Ziyao Zhou

First isolation of new canine parvovirus 2a from Tibetan mastiff and global analysis of the full-length VP2 gene of canine parvoviruses 2 in China.

Canine parvovirus 2 (CPV-2) was first identified in 1978, and is responsible for classic parvoviral enteritis. Despite the widespread vaccination of domestic carnivores, CPVs have remained important pathogens of domestic and wild carnivores. In this study, we isolated CPV-2 from Tibetan mastiffs and performed a global analysis of the complete VP2 gene sequences of CPV-2 strains in China. Six isolates were typed as new CPV-2a, according to key amino acid positions. On a phylogenetic tree, these six sequences formed a distinct clade. Five isolates occurred on the same branch as KF785794 from China and GQ379049 from Thailand; CPV-LS-ZA1 formed a separate subgroup with FJ435347 from China. One hundred ninety-eight sequences from various parts of China and the six sequences isolated here formed seven distinct clusters, indicating the high diversity of CPVs in China. Of 204 VP2 sequences, 183 (91.04%) encoded the mutation Ser297Ala, regardless of the antigenic type, implying that most Chinese CPV-2 strains contain the VP2 mutation Ser297Ala. However, the biological significance of this change from prototype CPV-2a/2b to new CPV-2a/2b types remains unclear. This study is the first to isolate new CPV-2a from the Tibetan mastiff. Our data show that new CPV-2a/2b variants are now circulating in China.

Emergence of Cryptosporidium hominis Monkey Genotype II and Novel Subtype Family Ik in the Squirrel Monkey (Saimiri sciureus) in China

A single Cryptosporidium isolate from a squirrel monkey with no clinical symptoms was obtained from a zoo in Ya’an city, China, and was genotyped by PCR amplification and DNA sequencing of the small-subunit ribosomal RNA (SSU rRNA), 70-kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein, and actin genes. This multilocus genetic characterization determined that the isolate was Cryptosporidium hominis, but carried 2, 10, and 6 nucleotide differences in the SSU rRNA, HSP70, and actin loci, respectively, which is comparable to the variations at these loci between C. hominis and the previously reported monkey genotype (2, 3, and 3 nucleotide differences). Phylogenetic studies, based on neighbor-joining and maximum likelihood methods, showed that the isolate identified in the current study had a distinctly discordant taxonomic status, distinct from known C. hominis and also from the monkey genotype, with respect to the three loci. Restriction fragment length polymorphisms of the SSU rRNA gene obtained from this study were similar to those of known C. hominis but clearly differentiated from the monkey genotype. Further subtyping was performed by sequence analysis of the gene encoding the 60-kDa glycoprotein (gp60). Maximum homology of only 88.3% to C. hominis subtype IdA10G4 was observed for the current isolate, and phylogenetic analysis demonstrated that this particular isolate belonged to a novel C. hominis subtype family, IkA7G4. This study is the first to report C. hominis infection in the squirrel monkey and, based on the observed genetic characteristics, confirms a new C. hominis genotype, monkey genotype II. Thus, these results provide novel insights into genotypic variation in C. hominis.

Transcriptional Regulation and Adaptation to a High-Fiber Environment in Bacillus subtilis HH2 Isolated from Feces of the Giant Panda

In the giant panda, adaptation to a high-fiber environment is a first step for the adequate functioning of intestinal bacteria, as the high cellulose content of the gut due to the pandas vegetarian appetite results in a harsh environment. As an excellent producer of several enzymes and vitamins, Bacillus subtilis imparts various advantages to animals. In our previous study, we determined that several strains of B. subtilis isolated from pandas exhibited good cellulose decomposition ability, and we hypothesized that this bacterial species can survive in and adapt well to a high-fiber environment. To evaluate this hypothesis, we employed RNA-Seq technology to analyze the differentially expressed genes of the selected strain B. subtilis HH2, which demonstrates significant cellulose hydrolysis of different carbon sources (cellulose and glucose). In addition, we used bioinformatics software and resources to analyze the functions and pathways of differentially expressed genes. Interestingly, comparison of the cellulose and glucose groups revealed that the up-regulated genes were involved in amino acid and lipid metabolism or transmembrane transport, both of which are involved in cellulose utilization. Conversely, the down-regulated genes were involved in non-essential functions for bacterial life, such as toxin and bacteriocin secretion, possibly to conserve energy for environmental adaptation. The results indicate that B. subtilis HH2 triggered a series of adaptive mechanisms at the transcriptional level, which suggests that this bacterium could act as a probiotic for pandas fed a high-fiber diet, despite the fact that cellulose is not a very suitable carbon source for this bacterial species. In this study, we present a model to understand the dynamic organization of and interactions between various functional and regulatory networks for unicellular organisms in a high-fiber environment.

