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Am. J. Trop. Med. Hyg., 71(4), 2004, pp. 373-379
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

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DETECTION OF CYCLOSPORA CAYETANENSIS IN ANIMAL FECAL ISOLATES FROM NEPAL USING AN FTA FILTER-BASE POLYMERASE CHAIN REACTION METHOD

DAN-MY T. CHU, JEEVAN B. SHERCHAND, JOHN H. CROSS, AND PALMER A. ORLANDI
Division of Microbiological Studies, and Division of Virulence Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland; Tribhuvan University Teaching Hospital, Department of Microbiology-Parasitology/Infectious and Tropical Diseases Research Center, Kathmandu, Nepal; Uniformed Services University of Health Science, Bethesda, Maryland


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The growth of international travel and the rapid global distribution of fresh fruits and vegetables have increased the likelihood of consumers acquiring a foodborne parasitic illness.1 Cyclosporiasis, a protracted diarrheal illness in both immunocompetent and immunocompromised individuals, is one such example. Prior to the 1990s, the causative agent, Cyclospora cayetanensis, was generally associated with environmental conditions and hygienic practices in predominately underdeveloped countries. However, in the 1990s, C. cayetanensis and cyclosporiasis emerged in the United States and Canada as an ill-defined foodborne illness. Public awareness was heightened as epidemiologic investigations into the nature of multiple outbreaks linked illness to the importation of contaminated produce.2 Cyclospora cayetanensis has now been recognized worldwide as a foodborne and waterborne pathogen usually associated with the consumption of fresh or under-prepared produce.3–5

The genus Cyclospora was first characterized and named by Schneider in 1881.6 Since that time, Cyclosporans have been described in vipers, reptiles, myriapods, insectivores, and rodents.7–10 Ashford and others may have observed the first cases of Cyclospora infection in humans.11,12 However, Ortega and others are credited with characterizing and naming the human pathogen C. cayetanensis.13

Morphologically, oocysts of C. cayetanensis are quite distinct from other species within the Cyclospora genera. Oocyts of C. cayetanensis are 8–10 µm in size and spherical with a characteristic blue or green outer ring-fluorescence when examined under ultraviolet (UV) light, in contrast to other Cyclospora spp. isolated from rodents and insectivores, which differ significantly in size and shape. Phylogenetic analysis of the 18S ribosomal RNA (rRNA) further distinguishes C. cayetanensis from other Cyclospora spp. Complete sequence analysis at this loci suggests that C. cayetanensis is more closely related to the Eimeria.14 Reinforcing this phylogenetic association is the observation that C. cayetanensis oocysts are noninfectious when shed by an infected host but require a period of time in the environment before sporulation occurs.15,16 As such, it is unlikely that human transmission of C. cayetanensis occurs via the conventional fecal-oral route. Rather, the ingestion of fecal-contaminated water or fresh produce is most likely the vehicle of transmission for cyclosporiasis.

Unlike Eimeria spp, very little is known regarding the environmental biology of C. cayetanensis. Although C. cayetanensis oocysts have been detected worldwide and cyclosporiasis is considered to be endemic in Haiti, Peru, and Nepal,17–19 no definitive animal reservoir for C. cayetanensis in the environment has been identified; humans may be the only natural host for C. cayetanensis.18,20 Multiple attempts to identify a non-human host and to establish a laboratory animal model to provide a sufficient source of oocysts have continually failed and have therefore hampered basic research efforts.20

In the search to identify an environmental reservoir for C. cayetanensis, several studies have identified Cyclospora-like oocysts in fecal samples collected from dogs, poultry, and primates.21–25 A limited body of molecular phylogenetic information (primarily from non-human primate- derived Cyclospora isolates) is available to establish a taxonomic relationship to C. cayetanensis.25,26 However, other reports have been based solely on morphologic examination and sporulation characteristics. Due to the limited availability, reliability, and use of molecular techniques to differentiate and speciate these isolates, results have been met with skepticism and uncertainty.

In an earlier study, Sherchand and Cross demonstrated the presence of Cyclospora-like oocysts in the fecal samples of chickens, dogs, rats, and mice from Nepal through microscopic evaluation.27 In the present report, we present a follow-up to that study in which we use a filter-based polymerase chain reaction (PCR) method28 in conjunction with a species-specific multiplex primer set29 to analyze 20 additional fecal specimens collected from Nepalese dogs, chickens, and monkeys for the presence of Cyclospora spp. and Eimeria spp. Our results confirmed the presence of C. cayetanensis in the fecal samples of two dogs, one chicken, and one monkey. Whether identification of C. cayetanensis in these copraphagic animals represents either a spurious infection, the passing of ingested oocysts through the gut, or a natural infection in an animal host other than humans remains to be determined.


MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of fecal isolates. During the time period between May and September 2002, fecal samples from dogs, chickens, and monkeys were collected from selected areas of the Kathmandu (Kathmandu district), Panchkhal (Kavre district) and Trishuli (Dhardhing district) valleys of Nepal. Fecal samples were taken from both domestic and street dogs, wild chickens inhabiting the forest regions by nearby villages, and Macaca mulatta rhesus monkeys that wander through the forest region and temples. Fecal specimens were collected immediately after animal defecation and placed in a solution of 2.5% potassium dichromate. Samples were shipped to the Food and Drug Administration’s Center for Food Safety and Applied Nutrition via Dr. John H. Cross (Uniformed Services University of the Health Sciences, Bethesda, MD). Purified DNA from C. colobi was provided by A. J. da Silva and M. L. Eberhard (Centers for Disease Control and Prevention, Atlanta, GA). Eimeria acervulina was obtained from R. Fayer (U.S. Department of Agriculture, Beltsville, MD) and C. cayetanensis was obtained from Dr. John Cross.

Microscopy. Fecal specimens were examined by differential interference contrast (DIC) microscopy for the presence of oocysts measuring approximately 8–10 µm in size. Cyclospora oocysts were further identified by UV epifluorescence microscopy using an Olympus (Mellville, NY) BF-51 fluorescence microscope. Photomicrographs of isolated oocysts were taken using a Spot Diagnostic Camera and accompanying software (version 3.5) (Diagnostic Instruments, Inc., Sterling Heights, MI).

Concentration of fecal samples. Two different methods were used to concentrate oocysts in fecal samples. One method used a 10% formalin-ethyl acetate concentration in conjunction with Fecal Parasite Concentrators (Evergreen Scientific, Los Angeles, CA). These samples were used for microscopic examination. Alternatively, samples were concentrated using a discontinuous sucrose density purification method similar to that described by Ortega and others.13

Preparation of DNA templates. Stool specimens (10–20 µL) were spotted directly onto FTA filters (Whatman, Inc., Newton, MA) and allowed to dry on a heat block at 56°C. The FTA disks were processed according to the method of Orlandi and Lampel.28 Washed FTA filters were then used as DNA templates in primary PCR amplifications.

Nested PCR amplification of Cyclospora. Conventional nested PCR amplifications were performed using the primer pairs F1E-R2B and F3E-R4B with slight modification as described by Orlandi and Lampel.28 Primary amplifications were performed in a total volume of 100 µL using the Hot-StarTaq Master Mix Kit (Qiagen, Valencia, CA) and containing 2.0 mM MgCl2, and 200 µM of each dNTP, 0.2 µM of F1E (5'-TACCCAATGAAAACAGTTT-3') and R2B (5'-CAGGAGAAGCCAAGGTAGG-3'). All PCR amplifications were performed in a PTC-200 DNA Engine (MJ Research, Waltham, MA). The amplification program began with an initial activation of the HotStarTaq DNA Polymerase at 95°C for 15 minutes. The cycling program consisted of 35 cycles of denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, and primer extension at 72°C for 90 seconds. A final extension cycle at 72°C for 10 minutes was followed by soaking at 4°C. The nested PCR was performed in a total volume of 50 µL using HotStarTaq Master Mix, 2.0 mM MgCl2, 200 µM of each dNTP, and 0.2 µM each of primers F3E (5'-CCTTCCGCGCTTCGCTGCGT-3') and R4B (5'-CGTCTTCAAACCCCCTACTG-3'). One to five micro-liters of primary PCR product was used as the template for the secondary nested PCR. Cycling parameters were identical to first round amplification with the exception of the annealing temperature, which was increased to 60°C. The nested reaction generated a 294-basepair (bp) amplicon for both Cyclospora spp and Eimeria spp. For some experiments, a second amplification using primer pair F1E-R2B was carried out in a total volume of 50 µL using 0.5–5 µL of primary amplification product as a template. One to five microliters of this re-amplified product was then used as template for nested amplification with F3E-R4B primer set as described earlier. The PCR products were visualized after electrophoresis on a 1.5% agarose gel and staining with 0.2 µg/mL of ethidium bromide.

