Mycoplasma gallisepticum (MG) poses a significant threat to Bangladesh's poultry industry, causing substantial economic losses every year. This study aimed to determine the prevalence of MG infection in turkeys using serum plate agglutination (SPA), enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) in Sylhet, Bangladesh from December 2019 to November 2020. In addition, we evaluated the diagnostic accuracy of these tests and identified potential risk factors associated with MG infection. A total of 250 blood samples and 250 tracheal swabs were collected from suspected turkeys across 25 farms from three sub‐districts of Sylhet namely Sylhet Sadar, Golapganj and Beanibazar. Blood samples were tested with SPA and ELISA, while tracheal swabs were analysed by PCR targeting the 16S rRNA gene of MG. The overall prevalence of MG was 35.2%, 29.2% and 25.6% for SPA, ELISA and PCR respectively. Higher infection rates were observed in turkeys aged 0–4 months (SPA 57.1%, ELISA 52%, PCR 42.8%), during winter (SPA 43.1%, ELISA 37.8%, PCR 30%) and among female turkeys (SPA 54.5%, ELISA 49.5%, PCR 45.5%). Geographically, the Beanibazar had the highest prevalence (SPA 54.2%, ELISA 48.6%, PCR 41.4%), compared to the Sylhet Sadar and Golapganj sub‐districts. Both SPA and ELISA tests showed 100% sensitivity, with specificity of 87.1% and 95.2%, respectively using PCR as a gold standard. Overall, these findings provide valuable insights for developing effective control measures for MG infections in the poultry industry of Bangladesh.

Fibroblasts are cells that reside within the fibrous or loose connective tissues of most mammalian organs. For research purposes, fibroblasts are often subjected to long-term culture under defined conditions, during which their properties can significantly change. It is essential to understand and document these changes to obtain reliable outcomes. For the quantification of specific gene expressions, the most reliable and widely used technique is quantitative real-time polymerase chain reaction (qRT-PCR). Here, we assessed the impact of a reference gene’s stability on a qRT-PCR analysis of long-term cultured canine skin fibroblasts. After successfully isolating the fibroblasts from canine skin tissues, they were cultured and evaluated for proliferation and β-galactosidase activity at different passage numbers. With extended culture, the fibroblasts showed a long doubling time and elevated β-galactosidase activity. Using three widely used algorithms, geNorm, Normfinder, and Bestkeeper, we identified HPRT1, YWHAZ, and GUSB as the most stable reference genes for both early- and late-passage fibroblasts. Conventional reference genes such as GAPDH were found to be less stable than those genes. The normalization of Vimentin by the stable genes showed statistical differences, whereas normalization by an unstable gene did not. Collectively, this study indicates that using stable reference genes is essential for accurately and reliably measuring gene expression in both early- and late-passage fibroblasts. These findings provide valuable insights into internal controls for gene expression studies and are expected to be utilized for analyzing gene expression patterns in molecular biology research.

Sang-Yun Lee, Seong-Ju Oh, Rubel Miah, Yong-Ho Choe, Sung-Lim Lee, Yeon Woo Jeong and Young-Bum Son. Journal of Animal Reproduction and Biotechnology 2024;39:95-104. https://doi.org/10.12750/JARB.39.2.95

Feline mesenchymal stem cells (MSCs) isolated from adipose (A-MSCs) and uterine (U-MSCs) tissues during routine ovariohysterectomy represent valuable therapeutic sources in veterinary regenerative medicine. While quantitative real-time PCR (qRT-PCR) remains the gold standard for transcriptional profiling in these cells, its analytical reliability relies on empirically validated reference genes. In this study, we assessed the expression stability of nine candidate reference genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, RPL7, TBP, and YWHAZ) in feline A-MSCs and U-MSCs using the geNorm and NormFinder algorithms. Both computational approaches identified TBP and HMBS as the most stably expressed genes across both cell types, whereas the historically popular GAPDH was the least stable. All geNorm pairwise variation values were below the commonly used threshold of 0.15 in both A-MSCs and U-MSCs. To illustrate the impact of reference gene choice, we evaluated the pluripotency marker OCT4. Normalizing using TBP and HMBS revealed significant differences in OCT4 expression between A-MSCs and U-MSCs (p < 0.05). In contrast, applying the unstable GAPDH masked this difference. These findings identify TBP and HMBS as promising reference gene candidates for qRT-PCR normalization in P3 undifferentiated feline A-MSCs and U-MSCs.