Moraxella nasicaprae sp. nov., Isolated from a Goat with Respiratory Disease.

A novel Gram-stain-negative, aerobic, irregular coccus designated as ZY201224T, was isolated from the nasal cavity of a goat with respiratory disease in a goat farm, located at Jianshui, Yunnan Province, PR China and its taxonomic position was clarified using a polyphasic approach. The strain grew optimally at 37 °C, at pH 8.0 and in the presence of 1% NaCl. Phylogenetic analysis based on 16S rRNA gene sequence and phylogenomic analysis based on 808 single-copy genes revealed that the strain is affiliated to the genus Moraxella and is distinct from the recognized species of the genus. The 16S rRNA gene sequence similarity analysis indicated that the strain is most closely related to Moraxella caviae CCUG 355T with sequence similarity of 98.1%. The genomic OrthoANI and digital DNA-DNA hybridization (dDDH) values between the strain and the type strains of Moraxella species were no higher than 74.7% (Moraxella pluranimalium CCUG 54913T) and 26.0% (Moraxella oblonga NBRC 102422T), respectively. The G + C content of the complete genome sequence was 43.6 mol%. The strain contained CoQ-8 as the major respiratory quinone, and C18:1ω9c, C17:1ω8c, C16:0 and summed feature 3 (C16:1 ω7c and/ or C16:1ω6c) as the predominant fatty acids (> 5%). The major polar lipids comprised phosphatidylglycerol (PG), cardiolipin (CL), monolysocardiolipin (MLCL), phosphatidylethanolamine (PE) and lysophosphatidylglycerol (LPG). Based on these taxonomic characterizations, strain ZY201224T represents a novel species of the genus Moraxella, for which the name Moraxella nasicaprae sp. nov. is proposed. The type strain is ZY201224T (= CCTCC AB 2021474T = NBRC 115473T).

Moraxella nasovis sp. nov., isolated from a sheep with respiratory disease.

A novel Gram-stain-negative, aerobic, coccus-shaped bacteria, designated ZY201115T, was isolated from the nasal cavity of a sheep with respiratory disease in Yunnan Province, south-west China, and its taxonomic affiliation was studied by applying a polyphasic approach. The strain grew at 18-41 °C (optimum, 37 °C), at pH 6.0-9.0 (optimum, pH 8.0) and in 0.5-3.0% (w/v) NaCl (optimum, 1.0 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain is affiliated to the genus Moraxella with highest similarity to Moraxella bovis ATCC 10900T (96.6 %). Phylogenomic analysis based on 811 single-copy genes also indicated that the strain represents a novel species in the genus Moraxella and formed a deep and separated clade with Moraxella caviae NCTC 10293T. The highest genomic orthologous average nucleotide identity and digital DNA-DNA hybridization values between the strain and the type strains in the genus Moraxella were 73.7% (M. caviae NCTC 10293T) and 25.3% (Moraxella osloensis CCUG 350T), respectively. The G+C content of the complete genome sequence was 42.1 mol%. The predominant fatty acids (>5 %) were C18:1 ω9c, C17:1 ω8c, C12:03OH and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The major polar lipids were phosphatidylglycerol, cardiolipin, monolysocardiolipin, phosphatidylethanolamine and hemibismonoacylglycerophosphate. The major respiratory quinone was CoQ-8. On the basis of the results of phylogenetic, phenotypic and chemotaxonomic characterizations, strain ZY201115T clearly represents a novel species of the genus Moraxella, for which the name Moraxella nasovis sp. nov. is proposed. The type strain is ZY201115T (=CCTCC AB 2021473T=CCUG 75922T).

Epidemiological investigation and phylogenetic analysis of Classical Swine Fever virus in Yunnan province from 2015 to 2021.

BACKGROUND: Classical swine fever virus (CSFV), the causative agent of classical swine fever (CFS), is a highly contagious disease that poses a serious threat to Chinese pig populations. OBJECTIVES: Many provinces of China, such as Shandong, Henan, Hebei, Heilongjiang, and Liaoning provinces, have reported epidemics of CSFV, while the references to the epidemic of CSFV in Yunnan province are rare. This study examined the epidemic characteristics of the CSFV in Yunnan province. METHODS: In this study, 326 tissue samples were collected from different regions in Yunnan province from 2015 to 2021. A reverse transcription-polymerase chain reaction (RT-PCR), sequences analysis, and phylogenetic analysis were performed for the pathogenic detection and analysis of these 326 clinical specimens. RESULTS: Approximately 3.37% (11/326) of specimens tested positive for the CSFV by RT-PCR, which is lower than that of other regions of China. Sequence analysis of the partial E2 sequences of eleven CSFV strains showed that they shared 89.0-100.0% nucleotide (nt) and 95.0-100.0% amino acid (aa) homology, respectively. Phylogenetic analysis showed that these novel isolates belonged to the subgenotypes 2.1c and 2.1d, with subgenotype 2.1c being predominant. CONCLUSIONS: The CSFV was sporadic in China's Yunnan province from 2015 to 2021. Both 2.1c and 2.1d subgenotypes were found in this region, but 2.1c was dominant.