Restriction fragment length polymorphism analysis. Restriction fragment length polymorphism (RFLP) analysis was performed on the nested amplicons to differentiate Cyclospora spp. from Eimeria spp as described.30 Briefly 20 µL of the secondary amplicon, 2.5 µL of 10x reaction buffer, and 2 units of Mnl I (New England Biolabs, Beverly, MA) were combined in a 25-µL total reaction volume and incubated for two hours at 37°C. Digested products were separated by electrophoresis on a 5% Nusieve 3:1 agarose gel (Biowittaker Molecular Applications, Rockland, ME) containing 0.2 µg/mL of ethidium bromide and visualized by UV transillumination.

Species-specific multiplex PCR analysis. To distinguish the presence of primate-derived Cyclospora and Eimeria from human C. cayetanensis, multiplex PCR was performed according to the method described by Orlandi and others.29 Briefly 1–5 µL of the primary PCR product or re-amplified primary product was added to a 50-µL reaction mixture containing HotStarTaq Master Mixture, 2 mM MgCl2, 0.2 µM of the common reverse primer CRP999 (5'-CGTCTTCAAACCCCCTACTGTCG-3') and 0.2 µM each of the species-specific forward primers: CC719 (5'-GTAGCCTTCCGCGCTTCG-3'); PDCL661 (5'-CTGTCGTGGTCATCTGT.CCGC-3'), and ESSP841 (5'-GTTCTATTTTGTTGGTTTCTAGGACCA -3'). The amplification program began with an initial activation step at 95°C for 15 minutes, followed by a 25-cycle program of 94°C for 15 seconds and 66°C for 15 seconds. Primer pair CC719-CRP999 will generate a 298-bp amplicon from the 18S rRNA gene of C. cayetanensis, primer pair PDCL661-CRP999 will generate a 361-bp amplicon from the 18S rRNA gene of non-human primate-derived Cyclospora spp, and primer pair ESSP841–CRP999 will generate a 174-bp amplicon from the 18S rRNA gene of Eimeia spp. All PCR products were analyzed after electrophoresis on a 1.5% agarose gel containing 0.2 µg/mL of ethidium bromide and visualized on a UV transilluminator.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 20 fecal samples from dogs, monkeys, and chickens were collected over a period of six months in 2002 from three geographically distinct districts of Nepal. Cyclospora-like oocysts were identified in at least one sample from each group of animal feces by DIC and fluorescence microscopy. All tentatively identified Cyclospora-like oocysts where spherical in shape (8–10 µm diameter). All displayed a brilliant blue fluorescent outer ring when viewed under UV light as is consistent with and characteristic of Cyclospora oocysts. Representative micrographs of oocysts in dog, chicken, and monkey fecal samples are shown in Figure 1Go. The vast majority of oocysts detected in these fecal samples were unsporulated. However, several sporulated oocysts were detected in one chicken isolate (Figure 2Go).



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  		FIGURE 1. Microscopic images of Cyclospora-like oocysts (arrows) found in dog, chicken, and monkey fecal isolates. Oocysts were observed and photographed with 40x differential interference contrast microscopy (A, B, and C) and ultraviolet fluorescence microscopy (D, E, and F). A and B, dog feces; B and E, chicken feces; C and F, monkey feces.

 


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  		FIGURE 2. A sporulated Cyclopsora-like oocyst (arrow) from a chicken fecal isolate. A representative field was observed and photographed with 40x differential interference contrast microscopy and shown to contain a sporulated, spherical Cyclospora-like oocyst from chicken feces that measured 8–10 µm in size.

 
All fecal samples were further analyzed by a PCR. A filter-based method was used to prepare DNA templates directly from unprocessed, crude fecal samples.28 Molecular analysis using a nested, conventional PCR/RFLP protocol was then used to confirm the presence or absence of Cyclospora spp and Eimeria spp. in each specimen.30 In close agreement with the microscopic data, those fecal samples determined to contain Cyclospora-like oocysts by microscopy were also positive by the PCR (Figure 3AGo). A 294-bp amplicon arising from DNA templates prepared from representative dog, chicken, and monkey fecal isolates suggested the presence of either a Cyclospora- or Eimeria-like species and were coincident with positive control amplicons derived from Eimeria and Cyclospora DNA templates. To distinguish between Cyclospora and Eimeria spp., RFLP analysis was subsequently performed on these PCR amplicons using the restriction endonuclease Mnl I.30 As shown in Figure 3BGo, the resulting banding pattern was consistent with the presence of a Cyclospora-like species in all fecal samples found to be positive by the PCR.



