Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.     

Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism

Cyclospora cayetanensis is a coccidian parasite which causes severe gastroenteritis in humans. Molecular information on this newly emerging pathogen is scarce. Our objectives were to assess genetic variation within and between human-associated C. cayetanensis and baboon-associated Cyclospora papionis by examining the internal transcribed spacer (ITS) region of the ribosomal RNA operon, and to develop an efficient polymerase chain reaction- (PCR)-based method to distinguish C. cayetanensis from other closely related organisms. For these purposes, we studied C. cayetanensis ITS-1 nucleotide variability in 24 human faecal samples from five geographic locations and C. papionis ITS-1 variability in four baboon faecal samples from Tanzania. In addition, a continuous sequence encompassing ITS-1, 5.8S rDNA and ITS-2 was determined from two C. cayetanensis samples. The results indicate that C. cayetanensis and C. papionis have distinct ITS-1 sequences, but identical 5.8S rDNA sequences. ITS-1 is highly variable within and between samples, but variability does not correlate with geographic origin of the samples. Despite this variability, conserved species-specific ITS-1 sequences were identified and a single-round, C. cayetanensis-specific PCR-based assay with a sensitivity of one to ten oocysts was developed. This consistent and remarkable diversity among Cyclospora spp. ITS-1 sequences argues for polyparasitism and simultaneous transmission of multiple strains.     

Cyclospora cayetanensis: biology, environmental distribution and transfer

Cyclospora cayetanensis is an apicomplexan protozoan that has emerged as an important pathogen causing endemic or epidemic diarrheal disease worldwide. In industrialized countries, the parasite has been recognized as the causative agent of several outbreaks of diarrheal illness mostly associated with produce imported from endemic areas. In developing countries, human cyclosporosis is widely distributed. Infection rates from 0% to 41.6% have been described in the general population. However, the epidemiology, biology, and ecology of C. cayetanensis are not fully understood. The life cycle is not completely characterized, although it appears to require a single human host to be accomplished. The role of animals as natural reservoirs of the parasite remains to be determined. Little information is available concerning the environmental distribution and vehicles of transmission of C. cayetanensis. Contaminated water, foods or soil can be vehicles of spread of the parasite. The significant uncertainties that remain in the knowledge of C. cayetanensis highlight the need for continuing research in several areas, including its basic biology and environmental distribution.     

PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.     

The pig as a host for Schistosoma mekongi in Laos.

A survey of helminths in domestic pigs was conducted in Khong District, Laos, to elucidate if these domestic animals could act as definitive hosts for Schistosoma mekongi and to obtain a general overview of their helminthological infection status. Fecal samples were collected from 98 pigs. Twelve pigs (12.2%) were found to excrete S. mekongi eggs. Infection was confirmed by detection of S. mekongi eggs in tissues of liver, rectum, and cecum of 2 pigs. A total of 75.8% of the pigs was infected with 1 or more helminth species. This study showed that pigs may act as a definitive host for S. mekongi.     

Co-infection of Giardia intestinalis and Cyclospora cayetanensis in an immunocompetent patient with prolonged diarrhea: case report

Cyclospora cayetanensis is an agent of emerging infectious disease, and a recognized cause of diarrhea in some patients. Also, the flagellated protozoan, Giardia intestinalis, induces a diarrheal illness of the small intestine. Cases of cyclosporiasis are frequently missed, primarily due to the fact that the parasite can be quite difficult to detect in human fecal samples, despite an increasing amount of data regarding this parasite. On the other hand, G. intestinalis can be readily recognized via the microscopic visualization of its trophozoite or cyst forms in stained preparations or unstained wet mounts. In this report, we describe an uncommon case of co-infection with G. intestinalis and C. cayetanensis in an immunocompetent patient with prolonged diarrhea, living in a non-tropical region of Turkey.     

Porcine reproductive and respiratory syndrome (PRRS) virus in wild boar and Iberian pigs in south-central Spain

Porcine reproductive and respiratory syndrome (PRRS) is a swine infectious disease causing major economic problems on the intensive pig industry. This virus has been reported worldwide in domestic pigs and there is evidence of PRRS virus (PRRSV) infection in wild boar (Sus scrofa). Nonetheless, the epidemiological role of wild boar and extensively kept domestic pigs remains unclear. The aim of this study was to determine the occurrence of PRRS in wild boar and Iberian pigs in the dehesa ecosystem of the Castile-La Mancha region of Spain, which boasts one of the most important free-roaming porcine livestock and hunting industries in the country. Using geo-spatial analysis of literature data, we first explored the relationship between domestic pig density and PRRS occurrence in wild boar in Europe. Results revealed that PRRS occurrence in wild boar may be influenced, albeit not significantly, by domestic pig density. Next, we analyzed sera from 294 wild boar and 80 Iberian pigs by indirect enzyme-linked immunosorbent assay for PRRSV antibodies. The sera and 27 wild boar tissue samples were analyzed by two real-time RT-PCR assays, targeting the most conserved genes of the PRRSV genome, ORF1 and ORF7. Seven wild boar (2.4 %) and one Iberian pig (1.3 %) were seropositive, while none of the animals tested positive for PRRSV by RT-PCR. Our results confirm the limited spread of PRRSV in free-roaming Iberian pigs and wild boar living in mutual contact. Further studies would be necessary to address whether this low seroprevalence found in these animals reflects transmission from intensively kept pigs or the independent circulation of specific strains in free-roaming pigs.     

Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.     

Genetic characterization of Trypanosoma brucei circulating in domestic animals of the Fontem sleeping sickness of Cameroon

To improve our knowledge on the transmission dynamics of trypanosomes, Trypanosoma brucei was identified in domestic animals of the Fontem sleeping sickness focus of Cameroon and their genetic characterizations were performed using seven polymorphic microsatellite markers. About 397 domestic animals including 225 pigs, 87 goats, 65 sheep and 20 dogs were sampled. The card agglutination test for trypanosomiasis was positive for 254 (63.98%) animals while the parasitological examinations (thin blood film and capillary tube centrifugation) revealed 86 (21.66%) trypanosome infections. The PCR based method revealed 140 (35.26%) infections of trypanosomes of the subgenus Trypanozoon. The genetic characterization of these 140 positive samples revealed 89 different alleles: 82 in pigs, 72 in goat, 60 in sheep and 48 in dog. Whatever the microsatellite marker used, most of positive samples were amplified. However, the sensitivity (percentage of samples amplified for each marker) of these markers varies significantly between them (χ(2) = 120.32; P < 0.0001). This study showed a high level (80.00%) of mixed genotypes as well as a wide range of T. brucei genotypes circulating in domestic animals of the Fontem sleeping sickness focus of Cameroon. This indicates that several T. brucei genotypes can naturally be transmitted simultaneously to tsetse flies during a single blood meal.     

Comparison of three microscopic techniques for diagnosis of Cyclospora cayetanensis

Cyclospora cayetanensis oocysts in the feces of humans from Kathmandu, Nepal were identified on the basis of their size and other morphological characteristics. We compared the detection of C. cayetanensis oocysts in the feces using three microscopic techniques such as formalin-ether sedimentation, sucrose centrifugal floatation, and direct smear. Standard procedures were used for the formalin-ether sedimentation and the sucrose centrifugal floatation techniques using 0.5 g of feces, however, the direct smear technique was performed using 10 microl of fecal suspension (0.005 g of feces) and observed under the fluorescent microscope. Of the 403 samples examined, 21 samples were positive for oocysts by all three techniques. Therefore, in these 21 samples, the number of oocysts recovered by the three techniques were compared. The highest number of oocyst was obtained by the sucrose centrifugal floatation technique. In contrast, the formalin-ether sedimentation technique was found to be the least reliable concentration technique for the detection of Cyclospora in human feces. Surprisingly, the direct smear technique was found to be an effective and rapid technique for diagnosis of C. cayetanensis making it a technique of choice for routine epidemiological investigation of the prevalence of this infection in human populations.     

Intragenomic sequence variation of the ITS-1 region within a single flow-cytometry-counted Cyclospora cayetanensis oocysts

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS-1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.     

Detection of Antibodies Against Paracoccidioides brasiliensis in Free-Range Domestic Pigs (Sus scrofa)

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.     

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.     

Molecular identification and prevalence of Isospora sp. in pigs in Western Australia using a PCR-RFLP assay

Little is known about the prevalence of Isospora in domestic pigs in Western Australia. A total of 289 pig faecal samples were collected from pre- and post-weaned pigs and sows from 1 indoor and 3 outdoor piggeries located in the south-west region of Western Australia. Faecal samples were screened using a PCR-RFLP assay based on the ITS-1 rDNA locus. An overall prevalence of 10.4% (30/289) was identified. Isospora was detected in 16.3% (20/123) of pre-weaned animals and 6.4% (10/156) of post-weaned animals. PCR-RFLP analysis confirmed the presence of Isospora suis in 86.7% of the positive Isospora isolates. Isospora was significantly associated with diarrhea and the findings of this study suggest that management factors such as cleaning practices, flooring types and stocking densities need to be investigated in the porcine host to find new and improved measures for control.     

Chromosome R-banding patterns and NOR homologies in the European wild pig and four breeds of domestic pig

Four European wild pigs and 27 domestic pigs were studied; three Landrace, 12 village pigs from Papua New Guinea, two Chinese pigs Meishan and 10 Creole pigs from the French Antilles. The R-banding patterns were identical for all domestic breeds despite their different history and geographical divergence. The European wild pigs showed a similar R-banding pattern and a centric fusion between pairs 15 and 17 (2n = 36). The nucleolar organizers (NORs) in the European wild pig and the four domestic breeds were localized on the secondary constriction of chromosomes 8 and 10. All animals exhibited in the majority of metaphases two NORs on both chromosomes 10. In some animals. the NORs were expressed only in one of the homologs of chromosome 8. The Chinese pigs had a high amount of silver precipitates on two homologs of chromosome 8. This study confirms several previous reports on the polymorphism of NOR patterns in different domestic pig breeds.     

