A real-time PCR tool for the surveillance of zoonotic Onchocerca lupi in dogs, cats and potential vectors.

The ocular onchocercosis is caused by the zoonotic parasite Onchocerca lupi (Spirurida: Onchocercidae). A major hindrance to scientific progress is the absence of a reliable diagnostic test in affected individuals. Microscopic examination of skin snip sediments and the identification of adults embedded in ocular nodules are seldom performed and labour-intensive. A quantitative real-time PCR (qPCR) assay was herein standardized for the detection of O. lupi DNA and the results compared with microscopic examination and conventional PCR (cPCR). The specificity of qPCR and cPCR was assessed by processing the most common filarial nematodes infecting dogs, skin samples from O. lupi infected (n = 35 dogs) or uninfected animals (n = 21 dogs; n = 152 cats) and specimens of potential insect vector (n = 93 blackflies; n = 59 mosquitoes/midges). The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of DNA from adult specimen and from a pool of microfilariae. The qPCR on skin samples revealed an analytical specificity of 100% and a sensitivity up to 8 x 10-1 fg/2μl O. lupi adult-DNA and up to 3.6 x 10-1 pg/2μl of mfs-DNA (corresponding to 1 x 10-2 mfs/2μl). Only 9.5% O. lupi-infected skin samples were positive for cPCR with a sensitivity of 8 x 10-1 pg/2μl of DNA. Out of 152 blackflies and mosquitoes/midges, eight specimens experimentally infected (n = 1 S. erythrocephalum; n = 1 S. ornatum; n = 6 Simulium sp.) were positive by qPCR. The qPCR assay herein standardized represents an important step forward in the diagnosis of zoonotic onchocercosis caused by O. lupi, especially for the detection and quantification of low number of mfs. This assay provides a fundamental contribution for the establishment of surveillance strategies aiming at assessing the presence of O. lupi in carnivores and in insect species acting as potential intermediate hosts. The O. lupi qPCR assay will enable disease progress monitoring as well as the diagnosis of apparently clinical healthy dogs and cats.

Introduction

Within the genus Onchocerca (Spirurida: Onchocercidae), Onchocerca volvulus and Onchocerca lupi parasitize humans and carnivores, respectively [1-5], the latter being a zoonotic agent [6,7]. While O. volvulus is a well-known parasite of humans transmitted by blackflies (Simulium spp.) [8,9], the epidemiology of O. lupi is far from being understood, particularly because the information about insect species acting as vectors is lacking. Only Simulium tribulatum was suggested as the putative vector of this filarial worm in California (USA), but proof of its intermediate host competence is currently absent [10]. Onchocerca lupi belongs to the spirurids in the Nematode clade III [11] was first detected from a Caucasian wolf (Canis lupus) in Georgia [12], and, only recently, diagnosed in dogs and cats from Europe (Greece, Portugal, Spain, Germany, Hungary) and USA [13-20]. The reports of O. lupi infection are mainly based on the presence of ocular nodules on the eyelids, conjunctiva, and sclera [3,21,22], though the localization of adult worms in the retrobulbar area of the canine patients may impair the assessment of its distribution in endemic areas [23]. The detection of microfilariae (mfs) in skin snip sediments is the only available tool for the diagnosis of the infection when nodules are not apparent in the eyes. The retrieval and identification of mfs in skin snip samples is a rather invasive and time-consuming method, highly dependent on the anatomical location of skin biopsy and mfs density [24]. Again, the detection of mfs may depend upon the prepatent period, previous microfilaricidal treatments, and on the operator’s skills in examining skin sediments, as described for O. volvulus [25,26].

Conventional PCR (cPCR) amplification and sequencing of mitochondrial NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit 1 (cox1) genes are available for the molecular identification of O. lupi adults and mfs [7,27,28]. The cPCR, however, may be relatively labour-intensive and exhibit low sensitivity, mainly for mfs detection, limiting the establishment of large-scale epidemiological studies in vertebrate hosts and putative vectors.

