First Study of Ascaris lumbricoides from the Semiwild Population of the Sumatran Orangutan Pongo abelii in the Context of Morphological Description and Molecular Phylogeny.

There is little evidence that the already described and accepted taxa of ascarids (Ascaris lumbricoides, A. suum, and A. ovis) infecting individuals of taxonomically distant groups (hominids, pigs, sheep, goats, and dogs) can be genetically or morphologically distinguished. However, despite described morphological differences, e.g., due to intraspecific variation, these are insufficient for species determination and may indicate differences amongst ascarids because of cross infections, hybrid production, and specific adaptations to hosts. Herein, the results of a molecular and morphological analysis of ascarids parasitising Sumatran orangutans (Pongo abelii Lesson, 1827) in native populations are presented. The research took place in the Bukit Lawang area, Indonesia, in 2009. Throughout the year, fresh faecal samples were collected regularly from 24 orangutans, and all were examined for the presence of nematode adults. Only five adult worms from two orangutan females were found during regular collection. Using the integrative taxonomic approach, the nematodes found were identified as A. lumbricoides. The significance of the find and its rarity is documented by the fact that this is the first confirmed finding of adult ascarids from an original orangutan site (not from a zoo) in more than 130 years (including the long-term study spanning the last 20 years focusing on orangutan parasites and natural antiparasitic drugs). More accurate morphometric parameters and genetic differences for the identification of ascarids were established. These parameters will be helpful for other findings in great apes and will also be suitable for further and precise determination of this parasite. The details distinguishing between male and female specimens are also stated and well defined. A comprehensive evaluation of the situation of Ascaris species parasitising orangutans, including a comparison with previously described orangutan parasite (i.e., A. satyri-species inquirenda), is discussed.

Genetic characterization of nodular worm infections in Asian Apes.

Parasitic nematodes of Oesophagostomum spp., commonly known, as 'nodular worms' are emerging as the most widely distributed and prevalent zoonotic nematodes. Oesophagostomum infections are well documented in African non-human primates; however, the taxonomy, distribution and transmission of Oesophagostomum in Asian non-human primates are not adequately studied. To better understand which Oesophagostomum species infect Asian non-human primates and determine their phylogeny we analysed 55 faecal samples from 50 orangutan and 5 gibbon individuals from Borneo and Sumatra. Both microscopy and molecular results revealed that semi-wild animals had higher Oesophagostomum infection prevalence than free ranging animals. Based on sequence genotyping analysis targeting the Internal transcribed spacer 2 of rDNA, we report for the first time the presence of O. aculeatum in Sumatran apes. Population genetic analysis shows that there is significant genetic differentiation between Bornean and Sumatran O. aculeatum populations. Our results clearly reveal that O. aculeatum in free-ranging animals have a higher genetic variation than those in semi-wild animals, demonstrating that O. aculeatum is circulating naturally in wildlife and zoonotic transmission is possible. Further studies should be conducted to better understand the epidemiology and dynamics of Oesophagostomum transmission between humans, non-human primates and other wild species and livestock in Southeast Asia.

Entamoeba histolytica infections in wild and semi-wild orangutans in Sumatra and Kalimantan.

Key to the success of orangutan conservation management practices is the prevention of the introduction of infectious diseases to the remaining populations. Previous reports of Entamoeba spp. positive orangutans are of concern as Entamoeba spp. infection has been linked to morbidity and mortality in primates. It remains to be determined if the Entamoeba species infecting orangutans is the pathogenic Entamoeba histolytica. Orangutan fecal samples have been collected from orangutans from sites in Sumatra (Bukit Lawang, Ketambe, and Suaq, 241 samples from 64 individuals), and two sites in Kalimantan (Sebangau and Tuanan, 129 samples from 39 individuals). All samples were from wild orangutans except for a proportion from Sumatra which were from semi-wild (108 samples, 10 individuals). E. histolytica-specific nested PCR assays were carried out on the fecal samples. A total of 36 samples from 17 individuals tested positive for E. histolytica. When compared with published sequences using NCBI BLAST the E. histolytica positive samples showed a 98-99% concordance. The majority (76%, n = 36) of the positive isolates came from semi-wild orangutans in Bukit Lawang. This study supports the growing body of evidence that contact with humans is an important risk factor for infection of wild primates with E. histolytica.

Limitations in the screening of potentially anti-cryptosporidial agents using laboratory rodents with gastric cryptosporidiosis.

