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Abstract Background Trichosporon is the dominant genus of epidermal fungi in giant pandas (Ailuropoda melanoleuca) 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 were 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. Results The twenty-nine isolates of Trichosporon spp. were identified as 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. Conclusions Multiple species of Trichosporon were identified on the skin surface of giant panda, which varied in morphological development and pathogenicity. Combination of ITS, D1/D2, and IGS1 loci analysis, and morphological development process can effectively identify the genotype of Trichosporon spp.

Plant-based natural products represent an alternative to chemical compounds for the control of mites in veterinary medicine. Here, the essential oil of Elsholtzia densa (E. densa) Benth was extracted using hydrodistillation at a rate of 1.2%. The chemical composition of the essential oil was determined by gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS analysis indicated that the principal compounds in the volatile oil of the sample were 4-Pyridinol (28.16%) and thymol (26.58%). The acaricidal activity of E. densa oil against Sarcoptes scabiei (S. scabiei) was tested in vitro. Toxicity test data were analysed using a complementary log-log (CLL) model. The E. densa oil was prepared in five concentrations by dilution with liquid paraffin (1, 2, 4, 8 and 16 mg/ml) and exhibited strong toxicity against S. scabiei with LT50 values of 16.637, 5.075, 2.884, 1.184 and 0.760 h, respectively. The LC50 values were 7.678, 4.623, 2.543, 1.502, 1.298 and 0.981 mg/ml for S. scabiei at 1, 2, 4, 8, 16 and 24 h, respectively. Compared to the control, the essential oil showed significant effects against S. scabiei in vitro. At 16 mg/ml, E. densa oil was found to kill all mites within a 16-h period. The results indicate that E. densa oil possesses potential acaricidal activity in vitro and may be exploited as a novel drug for the effective control of S. scabiei.

The giant panda is one of the rarest animals in the world. Skin diseases seriously endanger the health of giant panda and are considered the second major cause of its morbidity. Skin microbiota is a complex ecosystem, and the community structure and the pathogenic potential of bacteria on giant panda skin remain largely unclear. In order to understand the skin bacterial flora of captive giant pandas, the microbiota in giant panda skin samples collected during different seasons was profiled via 16S rRNA gene sequencing. In total, 522 genera from 53 bacterial phyla were detected, with Proteobacteria (40.5%), Actinobacteria (23.1%), Firmicutes (21.1%), Bacteroidetes (9.5%), Cyanobacteria (2.1%), and Thermi (1.2%) as the predominant phyla and Streptococcus (13.9%), Acinetobacter (9.2%), Staphylococcus (2.9%), Pseudomonas (5.9%), Dermacoccus (4.8%), Brachybacterium (2.9%), Escherichia (2.7%), Chryseobacterium (2.1%), Arthrobacter (1.6%), Kocuria (1.5%), Psychrobacter (1.2%), Deinococcus (1.1%), and Flavobacterium (1.1%) as the predominant genera. The results indicated that the diversity was lower in winter than in other seasons and higher in autumn than in other seasons, and the abundance in spring was significantly higher than that in other seasons. Several skin disease-associated bacteria were detected as opportunists in the skin microbiota of healthy giant pandas. In this study, the results indicated that the high diversity and abundance of the skin bacteria may have enhanced the occurrence of skin disease in autumn and spring and that skin disease-associated bacteria are the normal components of the skin microbiota.

It is widely accepted that maintenance of microbial diversity is essential for the health of the respiratory tract; however, there are limited reports on the correlation between starvation and respiratory tract microbial diversity. In the present study, saline/β-hydroxybutyric acid (BHBA) intravenous injection after dietary restriction was used to imitate different degrees of starvation. A total of 13 healthy male yaks were imposed to different dietary restrictions and intravenous injections, and their nasopharyngeal microbiota profiles were obtained by metagenomic shotgun sequencing. In healthy yaks, the main dominant phyla were Proteobacteria (33.0%), Firmicutes (22.6%), Bacteroidetes (17.2%), and Actinobacteria (13.2%); the most dominated species was Clostridium botulinum (10.8%). It was found that 9 days of dietary restriction and 2 days of BHBA injection (imitating severe starvation) significantly decreased the microbial diversity and disturbed its structure and functional composition, which increased the risk of respiratory diseases. This study also implied that oral bacteria played an important role in maintaining nasopharynx microbial homeostasis. In this study, the correlation between starvation and nasopharynx microbial diversity and its potential mechanism was investigated for the first time, providing new ideas for the prevention of respiratory diseases.

