Detection of Cyclospora in captive chimpanzees and macaques by a quantitative PCR-based mutation scanning approach

Cyclospora is a protistan parasite that causes enteritis in several species of animals including humans. The aim of this study was to investigate the presence of Cyclospora in captive non-human primates. A total of 119 faecal samples from Pan troglodytes, Macaca sylvanus, Cercopithecus cephus, Erythrocebus patas, Chlorocebus aethiops and Macaca fascicularis from a wildlife animal rescue center as well as from Macaca fascicularis from an experimental primate research center were tested for the presence of Cyclospora by quantitative real-time PCR (qPCR) and single-strand conformation polymorphism (SSCP) analysis. Cyclospora was detected in three Pan troglodytes (13.6%) and nine (9.3%) Macaca fascicularis. The present study represents the first record of Cyclospora in captive primates in Europe, suggesting the presence of Cyclospora cayetanensis, which is transmissible to humans.


Background
Enteritis caused by protistan parasites is a principal cause of substantial morbidity in humans, often resulting in mortality in under-privileged communities [1]. Protozoa belonging to the genus Cyclospora (Apicomplexa: Eimeriidae) are obligate, intracellular parasites that infect the epithelium of the intestines or bile ducts of a variety of hosts, mostly vertebrates [2]. Nineteen species of Cyclospora have been described in millipedes, reptiles, insectivores, rodents, and human and non-human primates [2]. In humans, Cyclospora was first identified in Papua New Guinea in the late 1970s [3] and then named Cyclospora cayetanensis [4,5] in the early 1990s. Cyclospora cayetanensis has since been recognized as an important cause of endemic or epidemic diarrhoeal illness in children and immuno-compromised or -suppressed individuals worldwide [6]. Human cyclosporosis is endemic in developing countries, but also in industrialized settings, mainly in people with no history of foreign travel [7] and sometimes implicated in communitywide outbreaks of diarrhoeal illness [7].
Cyclospora species appear to be underestimated as infectious agents, and very little is known about their epidemiology (including host and geographical ranges) and pathogenicity [7,8]. Infections are transmitted via the faecal-oral route through contaminated environmental water, food or soil, although oocysts need to sporulate outside of the mammalian host (from 7-15 days) to be infective [9].
Investigations of outbreaks and case-control studies have shown that people with cyclosporosis had contact with animals, suggesting a zoonotic role of C. cayetanensis. Cyclospora oocysts, morphologically resembling C. cayetanensis, have been detected in chickens, ducks, cattle, mice, rats and dogs [10][11][12][13][14], whereas morphologically and molecularly characterized C. cayetanensis have been identified in dogs, domestic birds and in monkeys (Mulatta mulatta) [10]. This evidence supports the possible zoonotic nature of C. cayetanensis, although whether animals from which this parasite have been isolated are natural hosts is still unknown.
Investigations of Cyclospora of animals, particularly those with a close genetic relationship with humans, have important implications for better understanding the epidemiology and host range of members of this genus. PCR-based tools employing various nuclear (e.g., ribosomal and heat shock protein) gene markers have been utilized to detect and/or identify Cyclospora spp. [15]. In the present study, a quantitative PCR (qPCR), combined with single-strand conformation polymorphism (SSCP)-based sequencing analysis, was used for the detection and genetic characterization of Cyclospora from various species of captive primates using the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as a marker.  Table 1). Primates were kept in enclosures strictly according to the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Health, Italy. None of the animals studied showed clinical signs, such as diarrhoea, at the time of sampling. Faecal samples (packed in individual plastic bags) were sent frozen to the Parasitology Laboratory, Department of Agriculture Science, Food and Environment, University of Foggia, Italy, where they were stored at −80°C. Subsequently, samples were thawed, and genomic DNAs were isolated from individual faecal samples using the Qiagen stool kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. DNA was eluted in 50 μl of H 2 0, quantified using a Qubit 2.0 fluorometer and stored at −20°C. The individual genomic DNA samples contained 0.2 to 100 ng per μl.

