Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools. METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity. RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested. CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

A review of Cyclospora cayetanensis in animals.

Cyclosporiasis is a global, emerging disease in humans caused by Cyclospora cayetanensis. The role of animals in the epidemiology of cyclosporiasis is not fully understood. We conducted a narrative review of the published literature on C. cayetanensis in animals. MEDLINE® (Web of Science™ ), Agricola (ProQuest), CABI Global Health (1979 to December 2020) and Food Science and Technology Abstracts (EBSCOhost) (1979 to February 2020) were searched. Studies of C. cayetanensis in or on any species of animal were eligible. Thirteen relevant studies were found. C. cayetanensis was found in wild and farmed Mediterranean mussels (Mytilus galloprovincialis), wild grooved carpet shell clams (Ruditapes decussatus) and in the faeces of dogs (domestic and street), wild chickens, wild rhesus macaques (Macaca mulatta), chimpanzees (Pan troglodytes) from a wildlife research centre, and Cynomolgus monkeys (Macaca fascicularis) from an experimental primate research centre. As the small intestines of the naturally exposed animals were not biopsied, existence of a natural animal reservoir of C. cayetanensis could not be confirmed. Animals shedding oocysts in their faeces may be paratenic hosts. Investigators were able to successfully infect the following animals with C. cayetanensis: oysters, Asian freshwater clams (Corbicula fluminea), Swiss albino mice and guinea pigs. Future non-laboratory studies of animals should use PCR coupled with DNA sequencing to confirm that the species found is C. cayetanensis. The potential role of animals in the transport of oocysts and contamination of food, water, and soil could be explored through future primary research.

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

BACKGROUND: 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. METHODS: 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. RESULTS: Cyclospora was detected in three Pan troglodytes (13.6%) and nine (9.3%) Macaca fascicularis. CONCLUSIONS: 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.

Identification and morphologic and molecular characterization of Cyclospora macacae n. sp. from rhesus monkeys in China.

Cyclospora spp. in nonhuman primates are most closely related to Cyclospora cayetanensis, an emerging human pathogen causing outbreaks of cyclosporiasis in North America. Studies thus far indicate the possible existence of host specificity in Cyclospora spp. In this study, 411 fecal specimens from free-range rhesus monkeys (Macaca mulatta) were collected and examined for Cyclospora by sequence analysis of the small subunit rRNA gene. A novel Cyclospora species was identified in 28 (6.8%) specimens and named Cyclospora macacae based on morphologic and molecular characterizations. The oocyst of C. macacae is spherical and measures 8.49 ± 0.55 × 8.49 ± 0.49 µm in diameter. Phylogenetic analysis grouped this species together with the other four Cyclospora species infecting primates, including C. cayetanensis in humans, forming a monophyletic group closely related to avian Eimeria species. In addition, C. cayetanensis was detected in one specimen, although whether rhesus monkeys can serve as a natural reservoir host of C. cayetanensis needs further investigation.

Cyclospora papionis, Cryptosporidium hominis, and human-pathogenic Enterocytozoon bieneusi in captive baboons in Kenya.

Cyclospora papionis, Cryptosporidium hominis, and Enterocytozoon bieneusi were detected in 42 (17.9%), 6 (2.6%), and 29 (12.3%) of 235 newly captured baboons in Kenya, respectively. Most C. hominis subtypes and E. bieneusi genotypes found have been detected in humans in the area, suggesting that cross-species transmission of cryptosporidiosis and microsporidiosis is possible.

Combined PCR-oligonucleotide ligation assay for detection of dairy cattle-derived Cyclospora sp.

A rapid and sensitive assay for the detection of Cyclospora species in dairy cattle faecal specimens has been developed. The method utilizes a nested PCR to amplify a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. and an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) to detect the amplified product. In this study, the OLA technique was compared with conventional gel electrophoresis for the detection of amplified product. In evaluating the PCR-OLA for Cyclospora sp. and non-Cyclospora parasites, A(405) reading value for Cyclospora species was significantly higher than those for non-Cyclospora control. At known concentrations of purified amplicons from cattle-derived Cyclospora sp., the OLA was able to detect more than 0.5 ng of the amplified DNA. Of 168 clinical specimens collected from four dairy cattle farms, 6 were positive by both PCR-gel electrophoresis and the PCR-OLA procedure, and 2 were positive only by PCR-OLA, indicating the PCR-OLA procedure was more sensitive than the common way with gel electrophoresis. The results indicated that the PCR-OLA is simple, rapid and suitable in clinical detection of cattle-derived Cyclospora species.

Molecular characterization of Cyclospora-like organism from dairy cattle.

Cyclospora cayetanensis was identified as the cause of large outbreaks of diarrhea in many parts of the world, but its host range and reservoirs remains poorly defined. Recently, oocysts resembling the C. cayetanensis were detected in dairy cattle fecal specimens from China. The 18S rDNA from two of these Cyclospora-like oocyst specimens from dairy cattle was amplified and sequenced. Phylogenetic analysis indicated that these cattle-associated Cyclospora-like organisms are nearly identical to each other and belong to the group of primate-derived Cyclospora, which are the closest known relatives of C. cayetanensis; while these cyclosporans constitute a coherent clade within the diverse group of Eimeria species. Moreover, on the basis of our finding that ruminant- and avian-associated Eimeria species are different in MnlI sites, a new PCR-restriction fragment length polymorphism protocol with primers NesCycF and NesCycR was developed to distinguish the Cyclospora species from ruminant-associated Eimeria species.

Detection of Cyclospora cayetanensis in animal fecal isolates from Nepal using an FTA filter-base polymerase chain reaction method.

Cyclospora cayetanensis is an emerging protozoan parasite capable of causing a protracted diarrheal illness in both immunocompromised and immunocompetent individuals. Ingestion of fresh produce and water sources contaminated with mature sporulated oocysts results in acquisition of cyclosporiasis. Currently, no animal model exists for the study of this pathogenic parasite and the only confirmed reservoir host for C. cayetanensis in nature is humans. Previously, Cyclospora-like oocysts had been detected by microscopy in several animals including non-human primates. However, their phylogenetic relationship to C. cayetanensis remained uncertain due to the limited availability of molecular techniques to differentiate and speciate these isolates. In the present study, we examined a series of fecal isolates obtained from dogs, chickens, and monkeys collected between May and September 2002 from several geographic regions of Nepal. All samples were examined by microscopy and a polymerase chain reaction (PCR) for the presence of C. cayetanensis. Both microscopic and conventional PCR/restriction fragment length polymorphism (RFLP) analysis demonstrated the presence of Cyclospora sp. in the fecal samples of two dogs, one chicken, and one monkey. Application of a species-specific multiplex PCR assay confirmed the presence of both Eimeria sp. and C. cayetanensis in the positive chicken sample and only C. cayetanensis in the dog and monkey samples. However, in the absence of tissue analysis, the assignment of these animals as a natural reservoir host for C. cayetanensis remains to be determined.