Background: Bluetongue disease of ruminants is a typical insect-borne disease caused by bluetongue virus (BTV) of the genus Orbivirus (family Reoviridae) and transmitted by some species of Culicoides (Diptera: Ceratopogonidae). Recently, the detection of BTV in yaks in high altitude meadows of the Shangri-La district of Yunnan Province, China, prompted an investigation of the Culicoides fauna as potential vectors of BTV. Methods: A total of 806 Culicoides midges were collected by light trapping at three sites at altitudes ranging from 1800 to 3300 m. The species were identified based on morphology and the DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in Culicoides spp. A phylogenetic analysis was used to analyze the cox1 sequences of some specimens. Results: Four species dominated these collections and cox1 barcoding revealed that at least two of these appear to belong to species new to science. Culicoides tainanus and a cryptic species morphologically similar to C. tainanus dominated low altitude valley collections while C. nielamensis was the most abundant species in the high-altitude meadow. A species related to C. obsoletus occurred at all altitudes but did not dominate any of the collections. BTV RT-qPCR analysis detected BTV RNA in two specimens of C. tainanus, in one specimen closely related to C. tainanus and in one specimen closely related to C. obsoletus by barcode sequencing. Conclusions: This study suggests that BTV in high altitude areas of Yunnan is being transmitted by three species of Culicoides, two of which appear to be new to science. This research may be useful in improving understanding of the effects of global warming on arboviral disease epidemiology and further study is important in research into disease control and prevention.

Contagious Eczema (CE), caused by ORFV, impacts sheep and goats globally, with severe symptoms and economic losses. The ORFV situation in Yunnan, China, was unclear before 2021–2023 study. Eleven scab samples from goats on small farms in three Yunnan municipalities were collected. Four ORFV strains were isolated and characterized using scanning electron microscopy, cytopathic effect observation, and PCR. Phylogenetic analyses of ORFV011 and ORFV059 genes showed significant results. For ORFV011, the nucleotide similarity of the four strains to D1701 strain was 98.4–99%. For ORFV059, it was 97.2–97.9% with OV-SA00 strain. These findings suggest gene rearrangements and interactions among strains during Yunnan’s ORFV outbreak, forming a unique evolutionary lineage. Our study is the first comprehensive one on Yunnan’s ORFV prevalence with in-depth phylogenetic analysis. It has important implications. In vaccine development, understanding genetic variances helps create better vaccines. For disease control, customized strategies like targeted quarantine and disinfection can be designed based on strain characteristics. From a public health aspect, as CE is zoonotic, closely monitoring ORFV in goats aids in predicting and preventing human infections, thus being significant for protecting goats against CE in Yunnan.

Introduction: Research on Mycoplasma canis remains limited, possibly because dogs in most regions are usually not raised on a large scale or in intensive farming. Furthermore, existing antibacterial drugs are often proven to be effective in inhibiting or eradicating M. canis , resulting in insufficient attention being paid to its pathogenic potential. However, with the widespread use of antibacterial drugs, an increase in drug resistance has been found in human mycoplasma pneumoniae , bovine mycoplasma and avian mycoplasma also faces similar challenges. Given the irreplaceable role of dogs as companion animals and work partners, it is crucial to enhance research on Mycoplasma canine, especially in the areas of epidemiology, diagnostic techniques, drug resistance mechanisms and vaccine development. This study found that M. canis inoculated into liquid Pleuropneumonia-Like Organisms (PPLO) medium at 37 °C could reach the growth peak within 24 h and maintain this level for approximately 24 h. The survival rate is higher in environments with PH values of 6.5, 7.0 and 7.5 respectively, and it is suitable for storage in neutral or weakly acidic environments. 5 mM of BEI inactivated agent can completely inactivate M. canis within 15 h, and the experimental dogs produced specific antibodies within 1 week after vaccination with the inactivated vaccine, with a positive rate of 100%. It provides a theoretical basis and experimental evidence for the subsequent in-depth exploration of the pathogenic mechanism of M. canis and the development of its vaccine. Methods: Researchers collected eight lung tissue samples from working dogs exhibiting typical respiratory symptoms in Kunming City, Yunnan Province. From these samples, one strain of canine mycoplasma was successfully isolated. To characterize this isolate comprehensively, a series of technical approaches were employed. The growth dynamics of the mycoplasma strain were assessed by monitoring color changes in the culture medium. Molecular identification was conducted using polymerase chain reaction (PCR). Additionally, preliminary studies were undertaken regarding its biological characteristics and the development of an inactivated vaccine; these efforts provide both theoretical foundations and experimental support for future investigations into the pathogenic mechanisms and vaccine development for M. canis. Results: The findings indicated that M. canis could grow stably within PPLO medium and was confirmed as belonging to the M. canis genotype through 16S rRNA gene sequence analysis. The freshly cultivated strain was inoculated into liquid PPLO medium at a concentration rate of 1%. It reached its peak growth within 24 h at 37 °C and maintained this level for approximately another 24 h. Subsequently, the number of viable bacteria gradually declined. In environments with pH values of 6.5, 7.0, and 7.5 respectively, the survival rate of viable bacteria was significantly higher than that observed at pH 8.0. Therefore, it is recommended that the M. canis strain preserved for seed use be maintained in a neutral or weakly acidic environment. Additionally, the BEI inactivating agent at a final concentration of 5 mM can completely inactivate the strain within 15 h. Four experimental dogs developed specific antibodies within 1 week after being vaccinated with the inactivated vaccine, achieving a positive rate of 100%, which preliminarily confirms that the vaccine elicits an immune response. Conclusion: This study successfully isolated and identified M. canis , investigated its growth characteristics in PPLO medium, and established a method for preparing inactivated vaccines along with preliminary evaluations thereof. These findings provide a foundation for further research into the infection dynamics and pathogenicity of M. canis while offering valuable insights for preventing and controlling M. canis infections.

