The infection of Toxoplasma gondii is demonstrable among man and animals. Ir. Taiwan, Toxoplasma gondii has been isolated from swine, pork, canine, and wild rat. Therefore, toxoplasmosis has been become an important problem in the view of public health point of view.
It was used to detect the toxoplasma in organs with Giemsa ,Wright, Hematoxylin and Eosin, when a sample of infected tissue contains a few organisms, these method was then insufficient, a most reliable method must be established.
The fluorescent antibody technique was first described by Coons et al in 1942 and almost completed by them in 1950. It was first applied to the detection of Toxoplasma gondii by Goldman in 1957.
The present study was to manufacture fluorescent antibody (FAb) and to examine the stain titer, nonspecific, and preservation of the manufactured「\b and also applied this FAb to examine the infected organisms, field cases, the multiplication of Toxoplasma gondii on PK-15 cell line, and the pathological changes of swine toxo. The results obtained are summaried as follows :
1.Artificially immuneds erum was collected from experimental pigs which previously were orally infected with mice containing toxoplasma. The serum was fractionated with ammonium sulfate in order to obtain globulin. The globulin then was conjugated with fluorescein Isothiocynate (FITC), for the purpose of removing the uncon」ugated fluorescent materials(UFM)and nonspecific staining substance, the fluorescein con」ugated antibody was absorbed with Sephadex and DEAE, so manufactured FAb was applied to examine the various organ of infected mice, field swine cases and inoculated PK-15 cell. The specific fluorescent Toxoplasma was easily recognized.
2.The manufactured FAb after absorption with DEAE was usable, when preserved at 1°C for 2 months. The toxoplasma contained in specimen of organs could be µreserved without loss of antigenicity at 4°C and -20°C for at least 2 months after fixation and at room temperature for one week, at 4°C for 3 weeks after staining. The frozendried products of FAd retain original stain titer and show ideal results.
3.The process controlled the fixation with aceton for 15min and staining with manufactute<l for 30 min. was applied to examine the the infected mouse organs. Toxoplasma was specifically recognized in the liver, spleen, lung, kidney, lymph nodes. and pancreas in large numbers and in ovary, testis, adrenal gland,heart, intestine in small numbers. From.the submitted field swine cases, the toxoplasma also was identified in the liver, lung, 如dney, lymph nodes.
4.The PK-15 cell line was inoculated with toxoplasma RII strain. After the period of cultivation for 3, 6, 9, 12, 24, 36, 48 th hours and 3, 4, 5th day, the infected PK-15 was stained with manufactured FAb The results showed that the toxoplasma was recognized after 3 hrs一cultivation. At 6 hrs after inoculation, paired toxoplasma was found in the nucleus. After incubation with 12 hrs, the number of toxoplasma <lid not show increasing alternation. After 24hrs incubation, pair-formed toxoplasma showed a slight increase.
After 36 hrs一incubation,a large amount of paired toxoplasma were noted. While incubat ion from 3-5 days, a focal accumulation of toxoplasma was observed, this showed a rapid multiplication. In this experiment, the serial dilution of toxo. was inoculated on PK-15 cell, the quantity of toxoplasma stained with fluorescein-labelled antibody was also studied, The results showed when the quantity of toxoplasma inoculated on PK-15 cell was small, the rate of multiplication was slow, the toxoplasma showed distinct multiplication on the third day with the serial dilution.
5.The pathological examination of the submitted pigs died of toxoplasmosis was carried out. Through gross examination, the lung was watery, it appeared interstitial edema, in the more severe cases, the out surfaces of it appeared as marble structure. The. lymph nodes at different location were swollen moderately with watery appearance. The ascite with slightly cloudy appearance was found in the peritoneum.
The alveolar walls of lung on microscopic examimtion were rich in cells consisted of small round cell and monocyte, the edema in lung was often found. while the focal necrosis of the liver, spleen, lymph nodes Kidney could be found. The brain was found with slightly nonsuppurative encephalitis.