Here, we developed a quantitative real-time PCR (qPCR) assay based on the hybridization probe to detect O. lupi DNA in host and putative vector samples. The diagnostic validity of qPCR assay was compared with microscopic examination and cPCR methods.

Methods

Ethics statement

All dogs’ and cats’ skin samples were collected in previous studies [17,29] and approved by the ethical committee of the Department of Veterinary Medicine of the University of Bari (Prot. Uniba 1/16) and by the ethical committee of the Faculty of Veterinary Medicine, Universidade Lusófona de Humanidades e Tecnologias.

Samples

Genomic DNA of adult specimens of O. lupi (n = 3), as well as DNA from single (n = 7) or pooled mfs (n = 10), collected from dogs in different geographical locations (Table 1) were used as control. All specimens were previously identified based on morphological and molecular analyses [18,30].

Table 1

Filarial nematodes used to assess the analytical specificity of the qPCR assay.

Species	Host	Collection locality	Source	ID sample
Onchocerca lupi	Canis lupus familiaris	USA (Minnesota)	Adult	100–14
	Canis lupus familiaris	USA (New Mexico)	Adult	132–14
	Canis lupus familiaris	USA (Colorado)	Adult	478–15
	Canis lupus familiaris	Portugal	Microfilariae	63–12
	Canis lupus familiaris	Greece	Microfilariae	62–12
	Canis lupus familiaris	Portugal	Skin	537–15
	Felis catus	Portugal	Skin	61–15
Onchocerca armillata	Bos taurus	Cameroon	DNA	54FKA2
Onchocerca bohemi	Equus caballus	Italy	Adult	409
Onchocerca fasciata	Camelus dromedaries	Iraq	Adult	200
Onchocerca gutturosa	Bos taurus	Cameroon	DNA	54FKG1
Onchocerca ochengi	Bos taurus	Cameroon	DNA	54FKO2
Dirofilaria immitis	Vulpes vulpes	Italy	Adult	377
Dirofilaria repens	Canis lupus familiaris	Italy	Adult	379
Cercopithifilaria grasii	Canis lupus familiaris	Portugal	Skin	81–16
Cercopithifilaria bainae	Canis lupus familiaris	Portugal	Skin	81–16
Cercopithifilaria sp. II	Canis lupus familiaris	Portugal	Skin	81–16
Acanthocheilonema reconditum	Canis lupus familiaris	Italy	Blood	496
Brugia malayi	Meriones unguiculatus (experimental cycle)	FR3 strain	DNA	8YT1
Brugia pahangi	Meriones unguiculatus (experimental cycle)	FR3 strain	DNA	46YT
Wuchereria bancrofti	Homo sapiens	Singapore	DNA	82YT FIL13/01

Primers and probe design and qPCR run protocol

Primers (O.l.F 5′-GGAGGTGGTCCTGGTAGTAG-3′; O.l.R 5′- GCAAACCCAAAACTATAGTATCC-3′) and a TaqMan-MGB hydrolysis probe (FAM-5’-CTTAGAGTAGAGGGTCAGCC-3’-non-fluorescent quencher-MGB; Applied Biosystems; Foster City, CA, USA), targeting partial cox1 gene (90bp), were designed by alignment of sequences from a wide range of closely related filarial nematodes available from GenBank database (Table 2), using Primer Express 2.0 (Applied Biosystems, Foster City, CA). Specificity of the primers and probe for O. lupi were confirmed in silico using the basic local alignment search tool (BLAST, GenBank, NCBI).

Table 2

GenBank accession numbers (AN) of mitochondrial cytochrome c oxidase subunit 1 sequences of filarial nematodes used for primers and TaqMan-probe design.