Targeting single-nucleotide polymorphisms in the 18S rRNA gene to differentiate Cyclospora species from Eimeria species by multiplex PCR

Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans. C. cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named. The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis. Since the amplicons generated from C. cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species. The current PCR-RFLP protocol, however, cannot distinguish between C. cayetanensis and these new isolates. The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources. Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene. The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer. Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C. cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species. As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method. In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens.     

Serosurveillance for selected infectious disease agents in wild boars (Sus scrofa) and outdoor pigs in Switzerland

The large abundance of free-ranging wild boars (Sus scrofa) and a trend towards animal friendly outdoor management of domestic pigs lead to an increasing probability of disease transmission between those animal populations. In 2001, an active monitoring was started for classical swine fever (CSF), Aujeszky’s disease (AD) and porcine brucellosis (PB) in wild boars in Switzerland. The objective of this programme was to document the serological status of wild boars regarding the selected pathogens. To continue this serosurveillance, 1,060 wild boar samples were collected during two regular hunting seasons in 2004–2005. Furthermore, in a pilot study, 61 outdoor pigs from 14 farms located in areas with high wild boar densities were sampled in 2004 and serologically tested for AD and PB. All wild boar samples were negative for CSF. Seroprevalence for AD was 2.83% (95% CI 1.91–4.02%). Seroprevalence for PB was 13.5% (95% CI 10.7–16.7%) for the Rose Bengal test and 11.05% (95% CI 8.82–13.61%) for the indirect ELISA. There was no serological evidence for AD in domestic pigs. All tested animals from 13 piggeries were seronegative for PB, but three pigs from the same farm showed doubtful results. Further investigations on the farm did not indicate the presence of PB in the herd. These findings urge the need for better diagnostic tools to obtain reliable results concerning PB prevalence. Since contact and following transmission of infectious agents between infected wild boars and outdoor pigs might occur in the future, it is advisable to include outdoor pigs in areas at risk in routine surveillance programmes.     

Potential use of transgenic domestic pigs expressing recombinant human erythropoietin in diabetes translation research

Recently, diabetes mellitus (DM) has shown rapid global increases with about five million deaths annually. Animal models are imperative to understand disease mechanisms and develop diagnostic, preventive, and therapeutic interventions in translational research. Rodent and mini-pig models have been established and widely used for DM research. However, domestic pig models are limited in spite of advantages such as pharmacokinetic and physiopathological availability. This study examines the potential use of domestic pigs expressing recombinant human erythropoietin (rhEPO) as disease and therapeutic response models for DM. We previously generated transgenic pigs (n?=?16, EPO Tg) in which rhEPO was expressed and circulated in all organs. Thirty-two pigs, including 16 controls, were fed high fat (HF) diets for 42 weeks. Subsequently, blood samples for chemical and metabolic analysis were collected after fasting for 24?h and glucose loading for oral glucose tolerance tests (OGTTs). We found increased activation of the PI3?K/Akt signaling pathway under hypoxic conditions after rhEPO treatment, and HF diet-inducible-obesity in the EPO Tg and control pigs. OGTTs showed lower fasting glucose levels in the EPO Tg pigs than in controls before and after the HF diet, suggesting that rhEPO may affect glucose concentrations. Insulin and C-peptide concentrations responded slowly to glucose administration and returned to initial levels after 2?h. The blood test results suggest that EPO might affect metabolic and chemical components such as glucose, high-density lipoprotein, glucagon, triglyceride, and free fatty acid. Our findings support the use of rhEPO transgenic domestic pigs as model animals for translational DM research.     

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation

Aims:  To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters.
Methods and Results:  Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l⁻¹ into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were ≥84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation.
Conclusion:  Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation.
Significance and Impact of the Study:  Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.     

Prevalence of Cryptosporidium genotypes in pre and post-weaned pigs in Australia

A total of 289 pig faecal samples were collected from pre-weaned and post-weaned piglets and sows from 1 indoor and 3 outdoor piggeries located in the south-west region of Western Australia and screened at the 18S rRNA locus for the presence of Cryptosporidium. An overall prevalence of 22.1% (64/289) was identified. Cryptosporidium was more prevalent in post-weaned animals (p < 0.05); 32.7% (51/156) versus 10.6% (13/123) for pre-weaned animals. Sequence analysis identified Cryptosporidium suis in all pre-weaned isolates genotyped (7/13). In post-weaned pigs that were genotyped (n = 38), the non-zoonotic Cryptosporidium species, pig genotype II was identified in 32 samples and C. suis in 6 samples. The zoonotic species Cryptosporidium parvum was not detected, suggesting that domestic pigs do not pose a significant public health risk. Pig genotype II was significantly (p < 0.05) associated with ‘normal’ stools, indicating an asymptomatic nature in the porcine host.