Inflammatory bowel disease (IBD) is known to cause inflammation, as well as intestinal flora and epithelial barrier dysfunction. We evaluated the function of Bacillus subtilis HH2 in a trinitrobenzenesulfonic acid (TNBS)-induced colitis rabbit model through the oral administration of this bacterium. Body weight, disease activity index (DAI) score, and histological changes in the rabbits were recorded. Colons were collected for ELISA, RT-PCR, and western blotting. Intestinal microflora were tested by 16S rRNA sequencing. Results showed that B. subtilis HH2 alleviated body weight loss and decreased the DAI and histological injury scores and also reduced the mRNA expression of TNF-α and IL-1β and enhanced the expression of IL-10. In addition, B. subtilis HH2 promoted the expression of tight junction proteins, such as occludin, and the expression of related mRNA to maintain the intestinal mucosal barrier function to attenuate colitis symptoms in rabbits. By studying colonic microbiota, we observed that B. subtilis HH2 increased colonic microbial diversity after it was reduced due to TNBS administration; it also increased the relative abundance of the Bifidobacteriaceae family, but the abundances of Bacteroidetes and Proteobacteria were reduced. These results indicated that B. subtilis HH2 could decrease the severity of colitis induced by TNBS by regulating inflammatory cytokines, increasing tight junction proteins, and modulating intestinal microbiota. These findings suggest that the probiotic B. subtilis HH2 might prevent tissue damage due to colitis.

Mitochondria are highly dynamic organelles that maintain the dynamic balance of split-fusion via kinetic proteins. This maintains the stability of their morphological functions. This dynamic balance is highly susceptible to various stress environments, including viral infection. After viral infection, the dynamic balance of the host cell mitochondria is disturbed, affecting the processes of energy generation, metabolism, and innate immunity. This creates an intracellular environment that is conducive to viral proliferation and begins the process of its own infection and causes further damage to the body. Herein, we discuss the mechanism of the virus-induced mitochondrial dynamics imbalance and its subsequent effects on the body, which will help to improve our understanding of the relationship between mitochondrial dynamics and viral infection and its importance.

Klebsiella pneumoniae is not only a worldwide human pathogen, it also effects wildlife, such as the giant panda (Ailuropoda melanoleuca), in which it has recently been evidenced to result in diarrhea, organ failure, and even death. A K. pneumoniae investigation was carried out at the Chengdu Research Base of Giant Panda Breeding in 2018. As part of the investigation, the pulsed-field gel electrophoresis (PFGE) typing, multilocus-sequence typing (MLST), antibiotic resistance profiles (ARPs), and antibiotic resistance genes (ARGs) were studied based on all isolates. Fecal samples were collected from 72 A. melanoleuca from May to December 2018, and a total of 90 K. pneumoniae were isolated from 153 fecal samples. The genotyping results showed that the isolates had high diversity, of which 84 clusters were obtained by PFGE and 57 STs by MLST. The overall trend of the similarity of isolates was the first sample period > second sample period > third sample period, which showed the increasement of genome variability of K. pneumoniae. In addition, 90 isolates showed high resistance to ampicillin, rifampicin, and compound sulfamethoxazole. Of the obtained isolates, 50% carried 6~8 ARPs, and the carrying volume increased during three sample periods, in which we found two isolates carrying 12 and 13 ARPs during the third sample period, respectively. Moreover, a total of 65 ARGs were detected (90.28%, 65/72) in 90 K. pneumoniae samples. Almost all bacteria sampled contained 17 ARGs that belonged to the β-lactamase, Multidrug, MGEs, Aminoglycoside, and Tetracycline, which may be the basis of ARPs of K. pneumoniae. Moreover, the types of Multidrug and MGEs had a greater impact on antibiotic susceptivity of K. pneumoniae. Our results showed that K. pneumoniae has a serious risk of transmission in A. melanoleuca and K. pneumoniae had a high possibility of genome diversity and the risk of drugs tolerance under the large antibiotic usage.