Parasite	AN	Host	Collection locality
Onchocerca lupi	KC686702	Canis lupus familiaris	Greece
	KC686701	Canis lupus familiaris	Portugal
	EF521408	Canis lupus familiaris	Hungary
Onchocerca armillata	KP760200	Bos taurus	Cameroon
Onchocerca boehmi	KX898458	Equus caballus	Italy
Onchocerca dewittei japonica	AM749267	Sus scrofa leucomystax	Japan: Oita
Onchocerca eberhardi	AM749268	Cervus nippon	Japan: Oita
Onchocerca ochengi	KC167350	Simulium damnosum sensu lato	Cameroon: northern
Onchocerca gibsoni	AJ271616	Bos taurus	Australia: Queensland
Onchocerca gutturosa	KP760201	Bos taurus	Cameroon
Onchocerca lienalis	KX853326	Bos taurus	United Kingdom: Wales
Onchocerca ramachandrini	KC167356	Simulium damnosum sensu lato	Cameroon: northern
Onchocerca suzukii	AM749277	Nemorhaedus crispus	Japan: Yamagata
Onchocerca skrjabini	AM749269	Cervus nippon	Japan: Oita
Onchocerca sp. ‘Siisa’	KC167354	Simulium damnosum sensu lato	Cameroon: northern
Onchocerca volvulus	KC167355	Simulium damnosum sensu lato	Cameroon: northern
Brugia malayi	KP760171	Meriones unguiculatus	FR3 strain
Brugia pahangi	EF406112	Homo sapiens	Malaysia
Wuchereria bancrofti	AM749235	Homo sapiens	Italy
Cercopithifilaria bainae	JF461457	Canis lupus familiaris	Italy: Sicily
Cercopithifilaria bulboidea	AB178834	Cervus nippon	Japan
Cercopithifilaria crassa	AB178840	Cervus nippon	Japan
Cercopithifilaria grassii	JQ837810	Canis lupus familiaris	Italy
Cercopithifilaria japonica	AM749263	Ursus thibetanus	Japan: Gifu
Cercopithifilaria longa	AB178843	Cervus nippon	Japan
Cercopithifilaria minuta	AB178846	Cervus nippon	Japan
Cercopithifilaria multicauda	AB178848	Cervus nippon	Japan
Cercopithifilaria rugosicauda	KF479370	Capreolus capreolus	Italy
Cercopithifilaria sp. II	JQ837809	Canis lupus familiaris	Italy
Cercopithifilaria shohoi	AB178850	Cervus nippon	Japan
Cercopithifilaria tumidicervicata	AB178852	Cervus nippon	Japan
Acanthocheilonema delicata	JQ289993	Meles anakuma	Japan
Acanthocheilonema odendhali	KF038145	Callorhinus ursinus	USA: Alaska
Acanthocheilonema reconditum	JF461456	Canis lupus familiaris	Italy: Sicily
Acanthocheilonema spirocauda	KF038155	Erignathus barbatus	USA: Alaska
Acanthocheilonema vitaea	KP760169	Meriones unguiculatus	FR3 strain
Dirofilaria immitis	EU169124	Ailurus fulgens	China
Dirofilaria repens	AM749230	Canis lupus familiaris	Italy

qPCR reactions were carried out in a final volume of 20μl, consisting of 10μl of IQ Supermix (Bio-Rad Laboratories, Hercules CA, USA), 7.1μl of Di-Ethyl Pyro-Carbonate (DEPC) treated pyrogen-free DNase/RNase-free water (Invitrogen, Carlsbad, CA, USA), 2μl of template DNA (except no-template controls), 5 pmol and 0.5 pmol for primers and probe, respectively.

The run protocol consisted of a hot-start at 95°C for 3 min, and 40 cycles of denaturation (95°C for 10 sec) and annealing-extension (64°C for 30 sec). All assays were carried out in duplicate and a no-template control was included in each run. The qPCR was performed in a CFX96 Real-Time System (Bio-Rad Laboratories, Inc., Hercules CA, USA) and the increase in the fluorescent signal was registered during the extension step of the reaction and analysed by the CFX Manager Software Version 3.1 (Bio-Rad).