Epidemiological Investigation and Genetic Analysis of Pseudorabies Virus in Yunnan Province of China from 2017 to 2021.

In recent years, the prevalence of pseudorabies virus (PRV) has caused huge economic losses to the Chinese pig industry. Meanwhile, PRV infection in humans also sounded the alarm about its cross-species transmission from pigs to humans. To study the regional PRV epidemic, serological and epidemiological investigations of PRV in pig populations from Yunnan Province during 2017-2021 were performed. The results showed that 31.37% (6324/20,158, 95% CI 30.73-32.01) of serum samples were positive for PRV glycoprotein E (gE)-specific antibodies via enzyme-linked immunosorbent assay (ELISA). The risk factors, including the breeding scale and development stage, were significantly associated with PRV seroprevalence among pigs in Yunnan Province. Of the 416 tissue samples collected from PRV-suspected pigs in Yunnan Province, 43 (10.33%, 95% CI 7.41-13.26) samples were positive for PRV-gE nucleic acid in which 15 novel PRV strains from these PRV-positive samples were isolated, whose gC and gE sequences were analyzed. Phylogenetic analysis showed that all 15 isolates obtained in this study belonged to the genotype II. Additionally, the gC gene of one isolate (YuN-YL-2017) was genetically closer to variant PRV strains compared with others, while the gE gene was in the same clade with other classical PRV strains, indicating that this isolate might be a recombinant strain generated from the classical and variant strains. The results revealed the severe PRV epidemic in Yunnan Province and indicated that PRV variants are the major genotypes threatening the pig industry development.

Emergence of a Novel Recombinant Pseudorabies Virus Derived From the Field Virus and Its Attenuated Vaccine in China.

The occurrence of pseudorabies (PR) caused by the PR virus (PRV) causes huge economic losses to the pig industry in China. Moreover, the potential threat of PRV to humans' health has received wide attention recently. The prevalence of two PRV genotypes and the application of their corresponding live attenuated vaccines increase the recombination possibility. In the present study, a novel recombinant PRV strain designed as HN-2019 was isolated from one sick piglet in Hunan province, China, its genetic features and pathogenicity were further investigated. The results showed that the glycoprotein E (gE) and gG genes of the HN-2019 strain displayed higher nucleotide homology with PRV classical strains (such as Ea and Fa) compared to others. However, its TK gene with continuous nucleotide deletions shared 100% nucleotide identity with the HB-98 vaccine strain, which was derived from the Ea strain. Moreover, the HN-2019 strain exhibited similar growth characteristics to that of the Ea strain, but its pathogenicity in mice was significantly lower than the latter one. The results above suggested that a naturally recombinant event might occur in the genome of the HN-2019 strain between the PRV classical strain and the HB-98 vaccine strain, which will provide useful guidelines for PRV vaccine design in the future.

Homologous recombination technology generated recombinant pseudorabies virus expressing EGFP facilitates to evaluate its susceptibility to different cells and screen antiviral compounds.

Pseudorabies virus (PRV) is considered as an emerging zoonotic pathogen since its isolation from a human case. Meanwhile, the disease caused by PRV infection has led huge economic losses to Chinese pig industry since 2011. In this study, we constructed a recombinant PRV stably expressing the enhanced green fluorescent protein (EGFP) by homologous recombination technology for evaluating its susceptibility to different human cell lines and screening antiviral compounds. Stably expressed EGFP by this designed rPRVHuN-EGFP virus was confirmed in the infected cells, moreover, the growth kinetics of which was similar to that of wild type strain. Importantly, the application of this rPRV allowed us to easily verify its infectivity in all tested human cell lines, although the infection efficiencies were lower than that in PK15 cells. Meanwhile, the antiviral activities of harmine and PHA767491 were also conveniently validated in vitro, as directly reflected by the reduced EGFP signals. These results demonstrate that this recombinant PRV virus should be a useful tool for basic virology researches and antiviral agent screening.