The emergence of cryptosporidiosis, a zoonotic disease of the gastrointestinal and respiratory tract caused by Cryptosporidium Tyzzer, 1907, triggered numerous screening studies of various compounds for potential anti-cryptosporidial activity, the majority of which proved ineffective. Extracts of Indonesian plants, Piper betle and Diospyros sumatrana, were tested for potential anti-cryptosporidial activity using Mastomys coucha (Smith), experimentally inoculated with Cryptosporidium proliferans Kvác, Havrdová, Hlásková, Danková, Kandera, Jezková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016. None of the plant extracts tested showed significant activity against cryptosporidia; however, the results indicate that the following issues should be addressed in similar experimental studies. The monitoring of oocyst shedding during the entire experimental trial, supplemented with histological examination of affected gastric tissue at the time of treatment termination, revealed that similar studies are generally unreliable if evaluations of drug efficacy are based exclusively on oocyst shedding. Moreover, the reduction of oocyst shedding did not guarantee the eradication of cryptosporidia in treated individuals. For treatment trials performed on experimentally inoculated laboratory rodents, only animals in the advanced phase of cryptosporidiosis should be used for the correct interpretation of pathological alterations observed in affected tissue. All the solvents used (methanol, methanol-tetrahydrofuran and dimethylsulfoxid) were shown to be suitable for these studies, i.e. they did not exhibit negative effects on the subjects. The halofuginone lactate, routinely administered in intestinal cryptosporidiosis in calves, was shown to be ineffective against gastric cryptosporidiosis in mice caused by C. proliferans. In contrast, the control application of extract Arabidopsis thaliana, from which we had expected a neutral effect, turned out to have some positive impact on affected gastric tissue.

The course of experimental giardiasis in Mongolian gerbil.

Fifteen Mongolian gerbils were inoculated with 10 × 106 viable trophozoites of Giardia intestinalis. Their faeces were examined daily by flotation method and the number of shed cysts was counted. Two animals (male and female) were euthanised at 4- to 5-day intervals (9, 14, 18 days post-infection (DPI)). The remaining nine gerbils were sacrificed and dissected at the end of the experiment (23 DPI). Their small intestinal tissues were processed for examination using histological sectioning and scanning electron microscopy and their complete blood count (CBC) was examined. The highest number of trophozoites at the total was observed in the duodenum in gerbils sacrificed on 14 DPI. Number of shed cysts was positively correlated with number of trophozoites rinsed from the intestine. Infected gerbils had lower body weight gain in comparison with control group and in three male gerbils; diarrhoea occurred during infection. Cyst shedding was negatively correlated with values of mean corpuscular haemoglobin concentration. Females showed another pattern in cyst shedding than males. This information needs to be taken into account while planning the experiments.

Effect of Piper betle on Giardia intestinalis infection in vivo.

Piper betle has been used as a medicinal plant in traditional medical systems throughout South and South East Asia. Experimental studies have revealed its wide and diverse biological and pharmacological effects. In this study, antigiardial activity of Piper betle was tested using experimental infections of Giardia intestinalis, the most common cause of protozoal diarrhoea worldwide, in Mongolian gerbils. Plants were extracted in water, methanol and methanol:tetrahydrofuran. Gerbils were treated for ten days intragastrically twice a day, with the dose of 40 mg of the extract per 100 g of body weight. Drug metronidazole was used as a negative control. Gerbils' faeces were taken every day and examined by flotation method, the number of shed cysts were counted using a haemocytometer. After gerbils' sacrifice and dissection, their duodena were then processed for examination using histological sectioning and scanning electron microscopy. The antigiardial activity was evaluated by the course of cyst shedding throughout the entire experiment. A significant decline in cyst shedding, evaluated by linear regression was found in gerbils treated with the aqueous extract. Our results indicate that the aqueous extract of P. betle shows giardicidal effects.

Effects of selected Indonesian plant extracts on E. cuniculi infection in vivo.

The present study was conducted to evaluate the methanolic extracts from several plant leaves widely used in traditional medicine to cure digestive tract disorders and in the self-medication of wild animals such as non-human primates, namely Archidendron fagifolium, Diospyros sumatrana, Shorea sumatrana, and Piper betle leaves, with regard to their antimicrosporidial activity against Encephalitozoon cuniculi in immunocompetent BALB/c mice determined using molecular detection of microsporidial DNA (qPCR) in various tissues and body fluids of infected, treated mice. Of the plant extracts tested, Diospyros sumatrana provided the most promising results, reducing spore shedding by 88% compared to untreated controls. Moreover, total burden per 1 g of tissue in the D. sumatrana extract-treated group reached 87% reduction compared to untreated controls, which was comparable to the effect of the standard drug, Albendazole. This data represents the baseline necessary for further research focused on determining the structure, activity and modes of action of the active compounds, mainly of D. sumatrana, enabling subsequent development of antimicrosporidial remedies.

Parasites of orangutans (primates: ponginae): An overview.