Abstract Background Enterocytozoon bieneusi, the most commonly identified microsporidian species in humans, is also identified in livestock, birds, rodents, reptiles, companion animals, even wastewater. However, there is no information available on occurrence of E. bieneusi in pet chipmunks. The aim of the present study was to determine the genotypes, molecular characterization of E. bieneusi in pet chipmunks, and assess the zoonotic potential. Results A total of 279 fecal specimens were collected from chipmunks from seven pet shops and one breeding facility in Sichuan province, China. The prevalence for E. bieneusi was 17.6% (49/279) based on nested PCR targeting the internal transcribed spacer (ITS) region. The prevalence of E. bieneusi in chipmunks

Abstract Background Giardia 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. Results In 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. Conclusions To 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.

Abstract Background Giardia 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. Results A 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. Conclusions To 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.

Canine brucellosis, a worldwide zoonotic disease, is mainly caused by Brucella canis. In the present study, we isolated a Brucella strain (CD3) from a subclinically infected pet dog in Sichuan Province, Southwestern China. Classical biotyping methods and molecular biological tests (BCSP31 and BcSS PCR) proved that the strain belonged to B. canis. Furthermore, B. canis CD3 and another two B. canis strains (WJ5 and YA4), which were all isolated from pet dogs in Sichuan, were genotyped using multilocus sequence typing (MLST). Our results showed that the three B. canis strains were identified as the same sequence type (ST21). The present study is the first to report B. canis strain from a subclinically infected pet dog in China, indicating a potential threat to public health posed by subclinical infections in pet dogs. We suggest that screening for B. canis should be incorporated into routine medical examination of pet dogs and other companion animals in areas with a history of animal or human brucellosis.

Abstract Background Most studies on Enterocytozoon bieneusi are conducted based on the internal transcribed spacer (ITS) region of the rRNA gene, whereas some have examined E. bieneusi population structures. Currently, the population genetics of this pathogen in giant panda remains unknown. The objective of this study was to determine the E. bieneusi population in captive giant pandas in China. Results We examined 69 E. bieneusi-positive specimens from captive giant pandas in China using five loci (ITS, MS1, MS3, MS4 and MS7) to infer E. bieneusi population genetics. For multilocus genotype (MLG) analysis of E. bieneusi-positive isolates, the MS1, MS3, MS4, and MS7 microsatellite and minisatellite loci were amplified and sequenced in 48, 45, 50 and 47 specimens, respectively, generating ten, eight, nine and five types. We successfully amplified 36 specimens and sequenced all five loci, forming 24 MLGs. Multilocus sequence analysis revealed a strong and significant linkage disequilibrium (LD), indicating a clonal population. This result was further supported by measurements of pairwise intergenic LD and a standardized index of association (I S A) from allelic profile data. The analysis in STRUCTURE suggested three subpopulations in E. bieneusi, further confirmed using right’s fixation index (F ST). Subpopulations 1 and 2 exhibited an epidemic structure, whereas subpopulation 3 had a clonal structure. Conclusions Our results describe E. bieneusi population genetics in giant pandas for the first time, improving the current understanding E. bieneusi epidemiology in the studied region. These data also benefit future studies exploring potential transmission risks from pandas to other animals, including humans.