Faecalibacter bovis sp. nov., isolated from cow faeces.

A Gram-stain-negative, non-spore-forming, yellow-pigmented, aerobic, pleomorphic rod-shaped bacterium, designated ZY171143T, was isolated from faeces of a cow with diarrhoea in Wenshan, Yunnan Province, south-west China and its taxonomic position was studied. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZY171143T belonged to the family Weeksellaceae and was most closely related to the only species of the genus Faecalibacter, Faecalibacter macacae CCTCC AB 2016016T with a sequence similarity of 97.8 %. The genomic OrthoANI and digital DNA-DNA hybridization values between the strain and F. macacae CCTCC AB 2016016T were 86.2 and 30.5 %, respectively. The genomic G+C content was 31.1 mol%. The predominant fatty acids (>5 %) were C15 : 0 iso, C17 : 0 iso 3OH, C16 : 0, C16 : 1 ω5c and summed feature 3 (C16 : 1 ω7c and/or 16 : 1 ω6c). The major polar lipids were phosphatidylethanolamine, triacylglycerol and sulfonolipid. The sole respiratory quinone was MK-6. These chemotaxonomic characterizations also revealed that strain ZY171143T was a member of the genus Faecalibacter. Based on the phenotypic, chemotaxonomic and genotypic data, strain ZY171143T represents a novel species within the genus Faecalibacter, for which the name Faecalibacter bovis sp. nov. is proposed. The type strain is ZY171143T (=CGMCC 1.13663T=KCTC 62642T).

Mannheimia bovis sp. nov., Isolated from a Dead Cow with Hemorrhagic Pneumonia.

Strain ZY190616T was isolated from lung of a dead cow with hemorrhagic pneumonia in Yunnan Province, China. The strain was Gram-stain-negative, facultatively anaerobic bacterium. Phylogenetic analysis based on 16S rRNA gene sequence indicated that the strain was closely related to species of the genus Mannheimia and formed an independent clade with M.varigena CCUG 38462 T (97.0% similarity). Phylogenetic analysis based on recN gene indicated that the strain formed a clade with M.caviae CCUG 59995 T (87.8% similarity). Phylogenetic analysis based on rpoB gene indicated that the strain formed a clade with M.varigena CCUG 38462 T (94.7% similarity). The genomic OrthoANI values between strain ZY190616T and M. ovis, M.haemolytica and M.granulomatis were 84.5%, 82.7% and 81.9%, respectively. The genomic G + C content was 39.8 mol%. The predominant fatty acids (> 5%) of the strain were C16:0, C14:0, C18:1ω7c, summed feature 3 (C16:1 ω7c and/ or C16:1ω6c) and summed feature 2 (C14:0 3OH/ C16:1 Iso). The major polar lipids were phosphatidylglycerol (PG), phosphatidylethanolamine (PE), monophosphatidylglycerol (MGDG), triacylglycerol (TAG) and diphosphatidylglycerol (DLCL). The sole respiratory quinone was CoQ-7. Based on evidence from the taxonomic study, strain ZY190616T represents a novel species of the genus Mannheimia, for which the name Mannheimia bovis sp. nov. is proposed. The type strain is ZY190616T (= CCTCC AB 2020168 T = KCTC 25018 T).

Mannheimia ovis sp. nov., Isolated from Dead Sheep with Hemorrhagic Pneumonia.

Two Gram-stain-negative, facultatively anaerobic bacteria, designated ZY170218T and ZY180512, were isolated from lungs of dead sheep with hemorrhagic pneumonia in Yunnan Province, China and their taxonomic positions were studied by a polyphasic approach. The two isolates grew optimally at 37 °C, pH 9.0 and 1.0% NaCl (w/v), and showed identical 16S rRNA, recN and rpoB gene sequences. Phylogenetic analysis based on 16S rRNA gene sequence showed that the two strains fell within the cluster of species in the genus Mannheimia and formed a separated lineage with comparatively low similarity to the closest related species M. granulomatis (96.5%). Phylogenetic analysis based on rpoB gene indicated that the strains formed a monophyletic evolutionary lineage, with low sequence similarity ≤ 89.0% to the species of the genus Mannheimia. The genomic OrthoANI values between strain ZY170218T and M. granulomatis and M. haemolytica were 80.4% and 83.1%, respectively. The genomic G + C content of strain ZY170218T was 39.1 mol%. The predominant fatty acids (> 5%) of the two strains were C16:0, C14:0, C18:1ω7c, summed feature 3 (C16:1 ω7c and/ or C16:1ω6c) and summed feature 2 (C14:0 3OH/ C16:1 Iso). The major polar lipids of strain ZY170218T were phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, bis(monoacylglycero)phosphate and diacylglycerols. The sole respiratory quinone of the two strains was CoQ-7. On the basis of phylogenetic, phenotypic and chemotaxonomic features, strain ZY170218T and ZY180512 clearly represents a novel species of the genus Mannheimia, for which the name Mannheimia ovis sp. nov. is proposed. The type strain is ZY170218T (= CGMCC 1.13620 T = KCTC 15731 T).