Quantitative qPCR and melting curve analysis
The qPCR assay and melting curve analysis were performed in a CFX-96 Real Time Instrument (BioRad, Italy) [16]. Briefly, PCR was carried out in a final volume of 20 μl, utilizing SsoFast™ EvaGreen® Supermix (cat. no. 172-5201; Bio-Rad, Italy) and 0.5 μM of each primer CCITS2-F (forward: 5′-GCAGTCACAGGAGGCATATATCC-3′) and CCITS2-R (reverse: 5′-ATGAGAGACCTCACAGCCAAAC-3′) to a region (116 bp) within ITS-2 of C. cayetanensis (cf. [17]). Genomic DNA (50 to 100 ng), cloned ITS-2 (0.5 pg; reference, positive-control) DNA or water (negative control) in 5 μl were added to the reaction. Cycling conditions were: initial denaturation at 98°C for 2 min, followed by 35 cycles at 98°C for 5 s, and 59°C for 15 s. Fluorescence data were collected at the end of each cycle as a single acquisition. Melting curve analysis was performed at the end of each PCR run (70°C to 95°C at 0.5°C/5 s). Each sample was analyzed in duplicate, and the amplification cycle threshold (Ct) and melting temperature (Tm) values were calculated. The diagnostic Tm peak for Cyclospora was 84.5°C. The criteria used to define a test-positive sample were: (a) a detectable amplification curve, (b) a Tm value of ± 0.5°C with reference to the Tm value of plasmid control, and (c) a dF/dT fluorescence value of > 2.
Raw data were normalized by applying curve-scaling to a line of best fit, so that the highest fluorescence value was 100 and the lowest was zero (standard normalized melt curve). Then, the curves were differentiated, and a composite median curve was constructed using the median fluorescence values for each sample. The melting traces for each sample were subtracted from this composite median curve to draw a residual plot (difference graph). The number of DNA copies per μl was calculated by relating the Ct mean value of each sample to a standard curve for the plasmid control, and the number of oocysts calculated, assuming that an oocyst contains 15 copies of rDNA [18].

qPCR performance
The analytical sensitivity of the assay was estimated using 10-fold serial dilutions (from 10 10 to 10 copies/μl) of the (cloned) reference (positive) control, which were each subjected (in triplicate) to qPCR and subsequent melting curve analysis, and the mean value of the threshold cycle (Ct) was plotted against the logarithm of DNA copies per μl. The standard curve was produced by a linear regression of the plotted points and the range of linearity and the lowest detectable amount of DNA were estimated. PCR efficiency (E) was calculated according to the equation: E = 10 −1 /slope -1 [19]. E of between 90 and 110% and a correlation (R 2 ) of < 1 value were considered acceptable. To determine repeatability, three standard points of (cloned) reference (positive) control (10 7 , 10 5 and 10 copies/μl) were tested in triplicate three times in the same experiment and the Ct mean values were recorded. The same standards were tested once a day for three more days to determine reproducibility. To analyze the reproducibility and repeatability, both intra-and interassay coefficients of variation (CV) were assessed. CV mean values were calculated following the formula: CV = σ(Ct)/μ (Ct).

Single-strand conformation polymorphism (SSCP) analysis and sequencing
All amplicons were subjected to single-strand conformation polymorphism (SSCP) analysis using protocol B [20]. Amplicons representing distinct banding profiles were selected and treated with exonuclease I and shrimp alkaline phosphatase (Fermentas), according to the manufacturer's instructions, and then subjected to direct, automated sequencing (BigDye Terminator v.3.1 chemistry, Applied Biosystems, USA) using the primer CCITS2-R. The quality of each sequence was assessed based on the corresponding electropherogram using the program BioEdit, and the sequences determined were compared with known reference sequences using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/ BLAST).

