Isolation and identification of specific Enterococcus faecalis phage C-3 and G21-7 against Avian pathogenic Escherichia coli and its application to one-day-old geese.

Colibacillosis caused by Avian pathogenic Escherichia coli (APEC), including peritonitis, respiratory tract inflammation and ovaritis, is recognized as one of the most common and economically destructive bacterial diseases in poultry worldwide. In this study, the characteristics and inhibitory potential of phages were investigated by double-layer plate method, transmission electron microscopy, whole genome sequencing, bioinformatics analysis and animal experiments. The results showed that phages C-3 and G21-7 isolated from sewage around goose farms infected multiple O serogroups (O1, O2, O18, O78, O157, O26, O145, O178, O103 and O104) Escherichia coli (E.coli) with a multiplicity of infection (MOI) of 10 and 1, respectively. According to the one-step growth curve, the incubation time of both bacteriophage C-3 and G21-7 was 10 min. Sensitivity tests confirmed that C-3 and G21-6 are stable at 4 to 50 °C and pH in the range of 4 to 11. Based on morphological and phylogenetic analysis, phages C-3 and G21-7 belong to Enterococcus faecalis (E. faecalis) phage species of the genus Saphexavirus of Herelleviridae family. According to genomic analysis, phage C-3 and G21-7 were 58,097 bp and 57,339 bp in size, respectively, with G+C content of 39.91% and 39.99%, encoding proteins of 97 CDS (105 to 3,993 bp) and 96 CDS (105 to 3,993 bp), and both contained 2 tRNAs. Both phages contained two tail proteins and holin-endolysin system coding genes, and neither carried resistance genes nor virulence factors. Phage mixture has a good safety profile and has shown good survival probability and feed efficiency in both treatment and prophylaxis experiments with one-day-old goslings. These results suggest that phage C-3 and G21-7 can be used as potential antimicrobials for the prevention and treatment of APEC.

Transcriptome analysis of the Bactrian camel (Camelus bactrianus) reveals candidate genes affecting milk production traits.

BACKGROUND: Milk production traits are complex traits with vital economic importance in the camel industry. However, the genetic mechanisms regulating milk production traits in camels remain poorly understood. Therefore, we aimed to identify candidate genes and metabolic pathways that affect milk production traits in Bactrian camels. METHODS: We classified camels (fourth parity) as low- or high-yield, examined pregnant camels using B-mode ultrasonography, observed the microscopic changes in the mammary gland using hematoxylin and eosin (HE) staining, and used RNA sequencing to identify differentially expressed genes (DEGs) and pathways. RESULTS: The average standard milk yield over the 300 days during parity was recorded as 470.18 ± 9.75 and 978.34 ± 3.80 kg in low- and high-performance camels, respectively. Nine female Junggar Bactrian camels were subjected to transcriptome sequencing, and 609 and 393 DEGs were identified in the low-yield vs. high-yield (WDL vs. WGH) and pregnancy versus colostrum period (RSQ vs. CRQ) comparison groups, respectively. The DEGs were compared with genes associated with milk production traits in the Animal Quantitative Trait Loci database and in Alashan Bactrian camels, and 65 and 46 overlapping candidate genes were obtained, respectively. Functional enrichment and protein-protein interaction network analyses of the DEGs and candidate genes were conducted. After comparing our results with those of other livestock studies, we identified 16 signaling pathways and 27 core candidate genes associated with maternal parturition, estrogen regulation, initiation of lactation, and milk production traits. The pathways suggest that emerged milk production involves the regulation of multiple complex metabolic and cellular developmental processes in camels. Finally, the RNA sequencing results were validated using quantitative real-time PCR; the 15 selected genes exhibited consistent expression changes. CONCLUSIONS: This study identified DEGs and metabolic pathways affecting maternal parturition and milk production traits. The results provides a theoretical foundation for further research on the molecular mechanism of genes related to milk production traits in camels. Furthermore, these findings will help improve breeding strategies to achieve the desired milk yield in camels.

Prevalence and pathology of Cephalopina titillator infestation in Camelus bactrianus from Xinjiang, China.