Wild orangutan populations exist in an increasingly fragile state. As numbers continue to decline and populations became fragmented, the overall health of remaining individuals becomes increasingly at risk. Parasitic infections can have a serious impact on the health of wild orangutans, and can be fatal. It has been reported that rehabilitated individuals demonstrate a higher prevalence of parasitic diseases, and it is possible that they may spread these infections to wild orangutans upon reintroduction. In order to ensure the success of reintroduction and conservation efforts, it is crucial to understand the potential risks by fully understanding what parasites they have been reported to be infected with. Using this knowledge, future conservation strategies can be adapted to minimize the risk and prevalence of parasite transmission in the remaining orangutan populations. There is still limited information available on orangutan parasites, with several still not identified to the species level. Based on comprehensive literature review, we found 51 parasite taxa known to infect wild, semi-wild, and captive orangutans, including newly reported species. Here, we summarize methods used to identify parasites and draw conclusions relative to their reported prevalence. We also recommend fecal sample preservation and analytical methods to obtain best result in the future.

Statistical comparison of excystation methods in Cryptosporidium parvum oocysts.

Excystation of sporozoites of Cryptosporidium parvum from oocysts is essential for successful in vitro assays. It has also been traditionally used as a measure for oocyst viability and infectivity. Laboratories use various excystation protocols so there is a need to clarify which method is the best. In this study, six different protocols for in vitro excystation of C. parvum oocysts were compared to find the most efficient excystation method (expressed as percentage excystation). Tested protocols differed in chemical pre-incubation steps, excystation media or time of incubation. There were significant differences in percentage of excysted oocysts among groups excysted by different methods. There were also significant differences in percentage of excysted oocysts between methods using pre-incubation with sodium hypochlorite and those without. The other variables examined; the presence of trypsin, kind of excystation medium and the incubation time, did not show statistical differences in percentage excystation among groups. Pre-incubation steps which included sodium hypochlorite, enhancing the permeability of the oocysts were found to increase the excystation ratio and methods using this step were the most effective.

Prevalence of Cryptosporidium spp., Enterocytozoon bieneusi, Encephalitozoon spp. and Giardia intestinalis in Wild, Semi-Wild and Captive Orangutans (Pongo abelii and Pongo pygmaeus) on Sumatra and Borneo, Indonesia.

BACKGROUND: Orangutans are critically endangered primarily due to loss and fragmentation of their natural habitat. This could bring them into closer contact with humans and increase the risk of zoonotic pathogen transmission. AIMS: To describe the prevalence and diversity of Cryptosporidium spp., microsporidia and Giardia intestinalis in orangutans at seven sites on Sumatra and Kalimantan, and to evaluate the impact of orangutans' habituation and location on the occurrence of these zoonotic protists. RESULT: The overall prevalence of parasites in 298 examined animals was 11.1%. The most prevalent microsporidia was Encephalitozoon cuniculi genotype II, found in 21 animals (7.0%). Enterocytozoon bieneusi genotype D (n = 5) and novel genotype Pongo 2 were detected only in six individuals (2.0%). To the best of our knowledge, this is the first report of these parasites in orangutans. Eight animals were positive for Cryptosporidium spp. (2.7%), including C. parvum (n = 2) and C. muris (n = 6). Giardia intestinalis assemblage B, subtype MB6, was identified in a single individual. While no significant differences between the different human contact level groups (p = 0.479-0.670) or between the different islands (p = 0.992) were reported in case of E. bieneusi or E. cuniculi, Cryptosporidium spp. was significantly less frequently detected in wild individuals (p < 2×10-16) and was significantly more prevalent in orangutans on Kalimantan than on Sumatra (p < 2×10-16). CONCLUSION: Our results revealed that wild orangutans are significantly less frequently infected by Cryptosporidium spp. than captive and semi-wild animals. In addition, this parasite was more frequently detected at localities on Kalimantan. In contrast, we did not detect any significant difference in the prevalence of microsporidia between the studied groups of animals. The sources and transmission modes of infections were not determined, as this would require repeated sampling of individuals, examination of water sources, and sampling of humans and animals sharing the habitat with orangutans.

Molecular phylogeny of anoplocephalid tapeworms (Cestoda: Anoplocephalidae) infecting humans and non-human primates.