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  		FIGURE 3. Molecular identification of Cyclospora-like species in chicken, dog, and monkey feces. A, Conventional nested polymerase chain reaction (PCR) amplification. Crude or partially purified fecal isolates were spotted onto FTA filters that were then used as DNA templates in subsequent PCR amplifications. A primary amplification was carried out using the F1E/R2B primer pair and 1 µL of this product was used in a nested amplification using the F3E/R4B primer pair to generate the 294-basepair (bp) amplicon shown. Nested amplicons from reference isolates include Cyclospora colobi (lane b), Eimeria acervulina (lane c), and Cyclospora cayetanensis (lane d). Lanes e, f, and g, are representative samples from dog, chicken, and monkey fecal isolates, respectively. Lanes a and h are 100-bp DNA ladders and lanes i and j are FTA filter and PCR controls, respectively. B, Restriction fragment length polymorphism (RFLP) analysis of nested PCR amplicons. Nested PCR amplicons were digested with Mnl I and analyzed by electrophoresis using 5% Nusieve 3:1 agarose containing ethidium bromide (0.2 µg/mL). Lanes a, b, and c are representative Mnl I digests from PCR amplicons of dog, chicken, and monkey fecal isolates, respectively. Lanes e, f, and g are standard Mnl I restriction digestion patterns from C. colobi, (lane e), E. acervulina (lane f), and C. cayetanensis (lane g). Lanes d and h are 25-bp DNA ladders. Asterisks denote distinct RFLP patterns for most Eimeria sp.

 
Although conventional PCR/RFLP analyses has been routinely used (Figure 3Go) to diagnose and distinguish the presence of Cyclospora and Eimeria spp, this method is unable to differentiate between C. cayetanensis and non-human primate-derived Cyclospora spp. Furthermore, this method lacks the sensitivity to distinguish a mixed population of coccidia. Microscopic analysis of the lone positive chicken fecal isolates suggested the presence of both Eimeria- and Cyclospora-like parasites. Data obtained from the PCR/RFLP analysis suggested that the chicken sample contained only Cyclospora spp. A recently described multiplex PCR protocol that takes advantage of single nucleotide polymorphisms (SNP) in the 18S rRNA genes of Eimeria spp and Cyclospora spp has been shown to rapidly distinguish between all of these parasitic pathogens.29 Fecal isolates that were found to contain Cyclospora spp and/or Eimeria spp. by microscopy were reexamined using the SNP multiplex protocol. A representative gel shown in Figure 4Go confirmed earlier microscopic analyses that both Eimeria spp and C. cayetanensis were present in the chicken fecal sample in question. The monkey fecal sample and dog samples however contained only C. cayetanensis.



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  		FIGURE 4. Analysis of animal fecal isolates by a multiplex poly-merase chain reaction (PCR) amplification using single nucleotide polymorphism (SNP) primers to identify Cyclospora-like species. Crude fecal material was spotted onto an FTA filter and prepared for the PCR. A primary amplicon was generated using the F1E/R2B primer pair by a conventional PCR and then used in subsequent nested amplifications using SNP primers. Lane a, monkey fecal isolate; lane b, chicken fecal isolate; lane c, dog fecal isolate. Lanes e, f, and g are SNP multiplex PCR amplicon standards: lane e, Cyclospora cercopitheci (360 basepairs [bp]), a non-human primate species; lane f, Cyclospora cayetanensis (300 bp); lane g, Eimeria tenella (173 bp). Lanes d and h are 100-bp DNA ladders.

 
A compilation of microscopic and PCR analyses on all fecal isolates examined can be found in Table 1Go.