BACKGROUND: In camels, nasopharyngeal myiasis is caused by the larvae of Cephalopina titillator, which parasitize the tissues of nasal and paranasal sinuses, pharynx, and larynx. C. titillator infestation adversely affects the health of camels and decreases milk and meat production and even death. However, the C. titillator infestation in Bactrian camels has not been widely studied. METHODS: The present study was conducted to determine the prevalence and risk factors of C. titillator in Bactrian camels of northwestern Xinjiang. Suspected larvae recovered from infested camels were evaluated for C. titillator by microscopy and polymerase chain reaction. Nucleotide sequences of the partial mitochondrial cytochrome c oxidase subunit I (COX1) and cytochrome b (CYTB) genes from the C. titillator of camels were aligned from the NCBI database. Furthermore, the gross and histopathological alterations associated with C. titillator infestation were evaluated via pathological examination. RESULTS: Of 1263 camels examined 685 (54.2%) camels were infested with suspected C. titillator larvae. Different larval stages were topically detected in the nasal passages and pharynx of the camel heads. Microscopy analysis of the pharyngeal mucosa tissue revealed necrotic tissue debris and some inflammatory cells. Molecular detection of the larval COX1 and CYTB genes indicated that pathogen collected in Bactrian camels was C. titillator. The epidemiological study demonstrated that the prevalence rate of C.titillator infestation was significantly higher in camels of Bestierek Town Pasture (67.2%) and Karamagai Town Pasture (63.6%) compared to Kitagel Town Pasture (38.7%) and Qibal Town Pasture (35.8%) (P < 0.05). No significant difference was observed between the prevalence rates in male (52.6%) and female (54.6%) camels (P > 0.05). The prevalence was higher in warm (64.2%) than that in cold (48.4%) seasons (P < 0.001). The prevalence in camels with non-nomadic method (67.2%) was significantly higher than in animals with nomadic method (47.5%) (P < 0.001). The prevalence of C.titillator infestation was significantly higher in animals of aged 5-10 (60.1%) and aged > 10 (61.1%) years old compared to those of aged < 5 (31.7%) years old camels (P < 0.001). CONCLUSION: Our results confirm that there is a high prevalence of C. titillator in Bactrian camels from Xinjiang, closely related to age, season, pasture environment, and husbandry methods. Developing prevention, diagnosis, and control programs to prevent transmission is necessary.

Lysobacter chinensis sp. nov., a cellulose-degrading strain isolated from cow dung compost.

A novel bacterial strain, TLK-CK17T, was isolated from cow dung compost sample. The strain was Gram-staining negative, non-gliding rods, aerobic, and displayed growth at 15-40 °C (optimally, 35 °C), with 0-5.0% (w/v) NaCl (optimally, 0.5) and at pH 6.5-8.5 (optimally, 7.0-7.5). The assembled genome of strain TLK-CK17T has a total length of 4.3 Mb with a G + C content of 68.2%. According to the genome analysis, strain TLK-CK17T encodes quite a few glycoside hydrolases that may play a role in the degradation of accumulated plant biomass in compost. On the basis 16S rRNA gene sequence analysis, strain TLK-CK17T showed the highest sequence similarity (98.9%) with L. penaei GDMCC 1.1817 T, followed by L. maris KCTC 42381 T (98.3%). Cells contained iso-C16:0, iso-C15:0, and summed feature 9 (comprising C17:1 ω9c and/or 10-methyl C16:0), as its major cellular fatty acids (> 10.0%) and ubiquinone-8 as the exclusively respiratory quinone. Diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol prevailed among phospholipids. Based on the phenotypic, genomic and phylogenetic data, strain TLK-CK17T represents a novel species of the genus Lysobacter, for which the name Lysobacter chinensis sp. nov. is proposed, and the type strain is TLK-CK17T (= CCTCC AB2021257T = KCTC 92122 T).

Characterization of Shiga toxin-producing Escherichia coli isolated from Cattle and Sheep in Xinjiang province, China, using whole-genome sequencing.

