AF24hT2
18 (6 to 7 weeks-old) SPF Beagle puppies were purchased and kept in Good Laboratory Practice (GLP) in National Seed Center of Experimental Dogs, according with GB 14922.2-2011. For ensuring each puppy was negative of T. canis infection, anti-T. canis IgG antibody was examined by indirect ELISA using larval ES antigen; for ensuring each puppy was free of intestinal parasitic infection, puppies stool were examined by floatation method with sugar solutions and routine blood examination was tested by automatic blood analyzer (XT2000 iv; Sysmex, Kobe, Japan). Puppies from the same litters were split equally to reduce background differences. Puppies were divided into three time-point groups: the 24 h group, the 96 h group and the 36 d group. Each time-point group was consisted with infection and control group, and 3 biological repeats were designed. In order to monitor the status of T. canis infection in puppies, fecal eggs of 36 dpi group dogs were examined from 30 to 36 dpi. Lung samples were collected from infection groups that infected orally with 300 infectious T. canis eggs and from control groups that mock infected with saline at 24h post infection (pi), 96 hpi and 36 dpi. Samples were grinded into powders by using liquid nitrogen for RNA extraction. Half lung of each puppy was shredded to recover T. canis larvae for 12 hours at 37 C with 1% mycillin by modified Baermann funnel method (K and K 1990). DNA samples of infectious eggs, and larvae and adults of T. canis recovered from infected puppies were used to amplify the ITS region (Mikaeili et al. 2017). Positive PCR products were sequenced by Sangon Biotech (Shanghai, China). Sequences of T. canis ITS region were aligned by Nucleotide BLAST