Specificity and sensitivity of qPCR

To investigate the analytical specificity of the assay, genomic samples of Onchocerca spp. and of the most common filarial nematodes infesting dogs (Table 1) were used. The specificity of the assay was tested by using DNA from skin samples of naturally infected dogs, which were positive for O. lupi (n = 35) at microscopic examination [29]. Skin samples were divided in five groups (G1-G5) according to their mfs load (Table 3), being 14 also co-infected with Cercopithifilaria bainae and Cercopithifilaria sp. II. Skin samples (dogs n = 21; cats n = 152), which did not test positive to any mfs [17,29], were used as negative control.

Table 3

Skin samples tested for Onchocerca lupi by qPCR, divided (Groups 1–5) according to the parasitic load (mfs) microscopically detected.

The mean, minimum, maximum and standard deviation (sd) values of the threshold cycle (Cq), parasite load (Starting Quantity (SQ) value, expressed as ng/μl of DNA for reaction) and microfilariae concentration, assessed by qPCR is reported.

Parasitic load (mfs)		Skin (n)	Cq			Mfs			DNA
						SQ			SQ
			Mean	Min-Max	SD	Mean	Min/Max	SD	Mean	Min/Max	SD
G1	1 < 5	16	33.49	32.12–35.89	1.2	1.9	2.6 x 10−1–3.8	1.2	6.1 x 10−2	9.5 x 10−3–1.3 x 10−1	4.3 x 10−2
G2	6 < 10	7	31.24	30.24–31.75	0.5	6.9	4.9–10	1.8	2.5 x 10−1	1.8 x 10−1–3.8 x 10−1	6.5 x 10−2
G3	11 < 25	8	29.92	29.06–31.3	0.8	19.1	6.7–29.8	8.6	6.9 x 10−1	3.1 x 10−1–1.1	3 x 10−1
G4	26 < 40	2	28.65	28.37–28.93	0.4	38.4	3.5 x 101–4.1 x 101	4	1.4	1.3–1.5	1.5 x 10−1
G5	> 40	2	27.52	27.41–27.63	0.1	96	1 x 102–8.9 x 101	10.2	3.4	3.2–3.7	3.7 x 10−1

Specimens of blackflies (n = 66) and mosquitoes/midges (n = 39) collected from 2011 to 2014 in Greece [31], and 27 blackflies and 20 Aedes albopictus (colony specimens) experimentally infected by intrathoracic microinjection with mfs of O. lupi (parasitic load of 20mfs/μl) were analyzed after death (i.e., from one to 10 days post infection) (Table 4).

Table 4

Blackflies and mosquitoes/midges specimens used to test the analytical specificity of qPCR assay.

Geographical origin	Blackflies species	Number(pos/tot)	Mosquitoes/ midges species	Number(pos/tot)
Greece	Simulium balcanicum	0/14	Culex pipiens pipiens	0/10
	Simulium erythrocephalum	0/6	Ochlerotatus caspius	0/10
	Simulium pseudequinum	0/10	Anopheles maculipennis	0/4
	Simulium reptans	0/23	Coquilletidia richiardii	0/2
	Simulium ornatum	0/4	Culiseta annulata	0/3
	Simulium velutium	0/9	Culicoides spp.	0/1
			Ceratopogonidae	0/5
			Psychodidae	0/4
Italy
Basilicata region	Simulium erythrocephalum	1/1*
	Simulium linatum	0/5*
	Simulium ornatum	1/4*
	Simulium pseudequinum	0/10*
	Simulium sp.	6/7*
Reggio Emilia			Aedes albopictus	0/20*
Total		8/93		0/59

* = Specimens experimentally infected by intrathoracic microinjection with microfilariae of Onchocerca lupi.

The analytical sensitivity of the qPCR assay was assessed using 10-fold serial dilutions of DNA from adult specimen (i.e., ranging from 8 × 104 to 8 × 10−3 fg/2μl of reaction) and from a pool of 10 mfs (i.e., ranging from 10 to 10 × 10−3 microfilariae/2μl of reaction, corresponding to 3.6 ×10−1 ng/2μl to 3.6 ×101 fg/2μl of DNA). Ten replicates of each serial dilution were submitted to the same run for assessment of intra-assay reproducibility.