Giardia duodenalis is a common enteric protozoan that infects a range of hosts including humans and other mammals. Multilocus genotyping of G. duodenalis in captive non-human primates (NHPs) from zoos in China is limited. In this study, we evaluated 302 NHP fecal samples collected from 32 different NHP species. The primates were from 12 zoos distributed across eight provinces and two municipalities (Chongqing and Beijing) of China. The overall infection rate was 8.3% (25/302). The six G. duodenalis-positive zoos and their infection rates were: Suzhou Zoo (40.0%, 4/10), Yangzhou Zoo (22.2%, 2/9), Dalian Zoo (16.7%, 4/24), Chengdu Zoo (12.8%, 6/47), Guiyang Forest Wildlife Zoo (12.1%, 7/58), and Changsha Zoo (4.7%, 2/43). Molecular analysis of three loci, beta-giardin (bg), triose phosphate isomerase (tpi), and glutamate dehydrogenase (gdh), showed high genetic heterogeneity, and seven novel subtypes (BIII-1, MB10-1, WB8-1, B14-1, MB9-1, DN7-1, and BIV-1) were detected within assemblage B. Additional analysis revealed 12 different assemblage B multilocus genotypes (MLGs), one known MLG and 11 novel MLGs. Based on phylogenetic analysis, 12 assemblage B MLGs formed two main clades, MLG-SW (10-12, 18) and MLG-SW (13, 14, 16, 17), the other four MLG-SW (15, 19, 20, 21) were scattered throughout the phylogenetic tree in this study. Using multilocus genotyping, this study expands our understanding of the occurrence of Giardia infection and genetic variation in Giardia in captive non-human primates from zoos in China.

Abstract Background Enterocytozoon bieneusi is one of the most prevalent causative species of diarrhea and enteric diseases in various hosts. E. bieneusi has been identified in humans, mammals, birds, rodents and reptiles in China, but few studies have reported E. bieneusi in horses. Therefore, the present study was conducted to assess the prevalence, molecular characteristics and zoonotic potential of E. bieneusi among horses in southwestern China. Findings Three hundred and thirty-three fecal specimens were collected from horses on five farms in the Sichuan and Yunnan provinces of southwestern China. The prevalence of E. bieneusi was 22.5 % (75/333), as determined by nested polymerase chain reaction and sequencing analysis of the internal transcribed spacer region of the ribosomal RNA gene of E. bieneusi. Altogether, 10 genotypes were identified among the 75 E. bieneusi-positive samples: four of these genotypes were known (horse1, horse2, SC02 and D) and six were novel (SCH1-4 and YNH1-2). Multilocus sequence typing using three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) revealed three, two, three and three genotypes at these four loci, respectively. In phylogenetic analysis, all the genotypes of E. bieneusi obtained in this study were clustered into three distinct groups: D, SC02 and SCH1-3 were clustered into group 1 (zoonotic potential); SCH4 was clustered into group 2 (cattle-hosted); whereas horse2, YNH1 and YNH2 were clustered into group 6 (unclear zoonotic potential). Conclusions This is the first report of E. bieneusi among horses in southwestern China. This is also the first multilocus genotyping analysis using microsatellite and minisatellite markers of E. bieneusi in horses. The presence of genotype D, which was previously identified in humans, and genotypes SC02 and SCH1-3, which belong to potential zoonotic group 1, these results indicate that horses are a potential source of human E. bieneusi infections in China.

Giardia duodenalis is a zoonotic parasitic protist and poses a threat to human and animal health. This study investigated the occurrence of G. duodenalis infection in post-weaned calves from Sichuan province, China. Faecal samples were collected from a total of 306 post-weaned calves (3-12 months old) from 10 farms, including 4 intensive feeding farms and 6 free-ranging farms. The overall infection rate of G. duodenalis was 41.2% (126/306) based on the PCR results at any of the three genetic loci: beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia duodenalis assemblages E (n = 115, 91.3%), A (n = 3, 2.4%), and A mixed with E (n = 8, 6.3%) were identified among the 126 positive specimens. Multilocus sequence typing of G. duodenalis revealed 34 assemblage E multilocus genotypes (MLGs), 1 assemblage A MLG and 7 mixed assemblage (A and E) MLGs. The eBURST data showed a high degree of genetic diversity within assemblage E MLGs. The phylogenetic tree revealed that MLG E3 was the primary MLG subtype in Sichuan province and also the most widely distributed in China.

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.

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.

