Isolation, characterization, and pathogenicity assessment of Corynebacterium pseudotuberculosis biovar equi strains from alpacas (Vicugna pacos) in China.

Corynebacterium pseudotuberculosis is a zoonotic pathogen that causes lymphadenitis in humans, livestock, and wildlife. In this study, C. pseudotuberculosis biovar equi strains were isolated from three alpacas. Antibiotic susceptibility tests and pathogenicity tests were also conducted. Moreover, one strain was sequenced using DNBSEQ and Oxford Nanopore technology. The three strains exhibited resistance to aztreonam, fosfomycin, and nitrofurantoin. The median lethal doses (LD50) of strains G1, S2 and BA3 in experimentally infected mice was 1.66 × 105 CFU, 3.78 × 105 CFU and 3.78 × 105 CFU, respectively. The sequencing of strain G1 resulted in the assembly of a chromosomal scaffold comprising 2,379,166 bp with a G + C content of 52.06%. Genome analysis of strain G1 revealed the presence of 48 virulence genes and 5 antibiotic resistance genes (ARGs). Comparative genomic analysis demonstrates a high degree of genetic similarity among C. pseudotuberculosis strains, in contrast to other Corynebacterium species, with a clear delineation between strains belonging to the two biovars (ovis and equi). The data of the present study contribute to a better understanding of the properties of C. pseudotuberculosis biovar equi strains and the potential risk they pose to alpacas and other livestock, as well as the necessity of ongoing surveillance and monitoring of infectious diseases in animals.

Genetic characterization and zoonotic potential of Blastocystis from wild animals in Sichuan Wolong National Natural Reserve, Southwest China.

Blastocystis is a prevalent eukaryotic parasite that has been identified in a wide range of hosts. Several species are considered potential sources of Blastocystis infection in humans, but little is known about the prevalence of Blastocystis in wild animals. In this study, the prevalence and subtypes of Blastocystis were investigated to assess the zoonotic potential of wild animals in Sichuan Wolong National Natural Reserve. A total of 300 fecal samples were collected from 27 wildlife species in three areas of the Reserve. The subtype (ST), genetic characteristics, and prevalence of Blastocystis were determined by PCR amplification of part (~600 bp) of the SSU rRNA gene. Thirty fecal samples (10.0%) were Blastocystis-positive. The highest prevalence of Blastocystis was found in Yinchanggou (18.3%), with significantly less found in Niutoushan (7.5%) and Genda (5.5%) (p < 0.05). No significant differences were associated with different orders of animals in prevalence, which may be because of the small number of positive samples obtained. Sequence analysis showed five subtypes (ST1, ST3, ST5, ST13, and ST14), with ST13 and ST14 being predominant (33% each), followed by ST1 (20%). This is the first molecular investigation of Blastocystis infection in the wild animals of southwestern China. Subtypes ST1, ST3, ST5, and ST14 have previously been identified in humans, suggesting that wild animals may be potential reservoirs of Blastocystis for humans.

TITLE: Caractérisation génétique et potentiel zoonotique de Blastocystis provenant d'animaux sauvages dans la réserve naturelle nationale du Sichuan Wolong, sud-ouest de la Chine. ABSTRACT: Blastocystis est un parasite eucaryote répandu qui a été identifié dans un large éventail d'hôtes. Plusieurs espèces sont considérées comme des sources potentielles d'infection par Blastocystis chez l'homme, mais on sait peu de choses sur la prévalence de Blastocystis chez les animaux sauvages. Dans cette étude, la prévalence et les sous-types de Blastocystis ont été étudiés pour évaluer le potentiel zoonotique des animaux sauvages dans la réserve naturelle nationale du Sichuan Wolong. Au total, 300 échantillons de matières fécales ont été prélevés de 27 espèces de la faune dans trois zones de la réserve. Le sous-type (ST), les caractéristiques génétiques et la prévalence de Blastocystis ont été déterminés par amplification PCR d'une partie (~ 600 pb) du gène 18S de l'ARNr. Trente échantillons fécaux (10,0 %) étaient positifs pour Blastocystis. La prévalence la plus élevée de Blastocystis a été trouvée à Yinchanggou (18,3 %), avec une prévalence significativement moindre à Niutoushan (7,5 %) et à Genda (5,5 %) (p < 0,05). Aucune différence significative de prévalence n'était associée à différents ordres d'animaux, ce qui peut être dû au petit nombre d'échantillons positifs obtenus. L'analyse des séquences a montré cinq sous-types (ST1, ST3, ST5, ST13 et ST14), et ST13 et ST14 étant prédominants (33 % chacun), suivis de ST1 (20 %). Il s'agit de la première étude moléculaire de l'infection à Blastocystis chez les animaux sauvages du sud-ouest de la Chine. Les sous-types ST1, ST3, ST5 et ST14 ont déjà été identifiés chez l'homme, suggérant que les animaux sauvages pourraient être des réservoirs potentiels de Blastocystis pour l'homme.

First identification and molecular subtyping of Blastocystis sp. in zoo animals in southwestern China.