Genomic DNA was isolated from all skin samples and from O. lupi adults and mfs, blackflies, mosquitoes and midges specimens using the commercial kits DNeasy Blood & Tissue Kit (Qiagen, GmbH, Hilden, Germany), respectively, following the manufacturers’ instructions. The amounts of purified DNA were determined spectrophotometrically using the Qubit (Applied Biosystems, Foster City, CA, USA).

Specificity and sensitivity of cPCR

The analytical specificity and sensitivity of the cPCR for the specific amplification of cox1 gene fragment (∼689bp; [32]) was assessed by testing genomic DNA of: i) skin samples with different parasitic load of O. lupi (Table 3), ii) serial dilution of O. lupi mfs DNA (i.e., from 3.6 ×101 pg/2μl to 3.6 ×10−3 pg/2μl) and iii) DNA of adult specimens (i.e., from 8 ×101 ng/2μl to 8 x 10−3 fg/2μl).

All cPCR products were resolved in 0.5x GelRed stained (Biotium, CA, USA) agarose gels (2%), purified using enzymatic purification (Exo I-FastAP; Thermo Fisher Scientific, MA, USA) and sequenced in an automated sequencer (3130 Genetic Analyzer). All sequences generated were compared with those available in GenBank using Basic Local Alignment Search Tool (BLAST) [33].

Results

All O. lupi naturally-infected dog positive at skin samples examination by microscopy, considered the gold standard method as true positives, were positive by the O. lupi qPCR herein assessed (specificity of 100%). Out of 21 skin samples microscopically and qPCR positive for O. lupi, two were positive by cPCR (parasite load of 8 and 25 mfs), revealing a low analytical cPCR specificity (i.e., 9.5%). None of cat’s skin samples were positive by qPCR.

A specific fluorescent signal was recorded for all O. lupi adult and mfs positive controls tested (Fig 1). No fluorescence was obtained for all other Onchocerca species or filarial nematodes examined as well as for skin samples used as negative control.

Fig 1

Assessment of the specificity of qPCR assay in the detection of Onchocerca lupi DNA.

The amplification plot is represented by the fluorescent signal accordingly to relative fluorescence units (RFU) and threshold cycle.

The analytical sensitivity of qPCR was confirmed by detection of up to 8 x 10−1 fg/2μl and 3.6 x 10−1 pg/2μl of DNA (i.e., corresponding to 1 x 10−2 mfs/2μl) of O. lupi adult worm and mfs, respectively (Fig 2A and 2B). qPCR efficiencies ranged from 108.7 to 115.3% with an R2 from 0.996 to 0.999 and Slope from -3.003 to -3.131, for both adult and mfs (Fig 2A and 2B). The mean parasite load detected for the positive skin samples, ranged from 1.9 to 96 mfs/2μl of reaction, corresponding to 6.1 x 10−2 ng/2μl (mean cycle threshold of 33.49) and to 3.4 ng/2μl DNA (mean cycle threshold of 27.52), respectively (Table 3). The results of mfs detection by qPCR overlapped the values obtained by the microscopic examination. The detection limit registered for cPCR was up to 8 x 10−1 pg/2μl for adult worms and up to 3.6 x 101 pg/2μl for mfs DNA (i.e., corresponding to 1 mf/2μl), respectively (Fig 3).

Fig 2

Standard curves generated from serial dilutions of (A) genomic DNA from adult (from 8 × 104 to 8 × 10−3 fg/2μl of reaction) and microfilariae (B) (from 3.6 ×10−1 ng/2μl to 3.6 ×101 fg/2μl of reaction) of Onchocerca lupi. Each point was tested in triplicate. Slope, efficacy and R2 are reported on the bottom.

Fig 3

Detection limit of the conventional PCR assay determined by 10-fold serial dilution of genomic DNA of microfilariae and adult of Onchocerca lupi.