Anoplocephalid tapeworms of the genus Bertiella Stiles and Hassall, 1902 and Anoplocephala Blanchard, 1848, found in the Asian, African and American non-human primates are presumed to sporadic ape-to-man transmissions. Variable nuclear (5.8S-ITS2; 28S rRNA) and mitochondrial genes (cox1; nad1) of isolates of anoplocephalids originating from different primates (Callicebus oenanthe, Gorilla beringei, Gorilla gorilla, Pan troglodytes and Pongo abelii) and humans from various regions (South America, Africa, South-East Asia) were sequenced. In most analyses, Bertiella formed a monophyletic group within the subfamily Anoplocephalinae, however, the 28S rRNA sequence-based analysis indicated paraphyletic relationship between Bertiella from primates and Australian marsupials and rodents, which should thus be regarded as different taxa. Moreover, isolate determined as Anoplocephala cf. gorillae from mountain gorilla clustered within the Bertiella clade from primates. This either indicates that A. gorillae deserves to be included into the genus Bertiella, or, that an unknown Bertiella species infects also mountain gorillas. The analyses allowed the genetic differentiation of the isolates, albeit with no obvious geographical or host-related patterns. The unexpected genetic diversity of the isolates studied suggests the existence of several Bertiella species in primates and human and calls for revision of the whole group, based both on molecular and morphological data.

Phylogenetic relationships between pinworms (Nematoda: Enterobiinae) parasitising the critically endangered orang-utan, according to the characterisation of molecular genomic and mitochondrial markers.

Pinworms (Nematoda: Enterobiinae) include 52 species parasitising primates throughout the world. In the present study, we performed the first ever molecular analysis to investigate the phylogenetic position of recently described pinworms parasitising the Sumatran orang-utan. The phylogenetic analysis of mitochondrial CO1 and chromosomal 18S rDNA and ITS1 regions could support the independent status of several Nematoda species. Our molecular data clearly suggest that Enterobius (Colobenterobius) buckleyi and Lemuricola (Protenterobius) pongoi together with Pongobius hugoti form separate clades among other studied species, which significantly supports the hypothesis of recently described new species parasitising the orang-utan (Pongo abelii and Pongo pygmaeus). The phylogenetic tree based on cytochrome oxidase 1 (CO1) gene variability showed possible close relationships between L. (Protenterobius) pongoi and P. hugoti; thus, we can assume that these species could have initially diverged in sympatry from a common ancestor.

Redescription and resurrection of Bertiella satyri (Cestoda, Anoplocephalidae) parasitizing the orangutan (Pongo abelii) in Indonesia.

The tapeworm species Bertiella satyri from a semi-wild Sumatran orangutan (Pongo abelii: Ponginae) is redescribed and the sequence of its 18S rDNA is presented. The tapeworms parasitizing the genera Pan, Pongo, Homo and Hylobates from Muséum National d'Histoire Naturelle, Paris and from Muséum d´Histoire Naturelle, Genève are also presented. The validity of B. satyri is confirmed. B. satyri (BSA) differs from the most similar species Bertiella studeri (BSTU) in the following characteristics: (1) testes number, BSTU 300-400; BSA 116-124, (2) genital opening, BSTU regularly alternate; BSA irregularly alternate, (3) Cirrus-sac, BSTU short, 0.250-0.320, does not reach excretory ducts; BSA long, 0.630 × 0.495, reaches excretory ducts, (4) egg size, BSTU 0.053-0.060; BSA 0.030-0.051, (5) host BSTU Pan troglodytes, Africa; BSA Pongo pygmaeus, P. abelii, Indonesia (Borneo, Sumatra). Both species differ according to our material and the name B. satyri (Blanchard, 1891) is resurrected.

Description of Lemuricola (Lemuricola) pongoi--male (Nematoda: Enterobiinae) parasitising orangutan Pongo abelii.

The study presents the first description of male specimen (allotype) of the pinworm species Lemuricola (Lemuricola) pongoi, parasitising Sumatran orangutan (Pongo abelii) from Sumatra (Indonesia). The male specimen morphology presents all features fully corresponding with diagnose of the genus and subgenus Lemuricola. We are agreeing that Protenterobius is synonymous with Lemuricola because the length of tail appendix in males is not a sufficient feature for separation of the genera or subgenera. For L. (L.) pongoi male are characteristic features: body length (4,803 microm), total oesophagus length (530), tail length (235), tip of tail length (188) and spicule length (90).

Two cases of mother-infant cannibalism in orangutans.

Observations of ape cannibalism have to this point been limited to chimpanzees (Pan troglodytes) where it is associated with infanticide and consumption by unrelated individuals (Watts and Mitani, Primates 41(4):357-365, 2000). Here we report for the first time observations of two unrelated female Sumatran orangutans (Pongo abelii) cannibalizing the remains of their infants on different occasion, a behavior never before reported in any ape species. The two orangutans were wild-born rehabilitated individuals, and had been reintroduced to an area hosting a largely unregulated primate tourism industry and experienced restricted ranging conditions. Though it is possible that this is a strategy to regain energy and nutrients or a result of individual history, comparative data suggest that this is an aberrant behavior which may be linked to environmental stressors within the area.