Abstract A group based survey on the presence of. Entamoeba histolytica and Entamoeba dispar using real- time PCR among 20 species of captive non-human.
Parasitol Res (2003) 90: 100–103 DOI 10.1007/s00436-002-0808-z
O R I GI N A L P A P E R
Jaco J. Verweij Æ Jan Vermeer Æ Eric A.T. Brienen Coby Blotkamp Æ Daphne Laeijendecker Lisette van Lieshout Æ Anton M. Polderman
Entamoeba histolytica infections in captive primates
Received: 14 October 2002 / Accepted: 29 October 2002 / Published online: 7 February 2003 Springer-Verlag 2003
Abstract A group based survey on the presence of Entamoeba histolytica and Entamoeba dispar using realtime PCR among 20 species of captive non-human primates was performed after diagnosis of E. histolytica dysentery in a spider monkey (Ateles belzebuth hybridus). E. histolytica DNA was detected in three species of New World primates and in three species of Old World primates. In five of six E. histolytica isolates, it was possible to amplify the SREHP gene. They all revealed the same pattern after AluI digestion, indicating a common source of infection. E. dispar DNA was detected in two species of New World monkeys and three species of Old World monkeys. The results demonstrate that E. histolytica is capable of causing symptomatic and non-symptomatic infections in Old World and New World non-human primates. To our knowledge, this is the first report of E. histolytica sensu stricto in non-human primates after the redescription separating it from E. dispar in 1993.
Introduction Entamoeba histolytica is the cause of human amoebic colitis, amoebic dysentery, and amoebic liver abscess, resulting in 100,000 deaths annually (Walsh 1988). Infections have been reported based on the microscopic examination of faeces from non-human primates
J.J. Verweij (&) Æ E.A.T. Brienen Æ C. Blotkamp Æ D. Laeijendecker L. van Lieshout Æ A.M. Polderman Department of Parasitology, Leiden University Medical Centre, P.O. Box 9600, 2300 RC Leiden, The Netherlands E-mail: j.j.verweij@ lumc.nl Tel.: +31-71-5265080 Fax: +31-71-5266907 J. Vermeer La Valle´e des Singes, Le Gureau, 86700, Romagne, France
(Collet et al. 1986; Sleeman et al. 2000). Also, invasive amoebiasis in non-human primates with clinical symptoms has been demonstrated based on the examination of histopathological preparations of the caecum, colon and liver (Amyx et al. 1978; Beaver et al. 1988; MarquezMonter et al. 1991). This suggests that non-human primates could serve as a zoonotic reservoir of E. histolytica (Muriuki et al. 1988). However, the biochemical, immunological and genetic differences between E. histolytica and Entamoeba dispar, previously known as the pathogenic and nonpathogenic E. histolytica, respectively, has led to their redescription as two separate species (Diamond and Clark 1993; Gonzales-Ruiz and Wright 1998). When diagnosis is made by microscopy, the cysts or trophozoites (without ingested red blood cells) of the two species are indistinguishable and should be reported as E. histolytica/E. dispar. In addition, the rapid postmortem invasion of the cecal mucosa of macaques by non-pathogenic Entamoeba chattoni has been reported (Vogel et al. 1996); consequently this makes histopathological findings of Entamoeba difficult to interpret. In most reports, infections with E. histolytica sensu lato in non-human primates are based on the microscopic examination of faeces or histopathological preparations. In non-human primates, the use of iso-enzyme analysis or specific PCR only revealed infections with the non-invasive E. dispar (Sargeaunt et al. 1982; Smith and Meerovitch 1985; Jackson et al. 1990; Rivera and Kanbara 1999; Tachibana et al. 2000, 2001) Here, we report the on the specific detection of invasive E. histolytica using PCR in both Old and New World nonhuman primates. This study was carried out as part of a group-based survey in a primate sanctuary after the diagnosis of E. histolytica dysentery in a spider monkey (Ateles belzebuth hybridus). Furthermore, an attempt was made to estimate the relationship between the E. histolytica isolates found using the restriction site polymorphism of the serine-rich antigen gene (SREHP) as described by Clark and Diamond (1993).
