《Table 2 PCR detection and isolation of Mycoplasma leachii from milk samples of the inoculated cows》

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《"Mycoplasma leachii causes bovine mastitis: Evidence from clinical symptoms,histopathology and immunohistochemistry"》

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CCU，color changing unit；+，weakly positive in PCR detection；++，moderately positive in PCR detection；++++，strongly positive in PCRdetection；–，negative in PCR detection and M.leachii isolation；NP，these cows were euthanized，and the corresponding detect

The quarters of all M.leachii-inoculated cows were monitored for clinical manifestations throughout the experimental period.Histopathology and IHC detection of the mammary tissues were performed on PIDs 3，6 and 9.The clinical signs and histopathology of the M.leachii-inoculated quarters exhibited a gradual progression to severe mastitis over the 9-day experimental period（Table 1 and Fig.1），and the M.leachii antigen was detected by IHC in mammary tissue from the inoculated quarters of the affected cows on PIDs 6 and 9（Table 1 and Fig.2）.The control quarters of the M.leachii-inoculated cows were histopathologically and immunohistochemically negative on PIDs 3 and 6，but they developed mild mastitis and histopathological changes on PID 9，and the M.leachii IHC signal was also detected in mammary gland epithelial cells of these control quarters（Table 1，Figs.1 and 2）.Throughout the experimental period，however，the quarters of the negative control cow were clinically and histopathologically normal，and the M.leachii antigen was not detected in the mammary tissues of the control cow on PID 9（Table 1，Figs.1 and2）.The clinical signs of the affected mammary glands were swelling and firmness，the produced milk was yellow and contained large ropy clots，and the milk yield of the inoculated quarters was markedly decreased compared to pre-inoculation.These results，particularly the detection of the M.leachii antigen in the mammary gland epithelial cells of the inoculated cows，indicated that the mammary gland lesions were directly caused by M.leachii.