[This corrects the article DOI: 10.1371/journal.pone.0184913.].

Giardia intestinalis, a cosmopolitan zoonotic parasite, is one of the most common causes of protozoal diarrhea in both humans and animals worldwide. Although G. intestinalis has been detected in many animals, information regarding its prevalence and genotype in Chinese racehorses is scarce. In the present study, we investigated the prevalence of G. intestinalis in racehorses and performed molecular characterization of the pathogen to assess its zoonotic potential. Two hundred and sixty-four racehorse fecal samples from six equestrian clubs located in different regions of the Sichuan province of southwestern China were examined. Nested polymerase chain reaction (PCR) analysis of the gene encoding triose-phosphate isomerase (tpi) showed the prevalence of G. intestinalis to be 8.3% (22/264), and the prevalence in different clubs varied from 3.6% to 13.5%. Three assemblages were identified in the successfully sequenced samples, including the potentially zoonotic assemblages A (n = 5) and B (n = 14), the mouse-specific assemblage G (n = 3), and a mixed A and B assemblage. Sequence analysis of tpi, glutamate dehydrogenase (gdh), and beta giardin (bg) loci revealed that the majority of sequences isolated from assemblage A were identical to the subtype AIV and assemblage B isolates showed variability among the nucleotide sequences of the subtype BIV. Using the nomenclature for the multilocus genotype (MLG) model, one each of multilocus genotypes A (MLG1) and B (MLG2) were identified, with MLG2 being a novel genotype. To the best of our knowledge, this is the first study to investigate G. intestinalis in Chinese racehorses. The presence of both animal and human assemblages of G. intestinalis in racehorses indicated that these animals might constitute a potential zoonotic risk to human beings.

Giardia duodenalis is a common human and animal pathogen. It has been increasingly reported in wild and captive non-human primates (NHPs) in recent years. However, multilocus genotyping information for G. duodenalis infecting NHPs in southwestern China is limited. In the present study, the prevalence and multilocus genotypes (MLGs) of G. duodenalis in captive NHPs in southwestern China were determined. We examined 207 fecal samples from NHPs in Sichuan and Guizhou provinces, and 16 specimens were positive for G. duodenalis. The overall infection rate was 7.7%, and only assemblage B was identified. G. duodenalis was detect positive in northern white-cheeked gibbon (14/36, 38.9%), crab-eating macaque (1/60, 1.7%) and rhesus macaques (1/101, 0.9%). Multilocus sequence typing based on beta-giardin (bg), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) revealed nine different assemblage B MLGs (five known genotypes and four novel genotypes). Based on a phylogenetic analysis, one potentially zoonotic genotype of MLG SW7 was identified in a northern white-cheeked gibbon. A high degree of genetic diversity within assemblage B was observed in captive northern white-cheeked gibbons in Southwestern China, including a potentially zoonotic genotype, MLG SW7. To the best of our knowledge, this is the first report using a MLGs approach to identify G. duodenalis in captive NHPs in Southwestern China.

Enterocytozoon bieneusi has been increasingly reported in non-human primates (NHPs) in recent years, and this has garnered attention. However, reports of E. bieneusi infections in NHPs are limited worldwide. To appreciate the genetic diversity and assess the zoonotic potential during the transmission of human microsporidiosis, we examined a total of 369 fecal samples from NHPs and performed PCR amplification of the ITS gene of E. bieneusi. An infection rate of 12.5% (46/369) was detected in NHPs, with three known genotypes (D, PigEBITS7, and SC02) and a novel genotype (SCM01) characterized. Phylogenetic analysis indicated that all four genotypes in our study were classified as zoonotic group 1. Multilocus genotyping of positive E. bieneusi strains revealed that 36, 37, 30, and 29 specimens were successfully amplified and sequenced to generate 16, six, four, and five types of MS1, MS3, MS4, and MS7 loci, respectively. Twenty-four specimens were successfully amplified and sequenced at all four loci, forming 13 multilocus genotypes (MLGs). The occurrence of zoonotic genotypes suggests that zoonotic transmission of E. bieneusi between humans and NHPs has probably occurred and NHPs could be a source of human microspordiosis.