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Materials and methods Subjects and sample collection In the group-based survey, stool samples were obtained from 20 species of non-human primate in ‘‘La valle´e des singes’’ primate park, Romagne, France. The scientific and common names and division between Old and New World monkeys are listed in Table 1. In addition, stool samples from individual monkeys were obtained from animals with symptoms of diarrhoea with or without blood loss: A. belzebuth (n=2), Colobus guereza (n=1) and Mandrillus sphinx (n=1). Stool collection from individual animals without clinical symptoms was not possible due to the policy of the zoo. Two portions were made from each sample . One was preserved with sodium acetate-acetic-acid-formalin (SAF) fixative for microscopy and the other was mixed with ethanol for DNA isolation.
Microscopy Wet mounts of all specimens were examined microscopically for the presence of protozoa and/or eggs.
DNA isolation, specific amplification and detection For DNA isolation, 200 ll of faecal suspension (0.5 g/ml ethanol) was centrifuged and the pellet was washed with 1 ml of PBS. After centrifugation, the pellet was resuspended into 200 ll of 2% polyvinylpolypyrolidone (PVPP) (Sigma) suspension and heated for 10 min at 100C. After sodium-dodecylsulphate-proteinase K treatment (2 h at 55C), the DNA was isolated with QIAamp Tissue Kit spin columns (Qiagen) (Verweij et al. 2001). We designed PCR primers and probes using Primer Express software (Applied Biosystems, USA) based on the known episomal repeat sequences for E. histolytica and E. dispar (Garfinkel et al. 1989), such that DNA would be amplified and detected for E. histolytica and E. dispar specifically. The E. histolytica specific primer and probe set consisted of forward primer histolytica-50F (5¢-CATTAAAAATGGTGAGGTTCTTAGGAA-3¢), reverse primer histolytica-132R (5¢-TGGTCGTCGTCTAGGCAAAATATT-3¢) and the E. histolytica specific double-labelled probe histolytica-78T (FAM 5¢-TTGACCAATTTACACCGTTGATTTTCGGA-3¢ TAMRA). The specific primers and probe for E. dispar were forward primer dispar-1F (5¢-GGATCCTCCAAAAAATAAAGTT TTATCA-3¢), reverse primer dispar-137R (5¢-ATCCACAGAACG ATATTGGATACCTAGTA-3¢) and the E. dispar specific doublelabelled probe dispar-33, containing 5-propyne-2¢-deoxyuridine in place of thymidine (HEX 5¢-UGGUGAGGUUGUAGCA GAGAUAUUAAUU-3¢ TAMRA). Amplification reactions were performed in a volume of 25 ll with PCR buffer (10·Real-Time Reaction buffer; Eurogentec, Belgium), 3.5 mM MgCl2, each nucleotide at 200 lM, 50 pmol of each specific primer, 25 pmol double-labelled probe, 1 U of Taq polymerase (Hot Goldstar TaqPolymerase; Eurogentec) and 2 ll of the DNA sample. Amplification consisted of 10 min at 95C followed by 40 cycles of 15 s at 95C, and 60 s at 60C and was performed with the ABI PRISM 7700 sequence detection system (Applied Biosystems).
followed by 35 cycles of 30 s at 95C, 30 s at 55C, and 45 s at 72C with a final extension of 5 min at 72C and was performed with the BioRad I-cycler (BioRad, USA). PCR products were submitted to digestion with AluI under the conditions recommended by the manufacturer (New England Biolabs, USA). Amplification and restriction enzyme digestion products were separated electrophoretically in 2% and 3% agarose gels, respectively, in Tris-borate-EDTA buffer, and were stained with ethidium bromide.