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TABLE 1
Summary of positive Cyclospora samples from animal fecal specimens*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Research into the mechanism of transmission and biology of C. cayetanensis has been hampered due to the availability of oocysts needed to perform experiments. Human fecal material presently serves as the only source of oocysts. Previous efforts to establish C. cayetanensis in animal models and to propagate the oocysts in vitro have proven unsuccessful, and a true animal reservoir host for C. cayetanensis in the environment has not yet been confirmed.20 However, the appearance of Cyclospora-like oocysts resembling C. cayetanensis has been noted in several animal fecal specimens including chickens, dogs, primates, mice, rats, and a duck.12,21–26

Identification of Cyclospora-like oocysts in animal fecal specimens has been performed primarily by traditional microscopic techniques. While microscopy serves as a useful tool in identification of C. cayatenensis in environmental samples (to include food matrices), several limitations exist that hamper the detection and identification of oocysts. These include low levels of oocysts in the samples, complexity of the sample matrix, and skills and training of the microscopist.28 Furthermore, the existence of other coccidial oocysts in environmental samples similar in size and shape such as Neospora caninum in dogs and some smaller sized Eimeria sp. in chickens may lead to erroneous identification of Cyclospora sp. in animal fecal specimens to the untrained microscopist.17 The PCR methodology has proven to be a useful alternative in identification of C. cayetanensis in the absence of microscopic isolation of the organism from contaminated food products.31,32

This study sought to confirm the presence of C. cayetanensis in fecal samples obtained from Nepalese dogs, chickens, and monkeys through both traditional microscopic and recent PCR methodologies. This is an expansion of a study initiated by Sherchand and Cross27 in which these investigators conducted a longitudinal examination on cyclosporiasis among Nepalese villagers and the distribution throughout the environment as evidenced by the presence of oocysts in animal fecal isolates, environmental samples around vegetable farms, and from vegetable washings during seasonal high and low periods of disease transmission. All animal-derived fecal isolates were from free-roaming animals and were not kept by families.

Here we identified Cyclospora-like oocysts measuring approximately 8–10 µm in size in fecal samples from two dogs, one chicken, and one monkey. Sporulated oocysts were identified only in the positive chicken isolate; all positive monkey and dog samples exhibited only immature, undifferentiated oocysts. However, we did not attempt as part of our preliminary microscopic screening to sporulate those tentatively positive samples to further our morphologic assessment of these microorganisms.

Conventional nested PCR in conjunction with RFLP analysis confirmed the presence of Cyclospora sp. in each animal sample that appeared positive by microscopy. While the conventional PCR protocol has been a valuable diagnostic resource, particularly for analyzing human specimens, its limitations may present a significant problem when examining environmental samples that may be contaminated with both parasites.33 Conventional PCR-RFLP analysis cannot detect Cyclospora sp. and Eimeria sp. in the same sample or differentiate between human and non-human, primate-derived Cyclospora spp. This was readily apparent from the analysis of chicken fecal samples and illustrated the value of SNP multiplex PCR protocols such as the one used in this study.

Reports of C. cayetanensis and Cyclospora-like oocysts in the feces of domestic and feral animals in a myriad of environmental surveys have been met with some degree of skepticism because many conflicting reports can be found in the literature. Yai and others described the identification of Cyclospora-like oocysts in two Brazilian dogs laden with watery diarrhea.23 In contrast, no evidence of Cyclospora oocysts were detected when a survey of 140 stray Brazilian dogs was conducted by Carollo and others.34 The significance of these reports also seems to be marginal in the absence of any evidence for true infectivity. Our studies detected only two dog fecal samples positive for C. cayetanensis by both microscopy and PCR. All were unsporulated. While we were able to demonstrate the presence of C. cayetanensis in the dog sample by molecular methods, we can provide no evidence to suggest that these dogs were infected. Similar to studies conducted by Garcia-Lopez and others21 and Sherchand and Cross,27 our study also identified Cyclospora-like oocysts in one chicken fecal samples by microscopy. However, in our study, PCR analysis was able to confirm the presence of C. cayetanensis. Cyclospora-like oocysts were previously noted in baboons and chimpanzees by Smith and others.22 Other investigators reported similar observations in non-human primates; however further examination of these isolates suggested that these isolates may be distinct species of Cyclospora.24–26 Our studies identified the presence of unsporulated C. cayetanensis oocysts in the stool sample of one M. mulatta monkey. Unlike those species described by Eberhard and others26 that were isolated in baboons, green monkeys, and colobus monkeys, we detected no other primate-related Cyclospora species either by microscopy or SNP multiplex PCR.