First identification and multilocus genotyping of Giardia duodenalis in pet chipmunks ( Eutamias asiaticus ) in Sichuan Province, southwestern China

BackgroundGiardia duodenalis is a flagellated parasite that causes diarrhea in humans and other animals. Although G. duodenalis is found in companion animals worldwide, information regarding the prevalence and genetic characteristics of G. duodenalis in pet chipmunks in China is limited. The present study therefore aimed to investigate the prevalence and genotypes of G. duodenalis in pet chipmunks in Sichuan province, southwestern China, as well as to assess zoonotic potential of revealed assemblages.ResultsA total of 279 fecal samples were collected from pet chipmunks in seven pet shops and one breeding facility in Sichuan province, southwestern China. The prevalence of G. duodenalis was 8.6% (24/279), as determined by nested PCR detection of the beta giardin (bg) gene. Giardia duodenalis assemblages and subtypes were determined using multilocus genotyping of the bg, triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh) loci. Two assemblages were identified: potentially zoonotic assemblage A (54.2%, 13/24) and rodent-specific assemblage G (45.8%, 11/24). A total of 24, 17 and 17 sequences of the bg, gdh and tpi loci, respectively, were successfully obtained, which formed four, four and three subtypes, respectively. Moreover, four assemblage A (MLGs A1-A4) and three assemblage G (MLGs G1-G3) multilocus genotypes were identified.ConclusionsTo our knowledge, this is the first study that investigated G. duodenalis in pet chipmunks in China. Detection of assemblage A in pet chipmunks and in previous studies in humans suggests a possible role of chipmunks as a reservoir for human giardiasis in Sichuan Province, China.

Occurrence and genetic characterization of Giardia duodenalis and Cryptosporidium spp. from adult goats in Sichuan Province, China.

Giardia duodenalis and Cryptosporidium spp. are common gastrointestinal protozoa in mammals. Many studies have been conducted on the distribution of G. duodenalis and Cryptosporidium spp. genotypes in sheep and cattle. However, in China, information about molecular characterization and genetic analysis of G. duodenalis and Cryptosporidium spp. in goats is limited. In this study, 342 fecal samples from adult goats were collected from 12 farms in Sichuan Province, China. The occurrence of G. duodenalis and Cryptosporidium spp. in adult goats was 14.9% (51/342) and 4.7% (16/342), respectively. All G. duodenalis were identified as assemblage E, with two novel genotypes (assemblages E17 and E18) being detected at the beta-giardin (bg) locus. Based on three loci—beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh)—multilocus sequence typing revealed three novel multilocus genotypes (MLGs) of assemblage E (MLG-E1, E2, E3 (sc)). Small Subunit (SSU) rRNA-based PCR identified two Cryptosporidium species, namely C. xiaoi (11/16) and C. suis (5/16). This study is not only the first to report C. suis infection in adult goats in China but is also the first to use the MLG approach to identify G. duodenalis in adult goats.

Identification, Genotyping, and Pathogenicity of Trichosporon spp. Isolated from Giant Pandas

Trichosporon is the dominant genus of epidermal fungi in giant pandas and causes local and deep infections. To provide the information needed for the diagnosis and treatment of trichosporosis in giant pandas, the sequence of ITS, D1/D2, and IGS1 loci in 29 isolates of Trichosporon spp. which isolated from the body surface of giant pandas were combination to investigate interspecies identification and genotype. Morphological development was examined via slide culture. Additionally, mice were infected by skin inunction, intraperitoneal injection, and subcutaneous injection for evaluation of pathogenicity. The twenty-nine isolates of Trichosporon spp. were identified as belonging to 11 species, and Trichosporon jirovecii and T. asteroides were the commonest species. Four strains of T. laibachii and one strain of T. moniliiforme were found to be of novel genotypes, and T. jirovecii was identified to be genotype 1. T. asteroides had the same genotype which involved in disseminated trichosporosis. The morphological development processes of the Trichosporon spp. were clearly different, especially in the processes of single-spore development. Pathogenicity studies showed that 7 species damaged the liver and skin in mice, and their pathogenicity was stronger than other 4 species. T. asteroides had the strongest pathogenicity and might provoke invasive infection. The pathological characteristics of liver and skin infections caused by different Trichosporon spp. were similar. So it is necessary to identify the species of Trichosporon on the surface of giant panda. Combination of ITS, D1/D2, and IGS1 loci analysis, and morphological development process can effectively identify the genotype of Trichosporon spp.

First report and multilocus genotyping of Enterocytozoon bieneusi from Tibetan pigs in southwestern China

Enterocytozoon bieneusi is a common intestinal pathogen and a major cause of diarrhea and enteric diseases in a variety of animals. While the E. bieneusi genotype has become better-known, there are few reports on its prevalence in the Tibetan pig. This study investigated the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in the Tibetan pig in southwestern China. Tibetan pig feces (266 samples) were collected from three sites in the southwest of China. Feces were subjected to PCR amplification of the internal transcribed spacer (ITS) region. E. bieneusi was detected in 83 (31.2%) of Tibetan pigs from the three different sites, with 25.4% in Kangding, 56% in Yaan and 26.7% in Qionglai. Age group demonstrated the prevalence of E. bieneusi range from 24.4%(aged 0 to 1 years) to 44.4%(aged 1 to 2 years). Four genotypes of E. bieneusi were identified: two known genotypes EbpC (n=58), Henan-IV (n=24) and two novel genotypes, SCT01 and SCT02 (one of each). Phylogenetic analysis showed these four genotypes clustered to group 1 with zoonotic potential. Multilocus sequence typing (MLST) analysis three microsatellites (MS1, MS3, MS7) and one minisatellite (MS4) revealed 47, 48, 23 and 47 positive specimens were successfully sequenced, and identified ten, ten, five and five genotypes at four loci, respectively. This study indicates the potential danger of E. bieneusi to Tibetan pigs in southwestern China, and offers basic data for preventing and controlling infections.