BACKGROUND: Blastocystis sp. is an anaerobic protozoan that parasitizes many animal hosts and the human gastrointestinal tract, and its pathogenicity is controversial. Captive wildlife may be potential reservoirs for human infection with Blastocystis sp. The present study was performed to investigate the prevalence and subtype distribution of Blastocystis sp. in zoo animals in Sichuan Province, southwestern China. METHODS: A total of 420 fresh fecal samples were collected from 54 captive wildlife species in four zoos in southwestern China between June 2017 and September 2019. The prevalence and subtype (ST) genetic characteristics of Blastocystis sp. were determined by PCR amplification of the barcode region of the SSU rRNA gene and phylogenetic analysis. RESULTS: Overall, 15.7% (66/420) of the animal samples and 20.7% (14/54) of the species tested were shown to be infected with Blastocystis sp. The highest prevalence of Blastocystis sp. was found in Panzhihua Zoo (24.3%), which was significantly higher than that in Chengdu Zoo (6.9%), and Xichang Zoo (2.9%) (P < 0.05). There are also significant differences in the prevalence of Blastocystis sp. among different species (P < 0.05), and the highest of Blastocystis sp. prevalence was observed in white-cheeked gibbon, black great squirrel, and red giant flying squirrel (100%). Subtype analysis of Blastocystis sp. revealed nine subtypes, including six zoonotic STs (ST1-5, and ST8) and three animal-specific STs (ST10, ST14, and ST17), with ST17 as the predominant subtype (26/66) in Blastocystis sp.-positive isolates. CONCLUSIONS: To our knowledge, this is the first report on the prevalence and subtype distribution of Blastocystis sp. among captive wildlife in zoos in southwestern China. This study highlights that these animals may serve as reservoirs for human Blastocystis sp. infections.

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 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.

Investigation of antibacterial activity of Bacillus spp. isolated from the feces of Giant Panda and characterization of their antimicrobial gene distributions.

Bacillus group is a prevalent community of Giant Panda's intestinal flora, and plays a significant role in the field of biological control of pathogens. To understand the diversity of Bacillus group from the Giant Panda intestine and their functions in maintaining the balance of the intestinal microflora of Giant Panda, this study isolated a significant number of strains of Bacillus spp. from the feces of Giant Panda, compared the inhibitory effects of these strains on three common enteric pathogens, investigated the distributions of six universal antimicrobial genes (ituA, hag, tasA, sfp, spaS and mrsA) found within the Bacillus group by PCR, and analyzed the characterization of antimicrobial gene distributions in these strains using statistical methods. The results suggest that 34 strains of Bacillus spp. were isolated which has not previously been detected at such a scale, these Bacillus strains could be classified into five categories as well as an external strain by 16S rRNA; Most of Bacillus strains are able to inhibit enteric pathogens, and the antimicrobial abilities may be correlated to their categories of 16S rRNA; The detection rates of six common antimicrobial genes are between 20.58 %(7/34) and 79.41 %(27/34), and genes distribute in three clusters in these strains. We found that the antimicrobial abilities of Bacillus strains can be one of the mechanisms by which Giant Panda maintains its intestinal microflora balance, and may be correlated to their phylogeny.

[Intestinal fungal diversity of sub-adult giant panda].

OBJECTIVE: The fungi diversity in the guts of five sub-adult giant pandas was analyzed. METHOD: We analyzed the fungal internal transcribed spacer sequences (ITS) using restriction fragment length polymorphism (RFLP). ITS regions were amplified with fungal universal primers to construct ITS clone libraries. The fingerprints were analyzed by restriction fragment length polymorphism using the Hha I and Hae III enzymes. The cloned PCR products were analyzed by sequencing and diversities were demonstrated by phylogenetic tree. RESULTS: The gut fungi of 5 sub-adult giant pandas were mainly composed of Ascomycota (average of 46.24%), Basidiomycota ( average of 15.79%), unclassified (average of 29.14%), uncultured fungus (average of 8.83% ). Ascomycota was mainly composed of Saccharomycetes (average of 63.74%) and Dothideomycetes ( average of 35.91%); Basidiomycota was mainly composed of Tremellomycetes (average of 65.80%) and Microbotryomycetes (average of 33.15%). Four classes were mainly composed of Candida and Debaryomyces; Pleosporales and Myriangium; Cystofilobasidium and Trichosporon; Leucosporidium, and Leucosporidiella, whereas the proportions were different for each sample. CONCLUSION: Fungal flora existing in the intestines of sub-adult giant pandas expand our knowledge on the structure of the giant panda gut microbes and also help us to further study whether fungal flora can help giant pandas digest high-fiber foods.

A new genotype of Cryptosporidium from giant panda (Ailuropoda melanoleuca) in China.

Fifty-seven fecal samples were collected from giant pandas (Ailuropoda melanoleuca) in the China Conservation and Research Centre for the Giant Panda (CCRCGP) in Sichuan and examined for Cryptosporidium oocysts by Sheather's sugar flotation technique. An 18-year-old male giant panda was Cryptosporidium positive, with oocysts of an average size of 4.60×3.99 µm (n=50). The isolate was genetically analyzed using the partial 18S rRNA, 70 kDa heat shock protein (HSP70), Cryptosporidium oocyst wall protein (COWP) and actin genes. Multi-locus genetic characterization indicated that the present isolate was different from known Cryptosporidium species and genotypes. The closest relative was the Cryptosporidium bear genotype, with 11, 10, and 6 nucleotide differences in the 18S rRNA, HSP70, and actin genes, respectively. Significant differences were also observed in the COWP gene compared to Cryptosporidium mongoose genotype. The homology to the bear genotype at the 18S rRNA locus was 98.6%, which is comparable to that between Cryptosporidium parvum and Cryptosporidium hominis (99.2%), or between Cryptosporidium muris and Cryptosporidium andersoni (99.4%). Therefore, the Cryptosporidium in giant pandas in this study is considered as a new genotype: the Cryptosporidium giant panda genotype.