Bluetongue is an insect-borne disease of ruminants caused by the Bluetongue virus (BTV). It is primarily transmitted through the bites of Culicoides midges and is widely distributed in tropical, subtropical, and temperate regions around the world. Due to its favorable geographical location and abundant population of BTV vector midges, Yunnan Province, China, has become a key region for research on BTV diversity. However, there are gaps in research on the genetic evolution of BTV in this region. Between January 2024 and December 2025, Culicoides midges were collected from three border counties in Yunnan Province. Researchers isolated BTV strains and sequenced the full-length segment 2 (Seg-2) gene. Subsequently, phylogenetic analysis, Bayesian evolutionary rate analysis, population dynamics analysis, and phylogeographic analysis were performed utilizing Seg-2 sequence data sourced from the NCBI database. Four BTV serotypes (BTV-1, BTV-4, BTV-5, and BTV-16) were identified. The estimated average evolutionary rate varied from 2.88 × 10⁻⁴ to 4.26 × 10⁻³ substitutions per site per year, with ancestral origins dating back to the period between 1872 and 1988. The effective population size displayed significant fluctuations over time. Phylogeographic reconstructions indicated that the pathogen entered Yunnan through various transcontinental transmission pathways, originating from Europe, South Asia, Oceania, and East Asia, with evidence of reciprocal transmission between Africa, Europe, and West Asia. The BTV serotypes circulating in Yunnan Province are highly diverse, and the population dynamics of different serotypes exhibit significant heterogeneity. Yunnan is simultaneously exposed to BTV introductions from multiple directions, including Europe, South Asia, Oceania, and East Asia.

Bluetongue (BT) virus (BTV) is an arthropod‐borne pathogen that causes substantial economic losses in ruminants globally. Yunnan Province, located in China’s tropical and subtropical border region, faces a high risk of BTV circulation. This study monitored BTV vectors, genetically characterized circulating BTV strains, and assessed seroprevalence to inform early warning efforts. In 2025, 46,000 Culicoides midges were collected from three counties, identified, and grouped into 460 pools. Additionally, 5934 cattle serum samples from 17 border counties were tested using C‐ELISA. Midge pools were screened by RT‐qPCR for BTV nucleic acids and then inoculated into C6/ 36 cells for virus isolation. Whole‐genome sequencing and phylogenetic analysis of VP2 and VP5 genes were performed on the isolates. C. oxystoma and C. trithecoides were the dominant midge species at all sites. RT‐qPCR identified 11 BTV‐positive midge pools, primarily involving C. oxystoma , C. imicola , C. tainanus , and C. jacobsoni . Four BTV isolates were identified as serotypes 1, 4, 5, and 16. Phylogenetic analysis of VP2 and VP5 showed all four strains likely belong to the Eastern topotype and are closely related to historical BTVs from China, Japan, India, and Australia. The overall BTV seroprevalence in cattle was 41.37% (95% confidence interval [CI], 40.12%−42.62%), with significantly higher rates in autumn (58.07%, 95% CI, 56.29%−59.85%) than in spring (24.87%, 95% CI, 23.32%−26.42%). Several BTV serotypes have been detected and isolated from diverse Culicoides in the border regions of Yunnan. Additionally, BTV seroprevalence has been observed in local cattle populations, indicating that this area may represent a significant BTV endemic hotspot. These findings underscore the need for ongoing vector surveillance and early warning systems to prevent BT outbreaks in susceptible livestock.