Results and discussion
Upon assessing assay performance, we established that intra-assay CV values for the 10 7 , 10 5 and 10 standard points were 1.8%, 1.5%, and 1.2%, and inter-assay CV values for the 10 7 , respectively 10 5 and 10 standard points were 0.80%, 0.60%, and 0.93%, respectively. The range of linearity was acceptable from 10 10 to 10 copies/μl, with an amplification efficiency (E) of 93.6% and a slope of −3,486 (R 2 = 0.995). The qPCR was then applied to the test samples.
Twelve of 119 (10.1%) faecal samples were qPCR testpositive for Cyclospora, including 3 (13.6%) chimpanzees (Pan troglodytes) from 22 primates from the WARC and 9 (9.3%) from 97 cynomolgus monkeys (syn. crab-eating macaque) (Macaca fascicularis) from the EPRC ( Table 1). The numbers of Cyclospora oocysts in test-positive samples were predicted to range from 80 to 498 per gram of faeces (Table 1). All 12 Cyclospora ITS-2 amplicons produced in qPCR were subjected to SSCP analysis, and one profile was displayed for both non-human primate species. The sequence representing all 12 amplicons was identical to the reference sequence with GenBank accession no. AF301386.
Although the sequence of these amplicons was consistent with those determined for C. cayetanensis, some caution is needed in assigning species status, for the following reasons: (i) it is not yet known whether ITS-2 provides unequivocal specific identification of Cyclospora and (ii) we did not detect oocysts of Cyclospora for morphological identification/description. Therefore, although Cyclospora DNA was amplified using primers designed to ITS-2 of C. cayetanensis [17], the magnitude of sequence variation within individual, recognized species of Cyclospora as well as the levels of difference among recognised species of Cyclospora have not yet been rigorously assessed. Although various non-human primates have been reported to harbour unique Cyclospora species, such as Cyclospora cercopitheci n. sp. in green monkeys, C. colobi n. sp. in colobus monkeys, and C. papionis sp. n. in baboons [21][22][23], the finding of Cyclospora-like oocysts in chimpanzees (P. troglodytes), baboons (Papio cynochephalus) [11] and in drills (Mandrillus leucophaeus) [14] raises the vexed question as to the specific identity of these parasites and whether they are transmissible to humans or not.
Given that attempts to infect different animal species (chickens, ducks, mice, gerbils, hamsters, rabbits, rats, sand rats, ferrets, pigs, dogs, monkeys and baboons) with C. cayetanensis have been unsuccessful [24], it is possible that Cyclospora species are predominantly hostspecific, like Eimeria [25]; thus, further investigation is warranted in this regard. The identification of Cyclospora in a chimpanzee supports the previous detection of this parasite in this primate species [11]; if C. cayetanesis is confirmed, the role of M. fascicularis as a reservoir of this protozoan might support its molecular detection in a related species of macaque (i.e., Macaca mulatta) in Nepal [10].
While coprophagia is a likely mode of transmission of parasitic infections in captive primates [26], the animals studied here might have become infected by Cyclospora through water, soil or food (mostly fruit). Indeed, contaminated tap water and fresh produce (fruits and leafy vegetables) [27][28][29] are recognized as key sources of cyclosporosis outbreaks in humans [7,30], while the role of contaminated soil has only been suspected [8,29] but not yet proven.
It is possible that cynomolgus monkeys from the EPRS premises had been infected by Cyclospora upon arrival from China, where Cyclospora-like organisms have been detected recently in golden snub-nosed monkeys (Rhinopithecus roxellanae) from the Breeding Research Center in Shaanxi province, China [23], and where cyclosporosis in humans has often been reported [31][32][33]. Regarding chimpanzees from WARC, it is possible that the contamination might have been acquired locally, since the animals were not recently moved or imported from other areas. In Italy, humans have been found to harbour C. cayetanensis [34][35][36], and this parasite has also been isolated from stored tap water [37].

Conclusions
Although the possible zoonotic role of Cyclospora detected herein cannot be confirmed, the high number of Cyclospora oocysts shed in faeces might be a public health concern, considering their resilience in the environment and to routine chemicals used to disinfect water [7,38], which might facilitate their dissemination via water and food and the environment to humans and/or other animals. Understanding the host and distributional ranges of Cyclospora species and transmission thereof should have important implications for preventing and controlling cyclosporosis. Whether the nonhuman primates studied here serve as natural reservoirs for C. cayetanensis remains unclear, and future investigations are warranted to confirm the specific status of Cyclospora detected in the present study.

Competing interests
The authors declare that they have no competing interest.
Authors' contributions MM and AVK undertook molecular work; ASZ, MTM and EB undertook collections; RBG and AG designed the study and wrote the paper. All authors read and approved the final version of the manuscript.