Human-Pathogenic Enterocytozoon bieneusi in Captive Giant Pandas ( Ailuropoda melanoleuca ) in China

Human and animal infections of Enterocytozoon bieneusi (E. bieneusi) have consistently been reported worldwide, garnering public attention; however, the molecular epidemiology of E. bieneusi in the giant panda remains limited. We surveyed captive giant pandas in China for the presence of E. bieneusi by using PCR and sequence analysis of the ribosomal internal transcribed spacer (ITS) revealing a 34.5% positive rate, with seven known genotypes (SC02, EpbC, CHB1, SC01, D, F, and Peru 6) and five novel genotypes (SC04, SC05, SC06, SC07, and SC08) identified. We similarly analyzed water samples, and E. bieneusi was detected in two samples, with genotype SC02 identified. Phylogenetic analysis revealed that CHB1 did not cluster with any recognized group, while the remaining genotypes belonged to group 1. The predominance of zoonotic group 1 genotypes indicates a public health threat that giant pandas could spread E. bieneusi to humans. The identification of E. bieneusi in water samples suggests giant pandas could contribute to water contamination. Effective control measures are therefore needed to minimize the contamination of the water and prevent a human microsporidiosis outbreak.

First report of Giardia duodenalis and Enterocytozoon bieneusi in forest musk deer (Moschus berezovskii) in China

BackgroundGiardia duodenalis and Enterocytozoon bieneusi are widespread pathogens that can infect humans and various animal species. Thus far, there are only a few reports of G. duodenalis and E. bieneusi infections in ruminant wildlife. Thus, the objective of this study was to examine the prevalence of G. duodenalis and E. bieneusi in forest musk deer in Sichuan, China, as well as identifying their genotypes.ResultsIn total, we collected 223 faecal samples from musk deer at the Sichuan Institute of Musk Deer Breeding in Dujiangyan (n = 80) and the Maerkang Breeding Institute (n = 143). Five (2.24%) faecal samples were positive for G. duodenalis; three belonged to assemblage E, and two belonged to assemblage A based on the sequence analysis of the β-giardin (bg) gene. One sample each was found to be positive based on the glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) gene, respectively. Thirty-eight (17.04%) faecal samples were found to be E. bieneusi-positive based on the internal transcribed spacer (ITS) sequence, and only SC03 genotype was identified, which belonged to the zoonotic group 1 according to the phylogenic analysis. The infection rates were significantly different among the different geographical areas and age groups but had no apparent association with gender or clinical symptoms.ConclusionsTo our knowledge, this was the first molecular characterisation of G. duodenalis and E. bieneusi in musk deer. Identification of the zoonotic genotypes indicated a potential public health threat, and our study suggested that the forest musk deer is an important carrier of these parasites.

Cellulose-dependent expression and antibacterial characteristics of surfactin from Bacillus subtilis HH2 isolated from the giant panda

Surfactin secreted by Bacillus subtilis can confer strong, diverse antipathogenic effects, thereby benefitting the host. Carbon source is an important factor for surfactin production. However, the mechanism that bacteria utilize cellulose, the most abundant substance in the intestines of herbivores, to produce surfactin remains unclear. Here, we used B. subtilis HH2, isolated from the feces of a giant panda, as a model to determine changes in surfactin expression in the presence of different concentrations of cellulose by quantitative polymerase chain reaction and high-performance liquid chromatography. We further investigated the antimicrobial effects of surfactin against three common intestinal pathogens (Escherichia coli, Staphylococcus aureus, and Salmonella enterica) and its resistance to high temperature (60–121°C), pH (1–12), trypsin (100–300 μg/mL, pH 8), and pepsin (100–300 μg/mL, pH 2). The results showed that the surfactin expressed lowest in bacteria cultured in the presence of 1% glucose medium as the carbon source, whereas increased in an appropriate cellulose concentration (0.67% glucose and 0.33% cellulose). The surfactin could inhibit E. coli and Staphylococcus aureus, but did not affect efficiently for Salmonella enterica. The antibacterial ability of surfactin did not differ according to temperature (60–100°C), pH (2–11), trypsin (100–300 μg/mL), and pepsin (100–300 μg/mL; P > 0.05), but decreased significantly at extreme environments (121°C, pH 1 or 12; P < 0.05) compared with that in the control group (37°C, pH = 7, without any protease). In conclusion, our findings indicated that B. subtilis HH2 could increase surfactin expression in an appropriate cellulose environment and thus provide benefits to improve the intestinal health of herbivores.