Results Microscopy Microscopic examination of the wet mount preparations of SAF preserved stools revealed cysts and/or trophozoites of E. histolytica/E. dispar in four of nine groups of Old World primates (Table 1). Giardia duodenalis was found in 6 of 11 groups of the New World primates whereas in primates of the Old World it was only found in Lemur catta. In the New World primates, some trematode eggs were found in Cebus apella, but no other parasites were seen. In Old World primates, Balantidium coli trophozoites and cysts were found in Macaca and M. sphinx.
E. histolytica and E. dispar specific PCR E. histolytica specific PCR amplification of DNA extracted from stools gave positive results in six groups of animals and E. dispar was detected in five groups (Table 1). These PCR positives included all four of the samples that were microscopically positive for E. histolytica/E. dispar cysts. Furthermore, E. histolytica specific PCR products were produced in three DNA samples in which no E. histolytica/E. dispar cysts or trophozoites were found with microscopy. Similarly, E. dispar specific PCR products were produced in three DNA samples of microscopically negative stools. All samples, in which no product was detected in either of the two PCR reactions (i.e., negative), were tested for PCR inhibition, which might be caused by faecal constituents. After spiking with E. histolytica DNA all samples produced the specific product after amplification using the E. histolytica PCR. Hence there was no evidence of inhibition in any of the PCR-negative samples.
SHREP amplification and AluI digestion SREHP amplification and AluI digestion SREHP amplification reactions were performed in a volume of 50 ll with PCR-buffer and Hotstart Taq-Polymerase (Hot Star Taq Master Mix; Qiagen, Germany), 12.5 pmol of each primer (SREHP5 5¢-GCTAGTCCTGAAAAGCTTGAAGAAGCTG-3¢ and SREHP3 5¢-GGACTTGATGCATCAAGGT-3¢) and 5 ll of the DNA sample. Amplification consisted of 15 min at 95C
No amplification of the SHREP gene was observed in samples in which no product was detected using the E. histolytica specific PCR. In samples from five primate species, which were E. histolytica-PCR positive, the expected fragment of ca. 550 bp was produced upon amplification of the SREHP gene repeat region. Digestion
102 Table 1 Scientific and common names of non-human primates with the number of animals in a group and their clinical symptoms with results from specific Entamoeba histolytica and Entamoeba dispar PCR done on DNA isolated from stool samples and microscopy on wet mount preparations Primate species
Common name
n
Symptoms
Real time PCR
Microscopy
Entamoeba histolytica
Entamoeba dispar
Old World Cercopithecus ascanius schmidti
Red tail monkey
2
No
Negative
Positive
Colobus guereza Eulemur fulvus albifrons Gorilla gorilla Hylobates syndactylus Lemur catta Macaca sylvanus
Black and white colobus White-fronted lemur Gorilla Siamang Ring-tailed lemur Barbary macaque
3 5 6 2 17 20
Diarrhoea No No No No No
Positive Negative Negative Negative Negative Positive
Negative Negative Negative Positive Negative Negative
Mandrillus sphinx
Mandrill
9
Diarrhoea
Positive
Positive
Ruffed lemur
10
No
Negative
Negative
None
Spider monkey Red titi White-fronted marmoset Brown capuchin
5 5 4
Dysentery No No
Positive Negative Negative
Negative Negative Negative
None G. duodenalis G. duodenalis
20
No
Negative
Negative
Woolly monkey Golden-headed tamarin Golden lion tamarin White-faced saki Emperor tamarin Cotton-top tamarin Squirrel monkey
9 4
No No
Negative Positive
Positive Negative
G. duodenalis Trematode egg G. duodenalis None
2 5 4 4 56
No No No No No
Negative Negative Negative Positive Negative
Negative Positive Negative Negative Negative
None G. duodenalis None G. duodenalis None
Endolimax nana B. coli Varecia variegata New World Ateles belzebuth hybridus Callicebus cupreus Callithrix geoffroyi Cebus apella Lagothrix lagotricha Leontopithecus chrysomelas Leontopithecus rosalia Pithecia pithecia Saguinus imperator Saguinus oedipus Saimiri boliviensis peruviensis
of the amplicons with the restriction enzyme AluI revealed comparable patterns in all five cases (Figure 1). However, no amplicon was detected upon amplification in the sample obtained from Leontopithecus crysomelas, although this sample was positive in the E. histolytica specific PCR.
