动物造模,药效评价,莱姆病 z-bio 2024-08-19 1409

　　1. The modeling method involves selecting dogs that are not infected with other pathogens, aged 6-8 weeks, and each animal has its own independent room. They are fed with commercial feed and sterile drinking water. Expose the puppy to an environment containing a full ditch tick carrying Borrelia burgdorferi, which is from a high-risk area for Lyme disease in China. After laboratory testing, it was found to carry the pathogen and was kept in an environment of 10 ℃ and 94% humidity before being used in the experiment. Cut off the hair on the left chest of the experimental animal, place the tick carrying the pathogen in that area, make its body completely congested by biting the host, and remove it on the 7th day. To ensure complete infection of the experimental animals, the above experiment was repeated on day 14. Daily clinical sign testing is conducted to check the temperature, weight, affected limbs, and swelling of the puppy. After the hard tick bites the experimental animal dogs, only a few model animals showed a temperature increase of 39.4 ℃ or above for 1 day, occasionally with a temperature increase lasting for 2 days. Dogs infected with Lyme disease may experience limb or one limb limp, which can last for 3-5 days without any medication. After approximately 71 days of infection in the model animal, limping appeared, mainly in the left front leg of the animal near the tick bite site, which is prone to arthritis symptoms. As the infection time prolongs, limping can be seen in all limbs of the experimental model, and it occurs every 2-14 days.

　　2. Model checking methods

　　(1) Serological testing: Two weeks after the initial infection, serum samples from the model are taken for testing of antibodies against Borrelia burgdorferi. Using computer-controlled dynamic enzyme-linked immunosorbent assay technology for testing. Serum samples from all animal models were tested together to avoid bias and error in experimental results. Within 90 days after infecting experimental dogs with Borrelia burgdorferi, there was a significant increase in the potency of antiviral spirochetes in the body, followed by only a slight increase in antibody potency thereafter.

　　(2) Pathogen isolation and culture: After 4 weeks of modeling, euthanize the animal model and collect tissues from 25 parts including skin and heart. To avoid cross infection, use a set of surgical instruments for each animal model. The collected tissues include skin tissue near the bite site, tissues from six synovial regions (shoulder, elbow, knee), forelimb muscles and fascia (triceps, forelimb fascia), neck skin, armpit skin, lymph nodes, pericardium, etc. 0.5g of the obtained tissue was cultured in 9ml BSK medium, and after 5 weeks, live B.burgdorferi spirochetes could be detected under a microscope. Place the skin and lymph nodes at the bite site of the model in BSK medium and culture at 32 ℃. After 5 weeks, live B.burgdorferi spirochetes can be detected under a microscope.

　　(3) Polymerase chain reaction (PCR) detection: PCR can be used to detect specific DNA fragments of Borrelia burgdorferi in animal blood and tissues. This method can be used to detect Borrelia burgdorferi DNA in blood, skin, tissue, and cerebrospinal fluid. The skin sample culture and PCR results of the model dog showed positive for Borrelia burgdorferi infection.

　　(4) Histopathological examination: Euthanasia was performed on experimental model dogs infected for 505-600 days. The tissues associated with the limbs (knees, ankle joints, and toe bones) were placed in a 10% formalin solution and dehydrated, embedded, and sliced. Standard hematoxylin and eosin staining was performed on the tissue slides, and under an inverted optical microscope, the joints of the model animals showed varying degrees of synovial thickening and proliferative synovitis, with mononuclear cell infiltration visible below the synovium; Mononuclear cells can be seen around the blood vessels near the joints.

　　(5) Joint synovial fluid and cerebrospinal fluid testing: After euthanizing experimental animals, 3ml of joint fluid from the dog's limb joints was taken for post-mortem examination, diluted with physiological saline at a ratio of 1:10, and the total number of cells was counted using a blood cell counter. Approximately 3ml of cerebrospinal fluid was collected from a percutaneous puncture model dog for cell counting and classification.

　　3. Model Characteristics: Due to the close relationship between dogs and humans, tick infections can be transmitted from dogs to humans, making them one of the main targets of tick bite infections. Animal models of Lyme disease infection in dogs are prone to typical acute and recurrent limping, accompanied by purulent arthritis of the joints, which can develop into chronic arthritis, and in severe cases, symptoms such as limping may occur. This model is easy to study the mechanism, manifestations, drug treatment, and other related research of Lyme disease infection and arthritis lesions.