Shiga toxin-producing Escherichia coli (STEC) is an important food-borne pathogen capable of causing severe gastrointestinal diseases in humans. Cattle and sheep are the natural reservoir hosts of STEC strains. Previously, we isolated 56 STEC strains from anal and carcass swab samples of cattle and sheep in farms and slaughterhouses. In this study, we performed whole-genome sequencing of these isolates and determined their serotypes, virulence profiles, sequence types (STs) and genetic relationships. Our results showed that the 56 isolates belong to 20 different STs, 29 O:H serotypes and 8 stx subtype combinations. The highly prevalent serotypes for bovine and ovine isolates were O8:H25 and O87:H16, respectively. Five serotypes of cattle or sheep isolates are novel. The majority (63%) of cattle isolates contain stx1 + stx2, subtyped into stx1a, stx2a and stx2c. In contrast, most of the sheep isolates contain stx1 only, primarily subtyped into stx1a and stx1c. None of the isolates tested eae-positive, but virulence factors such as ehxA and espP were present with variable prevalence rates. The prevalence of saa (19.6%) and espP (12.5%) in cattle isolates is much higher than that in sheep isolates, whereas that of subA (34%), katP (14.3%) and ireA (28.6%) in sheep isolates is considerably higher than that in cattle isolates. Core-genome SNP analysis revealed that the majority of isolates could be clustered based on their serotypes or STs, whereas some clustering is associated with more than one ST or serotype. Five sheep isolates (4 belonging to ST675 and serotype O76:H19 and 1 belonging to ST25 and serotype O128:H2) share STs, serotypes and stx profiles with two hemolytic uremic syndrome-associated enterohemorrhagic E. coli (HUSEC) isolates; a cattle isolate belonging to the same ST as HUSEC isolate HUSEC001 contains all the nine virulence genes tested. These data suggest a potential of the six isolates for causing severe human infections. Collectively, we described the characteristics of cattle and sheep STEC isolates from Xinjiang, China, which may be utilized in comparative studies of other geographic regions and sources of isolation, and for surveillance as well.

Molecular Characteristics and Virulence Gene Analysis of Non-O157 Shiga Toxin-Producing Escherichia coli from Cattle in Xinjiang.

Non-O157 Shiga toxin (stx)-producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Cattle are the principal reservoirs of STEC, although other animals can be carriers. Humans are mainly infected by consuming contaminated drinking water or food. This study aimed to evaluate the virulence potential of isolated bovine non-O157 STEC to humans in Xinjiang. During 2015-2017, 978 rectal swab samples collected from cattle of 5 farms were screened for the presence of Shiga toxin-encoding genes by polymerase chain reaction. Strains identified as STEC were isolated from rectal swab samples, and were characterized for stx subtype, virulence genes, O serogroup, phylogenetic group, and hemolytic phenotype. Among 125 non-O157 STEC isolates, the prevalence percentages of stx1 and stx2 were 22 and 21, respectively, and 57% of the isolates carried both Shiga toxins. The stx subtypes were mainly found in the combination of stx1a/stx2a (57%), stx2a (20%), stx1a (22%), stx1a/stx2a/stx2c (1%), and stx2a/stx2c (1%). The enterohemolysin (ehxA) gene was found in 94% of the isolates. No intimin (eae) was detected. Hemolysis was observed in 33% of the isolates. Two STEC serogroups O145 (17%) and O113 (2%) were found, which were reported to be associated with outbreaks of human disease. Phylotyping assays showed that most strains largely belong to groups A (91%) and B1 (7%). The results of this study can help improve our understanding of the epidemiological aspects of bovine STEC and devise strategies for protection against it.

First Isolation and Molecular Characterization of bla CTX-M-121 -Producing Escherichia coli O157:H7 From Cattle in Xinjiang, China.

The bovine Escherichia coli O157:H7 is a major foodborne pathogen causing severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. Cattle are recognized major reservoir and source of E. coli O157:H7. We investigated the antibiotic resistance, molecular profiles, and intrinsic relationship between 21 isolates of E. coli O157:H7 from cattle farms and slaughtering houses in Xinjiang. Using pulsed-field gel electrophoresis (PFGE) molecular typing, two types of PFGE were revealed through cluster analysis, including clusters I and II, with 66 and 100% similarity of PFGE spectra between 21 isolates. We also detected that 18 isolates (86%) carried at least one virulence gene, 16 isolates (76%) carried the eae gene, and 7 (33%) carried the stx1 + stx2 + eae + hly + tccp genes. Eighteen isolates were susceptible to antibiotics. Three isolates were resistant to antibiotics, and two were multidrug resistant. One of the two multidrug-resistant isolates detectably carried the bla CTX-M-121 gene. This is the first finding of the bla CTX-M-121 gene detected in E. coli O157:H7 isolated from cattle in Xinjiang. The bla CTX-M-121 gene is transferable between the bacterial strains via plasmid transmission. The results indicated that E. coli O157:H7 may have undergone clonal propagation in cattle population and cross-regional transmission in Xinjiang, China.

Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Cultured from Cattle Farms in Xinjiang Uygur Autonomous Region, China, During 2016-2017.

Most outbreaks of Shiga toxin-producing Escherichia coli (STEC) are attributed to consumption of contaminated foodstuffs including beef and dairy products. In this study, we evaluated the prevalence of non-O157 STEC cultured from beef and dairy cattle and collected in Xinjiang Uygur Autonomous Region in China. Results identified 67 non-O157 STEC recovered from the 793 samples including beef cattle (10.28%, 43/418) and dairy cattle (6.40%, 24/375). A total of 67 non-O157 STEC was sequenced allowing for in silico analyses of their serotypes, virulence genes, and identification of the corresponding multilocus sequence types (STs). Twenty-one O serogroups and nine H serotypes were identified and the dominant serotype identified was O22:H8. One stx1 subtype (stx1a) and four stx2 subtypes (2a, 2b, 2c, and 2d) were found in the 67 non-O157 STEC isolates. The results revealed that stx1a+stx2a-positive STEC isolates were predominant (32.83%, 22/67), followed by stx1a+stx2d (29.85%, 20/67) and stx2a alone (17.91%, 12/67). Non-O157 STEC isolates carried virulence genes ehxA (98.51%), subA (53.73%), and cdtB (17.91%). Of the four adherence-associated genes tested, eaeA was absent, whereas lpfA and iha were present in 67 and 55 non-O157 STEC isolates, respectively. The STEC isolates were divided into 48 pulsed-field gel electrophoresis patterns and 10 STs, and ST446 (O22:H8) was the dominant clone (22.38%). Our results revealed that there was a high genetic diversity among non-O157 STEC isolated from beef and dairy cattle, some of which have potential to cause human diseases.

Transcriptome analysis of Corynebacterium pseudotuberculosis-infected spleen of dairy goats.

Caseous lymphadenitis is a chronic disease of goats caused by Corynebacterium pseudotuberculosis (C.pseudotuberculosis) which causes great harm to the dairy goats industry. In order to obtain detailed information about the pathogenesis and host immune response in C.pseudotuberculosis-infected goats, in this study, the gene expression difference of spleen tissue after infection with C.pseudotuberculosis was analyzed by high-throughput sequencing. Transcripts obtained over 412 700 462 clean reads after reassembly were 21 343 genes detected, of which 14 720 were known genes and 7623 new genes were predicted. There were 448 up-regulated and 519 down-regulated differentially expressed genes (DEGs). Gene Ontology (GO) analysis indicated that all of the DEGs were annotated into biological process, cellular component and molecular function. Most of these unigenes are annotated in cellular processes, the cell and binding. KEGG analysis of the DEGs showed that a total of 8733 DEGs unigenes were annotated into 459 pathways classified into 6 main categories. Most of these annotated unigenes were related to immune system response to the infectious diseases pathways. In addition, 14 DEGs were verified by quantitative real-time PCR. As the first, in vivo, RNAseq analysis of dairy goats and C.pseudotuberculosis infection, this study provides knowledge about the transcriptomics of spleen in C.pseudotuberculosis-infected goats, from which a complex molecular pathways and immune response mechanism are involved in C.pseudotuberculosis infection.

First detection and genetic characterization of a novel kirkovirus from a dead thoroughbred mare in northern Xinjiang, China, in 2018.

BACKGROUND: In May 2018, a 8 year old thoroughbred mare died at an equestrian club in Changji, Xinjiang, China. The horse had been imported from the United States in 2013. She became pregnant in December 2016 but, after foaling, gradually lost weight and died in May 2018. This study aim to identify the pathogen, who cause of horse death, using virome. RESULTS: We have identified an Equ1-like virus from the fecal virome of a dead thoroughbred mare in China. Full genomic sequencing and phylogenetic analysis of the virus, tentatively named "kirkovirus Cj-7-7", showed that it was closely related to kirkovirus Equ1 and clustered together with po-circo-like viruses 21, 22, 41, and 51, suggesting that it should be assigned to the proposed family "Kirkoviridae". An epidemiological investigation showed that kirkovirus Cj-7-7 circulates in horses of northern Xinjiang and may specifically infect intestinal cells. CONCLUSIONS: Our findings demonstrate the genetic diversity and geographic distribution of Kirkoviruses, and the prevalence of Kirkovirus Cj-7-7 in Xinjiang, China.