The identification of C. cayetanensis oocysts in chicken feces is noteworthy. Based on the genetic relatedness of the18S rRNA gene, Relman and others predicted that certain traits might be present in Cyclospora similar to those seen in Eimeria, i.e., host-specificity.14 The genus Eimeria is known for multiparasitism of susceptible hosts.35 While chickens are known to harbor multiple Eimeria parasites at a given time, it may be possible for chickens to harbor both Eimeria and Cyclospora. Bern and others reported an association between people with ownership of chickens and an elevated risk for Cyclospora infection in Guatemala.36

Our limited survey of 20 animal fecal isolates from Nepal while definitive in its results provides some intriguing questions that still require a great deal of investigation. Whereas the finding of C. cayetanensis in animal fecal isolates from dogs, chickens, and monkeys using a combination of microscopic and PCR methodologies is conclusive, it does not unequivocally confirm that these animals were infected or can in fact serve as natural reservoir hosts. Within the animals surveyed, only one or two animals from each group actually tested positive for Cyclospora oocysts. Several possibilities exist to explain our findings. Due to the coprophagous nature of these animals, the possibility exists that the oocysts were ingested by these animals and passed through the gut undamaged. In Nepal, domestic and wild animals coexist with humans within the same living environments; thus, it is conceivable that an animal may ingest human fecal material containing C. cayetanensis and subsequently shed the same undamaged immature oocysts in their feces. Unfortunately, none of the fecal isolates examined were from animals kept by families; thus, no correlation could be made between an animal carrier and human infectivity. That a sporulated oocysted was identified in the feces of chicken may suggest such a scenario and the presence of C. cayetanensis oocysts in addition to Eimeria oocysts may be purely coincidental. Ortega and others identified the presence of both sexual and asexual stages of Cyclospora in biopsies of infected individuals, suggesting that C. cayetanensis might be able to complete its lifecycle within one host, thus not requiring an animal intermediate host.37 Since most members of the Eimeria family lead a homoxenous lifecycle,38 this may be a possibility with Cyclospora as well. An alternative possibility exists that animals may serve as transport hosts depositing and or shedding oocysts from one region to another thereby spreading infection via contamination of water sources and or the food supply. It is also conceivable that some animals may be asymptomatic carriers of C. cayetanensis helping in the spread of the disease, while they themselves remain largely unaffected. Poor sanitary conditions between chickens and humans may allow for cross-contamination. Similarly, humans have been shown to be both symptomatic and asymptomatic carriers of C. cayetanensis.39 The fecal samples collected in these studies were from both symptomatic and asymptomatic animals.

Lastly, geography, environmental conditions, the quality of living conditions, and the health of the animal population may dictate to some extent the probability of an animal acting as an unnatural transient host for C. cayetanensis. Collectively, our results obtained through molecular and microscopic examination of monkey, dog, and chicken feces in our study are in contrast to those from the extensive study conducted by Eberhard and others.17 These investigators were unable to detect C. cayetanensis oocysts in any animal fecal specimen including dogs, chickens, cattle, ducks, and pigs in Haiti. Furthermore, attempts to infect a wide range of animals including primates, dogs, and chickens with C. cayetanensis have been unsuccessful.20 It is unclear why such a discrepancy should exist between two endemic locations (Nepal and Haiti). While species closely related to humans (non-human primates) and those phylogenetically related to C. cayetanensis may not necessarily share a host-parasite relationship, certain as yet undefined conditions may provide for limited transient infections in animals other than humans and as such act as an unnatural intermediate host.

In conclusion, while this study has confirmed the presence of C. cayetanensis in fecal samples collected from animals in Nepal, the ability of these animals to serve as natural reservoir hosts remains to be determined. Much research is still required to identify and establish the role of animals as a part of the lifecycle development of C. cayetanensis. Only the examination of tissue biopsies from infected animals can provide definitive proof.

Received October 6, 2003. Accepted for publication April 14, 2004.

Authors’ addresses: Dan-My T. Chu, Division of Microbiological Studies, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD 20740. Jeevan B. Sherchand, Tribhuvan University Teaching Hospital, Department of Microbiology-Parasitology/Infectious and Tropical Diseases Research Center, Kathmandu, Nepal. John H. Cross, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Palmer A. Orlandi, Center for Food Safety and Applied Nutrition/OARSA/Division of Virulence Assessment, U.S. Food and Drug Administration, MOD 1 Research Facility, Room 3603 (HFS-025), 8301 Muirkirk Road, Laurel, MD 20708, E-mail: porlande{at}cfsan.fda.gov.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Y. R. Ortega and R. Sanchez
Update on Cyclospora cayetanensis, a Food-Borne and Waterborne Parasite
Clin. Microbiol. Rev., January 1, 2010; 23(1): 218 - 234.
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