Histopathological Observation And The Preparation Of Diagnostic Antigen Of Demodicosis Canine

Three dogs with severe demodicosis were euthanatized, and the parotid lymph node, mandibular lymph nodes, retropharyngeal lymph node, proper axillary lymph nodes, accessory axillary lymph nodes, superficial cervical lymph nodes, sternal lymph nodes, popliteal lymph node, superficial inguinal lymph nodes, tracheobronchial lymph nodes, renal lymph nodes, gastric lymph node, hepatic lymph nodes, and lung, liver, kidney, spleen, heart and skin, were sampled for determination of Demodex canis with 2 assays of macerating samples in sodium hydroxide and histological examination.The results showed that adults, nymphs, larvae, and eggs of Demodex canis were present in sampled lymph nodes but tracheobronchial lymph nodes, renal lymph nodes, gastric lymph node and hepatic lymph nodes examined with the 2 assays. All the sampled lymph nodes showed variable degrees of proliferative, hemorrhagic and catarrhal lymphadenitis.Demodex canis was observed for the first time in the capsular lymph vessels and the cortex lymphatic sinus of the examined lymph nodes, but not in the medulla. Small amount of Demodex canis adults, nymphs, larvae, and eggs were observed in lung and liver samples macerated in 5% sodium hydroxide, but not in blood clots presented in spleen, kidney and heart. No Demodex canis adults, nymphs, larvae, and eggs were found in any of the sampled internal organs by histological examination.Hyperkeratinization, hair follicle dilatation, cross section of the parasite body, inflammatory infiltration of perifollicular tissue mostly with lymphocytes, were found in skin section, and in some cases, rupture of the follicular wall, parasites invasion of dermis, and proliferation, dilatation and heperemia of the dermis capillary, and edema of the perivascular tissues were also observed.After measuring of the parasite size that parasitized in skin and lymph nodes, the adults in lymph nodes were found significantly smaller than that in skin (P<0.01 by t test), but no significant.50.size differences were observed between the nymphs and eggs parasitized in skin and those in lymph nodes(P>0.05 by t test). In lymph nodes, the mean size of the parasite is 216.5 U mX40.7 u m for adults, 154.7 u m X 35.5 u m for nymphs, 81.3 u m X 33.2 u m for eggs, and in skin, the size is 263.2 v. m X45.5 u m, 169.4 n m X 38.1 u m and 83.1 u m X 33.2 u m respectively.The skin scrapings in the location of serious skin lesion were harvested and macerated in 5% sodium hydroxide for 2h, then the parasites were purified. By freezing and thawing the purified parasites and subsequently grinding, ultrasonication, and centrifugation at 8000r/min for 20 min, the supernatant was harvested as crude sodium-soluble antigen, and the pellet were mixed with urea solution and then ultrasonicated again, and with subsequently centrifugation at 8000rpm/min for 20 min, the supernatant was harvested as crude urea-soluble antigen. By Bradford assay determination, the harvested protein concentration is 1.4mg/ml for sodium-soluble antigen and l.lmg/ml for urea-soluble antigen.By separating the two kinds of crude antigens with Sephadex G-100, 2 main proteins with molecular weight of 45.7KDa and 27.5KDa from sodium-soluble antigen, and 2 main proteins with molecular weight of 46.8KDa and 27.5KDa from urea-soluble antigen were obtained. The 46.8KDa protein has strong reactivity with positive canine serum, and has some reactivity with pig positive serum. By testing 12 clinical cases of demodicosis and 19 clinical healthy cases, the obtained antigen was found had potential application in diagnosis of demodicosis.