, Pais-

2.2. DNA extraction from mosquito abdomens ley, UK). All sequences were edited using Lasergene version 12

(DNASTAR, Inc, Madison, Wisconsin, USA) and assigned to a par-

≥

DNA extraction from engorged abdomens was performed fol- ticular vertebrate species when agreement was 98% to sequences

lowing the protocols of Brugman et al. (2015). Brieﬂy, abdomens of of known species in GenBank (Martínez de la Puente et al., 2013;

engorged mosquitoes were separated from the rest of the body on a Brugman et al., 2015).

chilled plate using forceps and placed into individual 1.5 ml Eppen-

dorf tubes containing 200 ␮l phosphate buffered saline (PBS). The 2.5. Piroplasm polymerase chain reaction

abdomens were pressed against the walls of the tube using the for-

ceps to release the blood meal. The remaining head and thorax of One hundred and ﬁve specimens were tested for the presence of

◦

each mosquito was kept and stored at −20 C as voucher specimens. piroplasms in their blood meal. Piroplasm genome was detected by

Each sample was incubated with 20 ␮l proteinase K and 200 ␮l quantitative real-time PCR. Samples were screened using primers

◦

buffer AL (QIAgen, Manchester, UK) for 30 min at 56 C in a water PIRO-A and PIRO-B that ampliﬁed a 423 bp piroplasm sequence

bath. DNA extraction was carried out using the DNeasy Blood and (Armstrong et al., 1998). The ﬁnal PCR reaction mix (ﬁnal volume

® TM

Tissue Kit (QIAgen, Manchester, UK), following the manufacturer’s 40 ␮l) consisted of: 13 ␮l H2O, 20 ␮l SYBR Green JumpStart Taq

◦ TM

instructions. All DNA extractions were stored at 4 C until process- Ready Mix (Sigma-Aldrich, Dorset, UK), 1 ␮l of each primer (at

M. Fernández de Marco et al. / Veterinary Parasitology 229 (2016) 31–36 33

Fig. 1. Images of potential larval habitats (A, B), livestock (C) and bird life (D) at Elmley National Nature Reserve, Isle of Sheppey, Kent, UK.

20 ␮M) and 5 ␮l of the same DNA extract used above. Ampliﬁcation

◦

was performed as follows: an initial denaturation step of 94 C for

◦ ◦ ◦

10 s followed by 45 cycles of 94 C for 30 s, 58 C for 30 s, and 72 C

◦ ◦

for 1 min, followed by one cycle of 95 C for 1 min, 55 C for 30 s

◦

and 95 C for 30 s. Samples were then sequenced using PIRO-A and

® ®

PIRO-B primers at 1 pmol/␮l using the ABI PRISM BigDye Ter-

minator v3.1 Cycle Sequencing Kit (Applied Biosystems), following

Fig. 2. Gel image showing an example of Theileria orientalis PCR ampliﬁcation prod-

the manufacturer’s protocols.

ucts in mosquito blood-fed (BF) samples. Lane (M) PhiX174 DNA marker, (1) BF882,

Piroplasms sequences were edited in Lasergene version 12.1

(2) BF1349, (3) BF1386, (4) BF1450, (5) BF1557, (6) BF1827, (7) BF1837, (8) BF1136,

(DNASTAR) and assigned to a particular species, by BLAST (NCBI)

(9) NTC, (10) BF946, (11) T. annulata APHA positive control.

search, when agreement was ≥98% to sequences of known taxa in

GenBank. Once identiﬁcation was achieved, the sequences were

showed that at Elmley, females of Cs. annulata feed primarily on

analysed in MEGA v.6. NJ analysis was conducted as described

cattle (Bos taurus) (n = 100), although other hosts such as the Euro-

above and trees were exported as JPG ﬁles in Adobe Acrobat 8

pean rabbit (O. cuniculus) (n = 1) and sheep (Ovis aries) (n = 3) were

Professional, and edited using Adobe Photoshop CS3 v.10.0.1.

also identiﬁed.

3.2. Detection of piroplasms

3. Results

Sixteen samples from the 100 specimens that fed on cows pro-

3.1. Mosquito species and blood meal host identiﬁcation

duced a clear amplicon of a size corresponding with the piroplasm

controls (Fig. 2); however, only 12 samples produced sequences

In this study, 105 blood-fed female Cs. annulata specimens were

suitable for further analysis. A sequence 378 bp in length was

selected for investigation. The resting traps yielded the majority of

obtained for these 12 samples with BLAST searches on each

specimens, although 23 were collected from man-made structures

sequence indicating that they all shared 100% identity with T. orien-

such as the barn at the site. In total, we obtained COI DNA barcodes

talis genomes from China (T. orientalis – GenBank accession number

for 103 specimens (98% PCR ampliﬁcation success), while two sam-

KJ850941, isolate BMY female-108) or Spain (T. buffeli – Gen-

ples failed to amplify despite several re-ampliﬁcation attempts.