Suicoccus acidiformans gen. nov., sp. nov., isolated from a sick pig.

A Gram-stain-positive, non-spore-forming, catalase-positive and facultatively anaerobic coccus, designated ZY16052T, was isolated from mesenteric lymph nodes of a sick piglet in Kunming, Yunnan Province, PR China and its taxonomic position was studied by following a polyphasic approach. Optimal growth was observed at 37 °C, pH 8.0 and 2 % NaCl (w/v) on Columbia agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZY16052T formed a separated evolutionary lineage from recognized genera of the family Aerococcaceae and shared low similarity to its closest related species Facklamiasourekii (93.8 %) and Ignavigranum ruoffiae (93.4 %). Phylogenetic analysis based on the housekeeping gene recA indicated that strain ZY16052T represented a deep and distinct evolutionary lineage, and was well separated from all genera in the family Aerococcaceae, with very low sequence similarity(≤73.2 %). Sequence analysis based on the housekeeping gene rpoA indicated that strain ZY16052T shared very low similarity ≤77.0 % to related genera. The genomic OrthoANI values between strain ZY16052T and type species of related genera in the family Aerococcaceae and species in the genus Facklamia were ≤67.77 and ≤68.11 %, respectively. The genomic G+C content was 42.3 mol%. The predominant fatty acids (>5 %) were C16 : 0, C18 : 1ω9c, C14 : 0 and summed feature 5 (C18 : 2ω6,9c and/or C18 : 0 ante). The major polar lipids were digalactosyldiacylglycerol, phosphatidylglycerol, diacylglycerols, triacylglycerol and phosphatidic acid. The peptidoglycan contained the amino acids lysine, glycine, alanine and glutamic acid, which is characteristic of peptidoglycan type A1a. Based on the phylogenetic and phenotypic evidence, we propose that the unknown bacterium be classified as Suicoccus acidiformans gen. nov., sp. nov. The type strain of Suicoccus acidiformans is ZY16052T (=CCTCC AB 2017017T=DSM 105755T).

Emergence and pathogenicity of highly pathogenic Porcine reproductive and respiratory syndrome virus in Vientiane, Lao People's Democratic Republic.

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) characterized by high fever, high morbidity, and high mortality in pigs of all ages emerged in China in 2006 and spread rapidly throughout Southeast Asia. In July 2010, a highly contagious swine disease with clinical signs similar to those of HP-PRRS was observed in the Lao People's Democratic Republic (Lao PDR). A field investigation covering 8 pig farms and 1 slaughterhouse in 7 different districts in the capital city of Vientiane was conducted to identify the disease. Total mortality rates ranged from 6.02% in boars to 91.28% in piglets (mean 54.15%) across the farms investigated. Emergence of the HP-PRRS virus (HP-PRRSV) in Lao PDR was confirmed using real-time reverse transcription polymerase chain reaction as well as virus isolation and identification. An animal inoculation study was performed to characterize the HP-PRRSV responsible for this outbreak. Isolate Laos 1.13 was inoculated into 70-day-old specific pathogen-free pigs to study pathogenicity. Clinical signs of high fever, rubefaction, respiratory distress, nervous symptoms, and diarrhea were observed in inoculated pigs, as well as pathological hemorrhagic lesions consolidated in the lungs. Morbidity and mortality were 100% and 60%, respectively, in inoculated pigs. HP-PRRSV was re-isolated from the inoculated pigs. Results suggested that the newly emerged HP-PRRSV was responsible for recent outbreaks of the swine disease in Lao PDR. The current report highlights the importance of continuous surveillance in neighboring countries to prevent introduction of PRRS to new regions.