Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains.

Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.

First report of zoonotic Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi in golden takins (Budorcas taxicolor bedfordi).

Genetic study of Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi at species/assemblage/genotype/subtype level facilitates understanding their mechanical transmissions and underpins their control. A total of 191 fresh faecal samples were collected from golden takins in China and examined using multilocus sequence typing (MLST). Cryptosporidium spp. was detected in 15 faecal samples (7.9%), including Cryptosporidium parvum (2/15) and Cryptosporidium andersoni (13/15). MLST tool identified C. andersoni subtypes (A1, A4, A4, A1) and (A4, A4, A4, A1), and C. parvum gp60 gene subtype IId A19G1. The prevalence of G. intestinalis infection was 8.9% (17/191) and assemblage analysis identified 14 assemblage E and three assemblage B. Intra-variations were observed at triose phosphate isomerase (tpi), beta giardin (bg) and glutamate dehydrogenase (gdh) loci within the assemblage E, showing seven, three and three new subtypes in respective locus. Ten and one multilocus genotypes (MLGs) were present in assemblages E and B, respectively. E. bieneusi infection was positive in 14.7% (28/191) of the examined specimens, with three genotypes known (BEB6, D and I) and four novel internal transcribed spacer (ITS) genotypes (TEB1-TEB4). The present study revealed, for the first time, the presence of zoonotic C. parvum IId A19G1, G. intestinalis assemblage B and E. bieneusi genotype D and four novel genotypes in golden takins in China. These findings expand the host range of three zoonotic pathogens and have important implications for controlling cryptosporidiosis, giardiasis and microsporidiosis in humans and animals.

First report of Enterocytozoon bieneusi from giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China.

Enterocytozoon bieneusi is an emerging and opportunistic enteric pathogen triggering diarrhea and enteric disease in humans and animals. Despite extensive research on this pathogen, the prevalence and genotypes of E. bieneusi infection in precious wild animals of giant and red pandas have not been reported. In the present study, 82 faecal specimens were collected from 46 giant pandas (Ailuropoda melanoleuca) and 36 red pandas (Ailurus fulgens) in the northwest of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an overall infection rate of 10.98% (9/82) was observed in pandas, with 8.70% (4/46) for giant pandas, and 13.89% (5/36) for red pandas. Two ITS genotypes were identified: the novel genotype I-like (n=4) and genotype EbpC (n=5). Multilocus sequence typing (MLST) employing three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) showed that nine, six, six and nine positive products were amplified and sequenced successfully at four respective loci. A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that the genotype EbpC fell into 1d of group 1 of zoonotic potential, and the novel genotype I-like was clustered into group 2. The present study firstly indicated the presence of E. bieneusi in giant and red pandas, and these results suggested that integrated strategies should be implemented to effectively protect pandas and humans from infecting E. bieneusi in China.

The expression dynamics of IL-17 and Th17 response relative cytokines in the trachea and spleen of chickens after infection with Cryptosporidium baileyi.

BACKGROUND: Cryptosporidium baileyi is the dominant Cryptosporidium species in birds causing emerging health problems in the poultry industry, and is also a model to study the biology of Cryptosporidium spp.. IL-17 (also called IL-17A) is a hallmark pro-inflammatory cytokine of Th17 cells that plays an important role in several human autoimmune diseases and microbial infection disease of many animals, and it may play a role in Cryptosporidium infection. METHODS: The present study examined the mRNA level of IL-17 and Th17 response relative cytokines in the trachea and spleen of C. baileyi-infected chickens at different time points using real-time quantitative PCR (qPCR). RESULTS: All examined cytokines in the trachea were up-regulated in the infected chickens compared with the uninfected control during C. baileyi infection. Significant increased IL-17 mRNA level in the trachea was observed as early as 12 h post infection (pi), peaking at 24 h pi and 10 d pi, and declining thereafter. The transcription levels of IL-17 and Th17 response relative cytokines in spleen were also significantly increased at different time points during the infection. CONCLUSIONS: IL-17 was indicated to participate in the induction of inflammation during infection of some intracellular protozoan parasites. The results in the present study suggest that IL-17 may play a role in immunity against Cryptosporidium infection, and provide basic information for determining the role of Th17 cell in Cryptosporidium infection.

Genetic variability in the three mitochondrial genes among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan Provinces, China.

The present study examined sequence variations in three mitochondrial DNA (mtDNA) regions, namely, NADH dehydrogenase subunit 5 (nad5), adenosine triphosphate subunit 6 (atp6) and cytochrome c oxidase subunit 3 (cox3), among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan provinces, China. The lengths for partial sequences of nad5 (pnad5), atp6 (patp6) and cox3 (pcox3) were 427 bp, 381 bp and 337 bp, respectively. The intra-specific sequence variations among all O. asperum samples were 0-2.11%, 0-1.84% and 0-1.48% for pnad5, patp6 and pcox3, respectively, while the inter-specific sequence differences among Oesophagostomum species in pig and small ruminants were 18-21.3% for pnad5, 18.3-24.5% for patp6 and 10.6-13.7% for pcox3. A phylogenetic analysis based on combined sequences of three mtDNA fragments indicated that all O. asperum isolates were grouped in one solid clade, and the Oesophagostomum spp. from pig were located in another clade. However, these mtDNA fragments could not reveal genetic relationships of geographical isolates of O. asperum in China. These results provided valuable information for studying population genetics of Oesophagostomum spp., and for controlling Oesophagostomum infection in animals as well as humans.

Characterization of Haemaphysalis flava (Acari: Ixodidae) from Qingling subspecies of giant panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by morphology and molecular markers.

Tick is one of important ectoparasites capable of causing direct damage to their hosts and also acts as vectors of relevant infectious agents. In the present study, the taxa of 10 ticks, collected from Qinling giant pandas (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains of China in April 2010, were determined using morphology and molecular markers (nucleotide ITS2 rDNA and mitochondrial 16S). Microscopic observation demonstrated that the morphological features of these ticks were similar to Haemaphysalis flava. Compared with other Haemaphysalis species, genetic variations between Haemaphysalis collected from A. m. qinlingensis and H. flava were the lowest in ITS2 rDNA and mitochondrial 16S, with sequence differences of 2.06%-2.40% and 1.30%-4.70%, respectively. Phylogenetic relationships showed that all the Haemaphysalis collected from A. m. qinlingensis were grouped with H. flava, further confirmed that the Haemaphysalis sp. is H. flava. This is the first report of ticks in giant panda by combining with morphology and molecular markers. This study also provided evidence that combining morphology and molecular tools provide a valuable and efficient tool for tick identification.

The complete mitochondrial genomes of Oesophagostomum asperum and Oesophagostomum columbianum in small ruminants.

Oesophagostomum spp., commonly known as 'nodule worms', is one of the important emerging nematode zoonoses worldwide. In the present study, we sequenced the complete mitochondrial (mt) genomes of two small ruminant nodule worms, Oesophagostomum asperum and Oesophagostomum columbianum, and compared them with that of pig nodule worms (Oesophagostomum dentatum and Oesophagostomum quadrispinulatum). The complete mt genomes of O. asperum andO. columbianum were 13,672 and 13,561 bp, respectively. Both mt genomes were circular, and consisted of 36 genes, including 12 genes coding for proteins, 2 genes for rRNA, and 22 genes for tRNA. The gene content and arrangement are identical to that of pig nodule worms. The availability of full mtDNA sequences of O. asperum and O. columbianum provide useful information for studying population genetics of Oesophagostomum spp., molecular epidemiology and control of O. asperum and O. columbianum infection in small ruminants.

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences.

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.