Bank accession number DQ287959). A NJ analysis of the sequences

The BLAST search results conﬁrmed that 102 specimens were Cs.

demonstrated the position of these sequences within the T. orien-

annulata (99%), while a single specimen was revised to Anopheles

talis group (Fig. 3).

messeae. All Cs. annulata sequences showed less than 1% variation

indicating that they belong to the same species (data not shown).

As expected, the intraspeciﬁc divergence ranged from 0 to 0.15% as 4. Discussion

conspeciﬁc individuals from similar sites often exhibit zero or very

low divergence (Cywinska et al., 2006). This study demonstrates that a single DNA extract from the

We obtained positive results for the analysis of the blood meals abdomen of a blood-fed mosquito can be successfully used for three

in 104 specimens (99% success). A sample for one specimen did not purposes in a sequential workﬂow as reported by Brugman et al.

amplify and a bloodmeal host could not be identiﬁed. The results (2015): (1) the molecular identiﬁcation of members of Cs. annulata

34 M. Fernández de Marco et al. / Veterinary Parasitology 229 (2016) 31–36

Fig. 3. Neighbour-joining tree constructed from sequences (378 bp) of the 18S rRNA gene of Theileria orientalis in blood meals of Culiseta annulata feeding on cattle collected

from Elmley National Nature Reserve, Isle of Sheppey, Kent, UK. Only bootstrap values higher than 80% are shown in the tree. BF APHA refers to numbers assigned to each

blood-fed sample submitted to Animal and Plant Health Agency. Babesia divergens 18S rRNA sequence was used as an outgroup.

by sequencing the invertebrate COI DNA barcoding region, followed This study provides evidence that at Elmley Cs. annulata feeds on

by (2) the identiﬁcation of the vertebrate origin of a blood meal by cattle, indicating that this mosquito could potentially play a signif-

sequencing of the corresponding region of the vertebrate COI gene, icant role in disease transmission in livestock. A previous study by

then (3) the detection of a pathogen, in this case a species of piro- Service (1971) showed that 37.5% of Cs. annulata blood meals were

plasm, subsequently shown to belong to the T. orientalis complex. taken from cattle, but also that 20% of blood meals were taken from

DNA barcoding is currently widely used in the identiﬁcation birds. In the current study, Cs. annulata did not feed on birds despite

of mosquito species (Cywinska et al., 2006; Kumar et al., 2007; the presence of many bird species in the area (Fig. 1). Only one spec-

Versteirt et al., 2015), as well as many other groups of animals imen (<1%) fed on rabbits, a common host in the study area and one

and plants (Hebert et al., 2003a,b). The DNA barcoding approach which is fed on preferentially by Anopheles atroparvus (Brugman

was an effective method of conﬁrming morphological identiﬁcation et al., 2015). The reasons underlying this difference are unclear,

of the blood-fed females of Cs. annulata. Specimens are frequently but could potentially relate to differences in the host availability

damaged when collected by mosquito traps or during transporta- between ﬁelds, the grazing activities at the reserve during times of

tion, thus making it difﬁcult to identify them using morphological peak mosquito feeding or innate feeding behaviour between dif-

features on which identiﬁcation keys are based (Cranston et al., ferent mosquito species. A key limitation of this approach is that

1987). Moreover, the large numbers of specimens of different ability to capture sufﬁcient blood-fed mosquitoes. In this study, Cs.

species that can be found in a given trap can make the identiﬁca- annulata were captured in sufﬁcient numbers to detect a blood-

tion of all specimens a challenging exercise (Murugan et al., 2015). borne pathogen. Lower numbers would severely limit the ability to

Therefore, the combination of morphological and molecular identi- detect a pathogen at low prevalence. Also, blood-fed specimens of

ﬁcation methodologies, together with the additional data provided other mosquito species present at the site, such as Culex modestus,

by sequencing, enhances the taxonomic resolution of our study by were not captured at all.

detecting a labelling error within our dataset. The primer cocktail used in this study (Ivanova et al., 2007)

successfully identiﬁed the blood meal host of 99% of the captured

M. Fernández de Marco et al. / Veterinary Parasitology 229 (2016) 31–36 35

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