Enterocytozoon bieneusi is the most common pathogen of microsporidian species infecting humans worldwide. Although E. bieneusi has been found in a variety of animal hosts, information on the presence of E. bieneusi in captive kangaroos in China is limited. The present study was aimed at determining the occurrence and genetic diversity of E. bieneusi in captive kangaroos. A total of 61 fecal specimens (38 from red kangaroos and 23 from grey kangaroos) were collected from Nanjing Hongshan Forest Zoo and Hongshan Kangaroo Breeding Research Base, Jiangsu province, China. Using the nested PCR amplification ITS gene of rRNA of E. bieneusi, totally 23.0% (14/61) of tested samples were PCR-positive with three genotypes (i.e. one known genotype, CHK1, and two novel genotypes, CSK1 and CSK2). Multi-locus sequence typing using three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4) revealed one, five, two, and one types at these four loci, respectively. In phylogenetic analysis, the two genotypes, CHK1 and CSK1, were clustered into a new group of unknown zoonotic potential, and the novel genotype CSK2 was clustered into a separate clade with PtEb and PtEbIX. To date, this is the first report on the presence of E. bieneusi in captive red kangaroos in Jiangsu province, China. Furthermore, a high degree of genetic diversity was observed in the E. bieneusi genotype and seven MLGs (MLG1-7) were found in red kangaroos. Our findings suggest that infected kangaroo may act as potential reservoirs of E. bieneusi and be source to transmit infections to other animal.

Growth performance and meat quality are important traits for the pig industry and consumers. Adipose tissue is the main site at which fat storage and fatty acid synthesis occur. Therefore, we combined high-throughput transcriptomic sequencing in adipose and muscle tissues with the quantification of corresponding phenotypic features using seven Chinese indigenous pig breeds and one Western commercial breed (Yorkshire). We obtained data on 101 phenotypic traits, from which principal component analysis distinguished two groups: one associated with the Chinese breeds and one with Yorkshire. The numbers of differentially expressed genes between all Chinese breeds and Yorkshire were shown to be 673 and 1056 in adipose and muscle tissues, respectively. Functional enrichment analysis revealed that these genes are associated with biological functions and canonical pathways related to oxidoreductase activity, immune response, and metabolic process. Weighted gene coexpression network analysis found more coexpression modules significantly correlated with the measured phenotypic traits in adipose than in muscle, indicating that adipose regulates meat and carcass quality. Using the combination of differential expression, QTL information, gene significance, and module hub genes, we identified a large number of candidate genes potentially related to economically important traits in pig, which should help us improve meat production and quality.

Enterocytozoon bieneusi is an obligate eukaryotic intracellular parasite that infects a wide variety of vertebrate and invertebrate hosts. Although considerable research has been conducted on this organism, relatively little information is available on the occurrence of E. bieneusi in captive Asiatic black bears. The present study was performed to determine the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in captive Asiatic black bears in zoos in southwestern China. Fecal specimens from Asiatic black bears in four zoos, located in four different cities, were collected and analyzed for the prevalence of E. bieneusi. The average prevalence of E. bieneusi was 27.4% (29/106), with the highest prevalence in Guiyang Zoo (36.4%, 16/44). Altogether, five genotypes of E. bieneusi were identified among the 29 E. bieneusi-positive samples, including three known genotypes (CHB1, SC02, and horse2) and two novel genotypes named ABB1 and ABB2. Multi-locus sequence typing using three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4) revealed V, III, V, and IV genotypes at these four loci, respectively. Phylogenetic analysis showed that the genotypes SC02 and ABB2 were clustered into group 1 of zoonotic potential, the genotypes CHB1 and ABB1 were clustered into a new group, and the genotype horse2 was clustered into group 6 of unclear zoonotic potential. In conclusion, this study identified two novel E. bieneusi genotypes in captive Asiatic black bears, and used microsatellite and minisatellite markers to reveal E. bieneusi genetic diversity. Moreover, our findings show that genotypes SC02 (identified in humans) and ABB2 belong to group 1 with zoonotic potential, suggesting the risk of transmission of E. bieneusi from Asiatic black bears to humans and other animals.