Discussion Several studies have demonstrated E. histolytica/E. dispar infection in non-human primates. Isolates from Old World non-human primates were distinguished from E. histolytica and identified as E. dispar (non-pathogenic E. histolytica) by their zymodeme patterns (Sargeaunt et al. 1982; Smith and Meerovitch 1985; Jackson et al. 1990). In recent studies on groups of captive macaques and chimpanzees in Japan, E. dispar infection but no E. histolytica infection was observed using specific PCR (Rivera and Kanbara 1999; Tachibana et al. 2000, 2001). Invasive amoebiasis due to natural infection has been described in spider monkeys (Ateles sp.) (Amyx et al.
E. histolytica/E. dispar Entamoeba coli Iodamoeba butschlii E. histolytica/E. dispar None None None Giardia duodenalis E. histolytica/E. dispar Balantidium coli I. butschlii E. histolytica/E. dispar E. coli Entamoeba hartmanni
1978; Marquez-Monter et al. 1991) and in some other species of monkeys as well (Beaver et al. 1988). However, these results were based on histopathological findings only, without zymodeme or genetic identification of the Entamoeba species. In this study E. histolytica DNA was detected in samples from two spider monkeys (A. b. hybridus) presenting with amoebic dysentery. Furthermore, E. histolytica DNA was detected in a sample obtained from a mandrill with diarrhoea. Only E. dispar DNA was detected in the sample from the mandrill monkeys in the group without diarrhoea. Furthermore, E. histolytica DNA was detected in three groups of Old World primates of which C. gureza and M. sphinx had mild diarrhoea and Macaca sylvanus had no symptoms at all. In the two New World primate groups (L. chrysomelas and Saguinus oedipus) in which E. histolytica DNA was detected, the animals displayed no clinical symptoms. Five of six E. histolytica isolates showed a comparable pattern after AluI digestion of the amplification products upon SHREP PCR. This suggests the spread of one E. histolytica strain between these groups. It is
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Fig. 1 Agarose gel showing SREHP after AluI digestion of Entamoeba histolytica isolates from Ateles belzebuth (lanes 1 and 2), Saguinus oedipus (lane 3), Macaca (lanes 4 and 5, same isolate), Colobus guereza (lane 6), Mandrillus sphinx (lane 7), HM1 strain, and the 100 bp DNA marker (lane M)
unclear whether the lack of a SHREP PCR product in the sample obtained from L. crysomelas is due to strain differences or the lack of sensitivity of the SHREP amplification. We think that the infection was brought into the park by the spider monkeys and spread to the other monkey groups through faecal contamination of the shoes or clothes of the keepers, as diarrhoeal complaints started after the introduction of the A. belzebuth. The transmission of amoebae from non-human primates to humans has been reported (Sargeaunt et al. 1982), consequently the possibility of introduction and spread of the parasite through the separate groups by infected (asymptomatic) keepers cannot be ruled out. Unfortunately, it was not possible to examine faecal samples of the keepers for the presence of E. histolytica. This study clearly demonstrates that Old World as well as New World non-human primates can host E. histolytica. The actual role of this potential reservoir should be investigated in endemic areas using techniques that are capable of differentiation of non-invasive E. dispar and invasive E. histolytica. Acknowledgement We thank Dr. Kate Templeton for critical reading of the manuscript and Dr. Graham Clark for his comments on the SREHP result.
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