Lanes 1–4, from 3.6 ×101 pg/2μl to 3.6 ×10−3 pg/2μl of O. lupi mfs DNA (i.e., from 1 to 1×10−4 mfs); Lanes 5–15, from 8 ×101 ng/2μl to 8 x 10−3 fg/2μl of O. lupi adult DNA; Line 16, no-DNA control; M, 100 bp DNA marker.

Out of 152 blackflies, mosquitoes and midges, eight Simulim spp. (n = 1 S. erythrocephalum; n = 1 S. ornatum; n = 6 Simulium sp.), experimentally infected and died from 1 to three days post infection, returned positive signal for O. lupi DNA (Table 4). All field-collected blackflies and mosquitoes were negative for O. lupi DNA using qPCR (Table 4). All blackflies positive for qPCR scored positive also for cPCR.

Sequences derived from all amplicons of cPCR matched with 99–100% nucleotide identity appropriate reference sequences of O. lupi available from GenBank (accession numbers KC686702, KC686701).

Discussion

A qPCR assay has been developed for the detection of O. lupi in animal skin snip samples and potential vectors and proved to be a sensitive and specific tool for the diagnosis of this parasite, with a mean detection limit as low as 1.9 mfs per reaction. In addition, the high sensitivity of the qPCR protocol has been demonstrated by detecting a small amount of DNA (up to 8 x 10−1 fg/2μl for adult and up to 3.6 x 10−1 pg/2μl for mfs), by the slope value of standard curve (−3.131), the efficiency (115.3%) and the coefficient of determination (R2 = 0.999). These features of the assay are due to the selection of a stable hydrolysis probe designed (100% specific for O. lupi DNA), as well as to the choice of the target gene used. Indeed, cox1 gene of the mitochondrial DNA has been well recognized as a “barcode” for filarial nematodes [34], with a high amplification efficiency, also due to the large copy numbers enabling the detection of minimum amounts of DNA [35-37]. Though few Onchocerca species DNA were herein tested, which may represent a limitation of the qPCR assay, this new tool provides an alternative to the labor intensive microscopic examination of skin snip samples and to cPCR for the diagnosis of O. lupi [38]. The qPCR assay was highly specific in revealing O. lupi DNA both in co-infected samples from dogs as well as in potential vector species, avoiding the sequencing confirmation needed using cPCR with filarioid generic primers [32]. Overall, the positive fluorescent signal from samples of O. lupi, from different geographical areas (i.e., Europe and USA), which displayed genetic intraspecific variability [18], indicates the usefulness of the qPCR also for the surveillance of O. lupi where the parasite has been reported [13,14,16,17,19,39-41]. Similarly, even if the qPCR cannot discriminate between viable and nonviable parasites or immature and infective larvae, the assay could be useful for detecting O. lupi in blackfly, mosquito and/or midge species, potentially involved in the transmission of this parasite. Indeed, the specificity of the qPCR to amplify exclusively the DNA of the pathogen in potential insect vectors herein tested, may ultimately assist in the quest to identify the elusive vector of O. lupi.

The newly designed assay represents an improvement in the diagnosis of onchocercosis, by the detection and quantification of low mf densities from tissue samples and could provide a contribution to disease progress monitoring and to the surveillance of O. lupi-infected dogs, avoiding the introduction and/or spread of this life-threatening parasitic nematode, as well as to the identification of apparently healthy animals [29, 42].

The qPCR may speed-up time of diagnosis and prompt treatments of infected animals, which may avoid the appearance of nodular lesions in the eyes or in other anatomical localizations [43].

A TaqMan-based specific and sensitive assay without sequencing is expected to assist high-throughput analysis of samples, eventually leading to improve disease monitoring under the frame of a Public Health perspective. This would be particularly relevant considering that, since its first description of its zoonotic potential [7], cases of zoonotic onchocercosis are being detected increasingly in people from Europe, Iran and the USA [44-47].