[This corrects the article DOI: 10.1371/journal.pone.0183249.].

Enterocytozoon bieneusi is a common opportunistic pathogen causing diarrhea and enteric disease in a variety of animal hosts. Although it has been reported in many animals, there is no published information available on the occurrence of E. bieneusi in red-bellied tree squirrels. To understand the occurrence, genetic diversity, and zoonotic potential of E. bieneusi in red-bellied tree squirrels, 144 fecal specimens from Sichuan province, China, were examined by PCR amplification and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi. The overall infection rate of E. bieneusi 16.7% (24/144) was observed in red-bellied tree squirrels. Altogether five genotypes of E. bieneusi were identified: three known genotypes D (n = 18), EbpC (n = 3), SC02 (n = 1) and two novel genotypes CE01, CE02 (one each). Multilocus sequence typing (MLST) analysis employing three microsatellite (MS1, MS3, MS7) and one minisatellite (MS4) revealed 16, 14, 7 and 14 positive specimens were successfully sequenced, and identified eight, three, three and two genotypes at four loci, respectively. In phylogenetic analysis, the three known genotypes D, EbpC, and SC02 were clustered into group 1 with zoonotic potential, and the two novel genotypes CE01 and CE02 were clustered into group 6. The present study firstly reported the occurrence of E. bieneusi in red-bellied tree squirrels in China, and the E. bieneusi genotypes D and EbpC were found in humans previously. These results indicate that red-bellied tree squirrels may play a potential role in the transmission of E. bieneusi to humans.

[This corrects the article DOI: 10.1371/journal.pone.0163605.].

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 panda's 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.

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.

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.

Breast milk is the primary source of nutrition for newborns, and is rich in immunological components. MicroRNAs (miRNAs) are present in various body fluids and are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and could regulate target gene expression and recipient cell function. Here, we analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth) by a deep sequencing. We found that immune-related miRNAs are present and enriched in breast milk exosomes (p

BACKGROUND: Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage. CONCLUSIONS/SIGNIFICANCE: These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) is regarded as one of the most important priority pathogens within the One Health interface. However, few studies have investigated the occurrence of ESBL-EC in giant pandas, along with their antibiotic-resistant characteristics and horizontal gene transfer abilities. In this study, we successfully identified 12 ESBL-EC strains (8.33%, 12/144) out of 144 E. coli strains which isolated from giant pandas. We further detected antibiotic resistance genes (ARGs), virulence-associated genes (VAGs) and mobile genetic elements (MGEs) among the 12 ESBL-EC strains, and the results showed that 13 ARGs and 11 VAGs were detected, of which blaCTX-M (100.00%, 12/12, with 5 variants observed) and papA (83.33%, 10/12) were the most prevalent, respectively. And ISEcp1 (66.67%, 8/12) and IS26 (66.67%, 8/12) were the predominant MGEs. Furthermore, horizontal gene transfer ability analysis of the 12 ESBL-EC showed that all blaCTX-M genes could be transferred by conjugative plasmids, indicating high horizontal gene transfer ability. In addition, ARGs of rmtB and sul2, VAGs of papA, fimC and ompT, MGEs of ISEcp1 and IS26 were all found to be co-transferred with blaCTX-M. Phylogenetic analysis clustered these ESBL-EC strains into group B2 (75.00%, 9/12), D (16.67%, 2/12), and B1 (8.33%, 1/12), and 10 sequence types (STs) were identified among 12 ESBL-EC (including ST48, ST127, ST206, ST354, ST648, ST1706, and four new STs). Our present study showed that ESBL-EC strains from captive giant pandas are reservoirs of ARGs, VAGs and MGEs that can co-transfer with blaCTX-M via plasmids. Transmissible ESBL-EC strains with high diversity of resistance and virulence elements are a potential threat to humans, animals and surrounding environment. [ABSTRACT FROM AUTHOR]