EVALUATION OF VITALITY OF SARCOCYSTS IN BEEF BY THE DAPI FLUORESCENCE TEST

Koudela B., L. Steinhauser: Evaluation of Vitality of Sarcocysts in Beef by the DAPI Fluorescence Test. Acta vet. Brno, 53,1984: 193-197. Vitality of Sarcocystis sp. cystozoites in beef was evaluated by means of the DAPI fluorescence test. Results were verified by biological assays in definitive hosts. Sarcocysts remained vital in beef kept at refrigerator temperature over a period of 20 days after slaughter. They did not survive in frozen ( -18°C, 48 hours), cooked (70 °C, 10 min.), and pickled (12 % sol. curing mixture, 12°C, 72 hours) meat. Preliminary experiments with other protozoa indicate that the DAPI fluorescence test will meet application in different parasitological topics. Cystozoite, fresh meat, chilled meat, frozen meat, cooked meat, pickled meat. There are three species of the genus Sarcocystis known in muscle tissue of cattle. Their definitive hosts are different and so are apparently their pathogenic effects on the intermediate host: S. cruzi has the dog as definitive host and acts as pathogenic organism for cattle, S. hirsuta with the cat as definitive host is less pathogenic for the ox; S. hominis who has the man as definitive host, seems not to be pathogenic for the intermediate host. Several ways have been designed how to evaluate vitality and infectivity of sarcocysts in beef meat. Gestrich and Heydorn (1974), Fayer (1975), Golubkov (1978), and Leek and Fayer (1978) performed assays in definitive hosts. Beside biological experiments, in vitro cultivation and dye-colouring by methylen or trypan blue were recommended (Bergler et aI., 1980). Our goal was to examine the availability of the DAPI fluorescence test fot the same purpose. DAPI (4,6-diamidino-2-phenylindol) (SERVA FEINBIOCHEMICA) is a substance with soecific affinity to intracellular DNA and emits intensive fluorescence in the UV light (Russel et aI., 1975). Schilling et al. (1979) and Niemann et al. (1981) worked with the DAPI fluorescence test to determine vitality of early embryonic stages in cattle and rabbit. After incubation of embrya with DAPI, nuclei of dead cells emitted fluorescence whereas the live ones did not. Russel et al. (1975) and Hyman and MacInnes (1979) based detecting of intracellular parasites, mycoplasms and viruses on the specific activity of DAPI to DNA. Materials and Methods Meat samples From 10 at random chosen cows aged 3 to 5 years, samples of diaphragmal muscles and m. longissimus dorsi were collected at each stage of meat processing: A. Fresh meat Samples of 100-150 g were collected one hour after slaughter. B. Chilled meat As the next step in meat processing, beef halves were cooled down to 16°C in the core by a stream of cold air in a quick-chilling tunnel for 10 hours, then cut to quarters and stored in the chilling

There are three species of the genus Sarcocystis known in muscle tissue of cattle.Their definitive hosts are different and so are apparently their pathogenic effects on the intermediate host: S. cruzi has the dog as definitive host and acts as pathogenic organism for cattle, S. hirsuta with the cat as definitive host is less pathogenic for the ox; S. hominis who has the man as definitive host, seems not to be pathogenic for the intermediate host.
Our goal was to examine the availability of the DAPI fluorescence test fot the same purpose.DAPI (4,6-diamidino-2-phenylindol) (SERVA FEINBIOCHEMICA) is a substance with soecific affinity to intracellular DNA and emits intensive fluorescence in the UV light (Russel et aI., 1975).Schilling et al. (1979) and Niemann et al. (1981) worked with the DAPI fluorescence test to determine vitality of early embryonic stages in cattle and rabbit.After incubation of embrya with DAPI, nuclei of dead cells emitted fluorescence whereas the live ones did not.Russel et al. (1975) and Hyman and MacInnes (1979) based detecting of intracellular parasites, mycoplasms and viruses on the specific activity of DAPI to DNA.

Meat samples
From 10 at random chosen cows aged 3 to 5 years, samples of diaphragmal muscles and m. longissimus dorsi were collected at each stage of meat processing: A. Fresh meat Samples of 100-150 g were collected one hour after slaughter.

B. Chilled meat
As the next step in meat processing, beef halves were cooled down to 16°C in the core by a stream of cold air in a quick-chilling tunnel for 10 hours, then cut to quarters and stored in the chilling room at 12 cC.To register the progress of meat ageing, pH -values were measured by a needle electrode (ORION pH/temp.,model 221).Meat samples were collected 6, 24, 48, 72 and 120 hours after slaughter.From day 5 to 20, the samples were stored in a refrigerator at 4°C, and, examined in intervals of 3 days.
C. Frozen meat Meat samples of 200 -300 g each were sealed in PE-bags, frozen to --18°C and kept at this temperature fo 48 hours.Thawing took place in a refrigerator at 4 ac.
D. Heat-processed meat Samples were considered cooked when temperature in the core remained at 70 "C for at least 10 minutes.
E. Pickled meat Meat samples of 50-100 g each were pickled in a 12°~ solution of the standard curing mixture (94 % NaCl, 0.5 NaNO,) over 72 hours ast 12°C.

Isolation and purification of Sarcocystis sp. cystozoites
In order to isolate cystozoites, meat samples were exposed to digestion in artificial peptic juice (Sharma and Dubey 1981) for 10 minutes, and, purified by means of the chromatographic gel Spheron (Koudela in press).
Effect of peptic juice on Sarcocystis sp.cystozoites Vitality of cystozoites was evaluated after 60, 90, and 120 minutes of exposure to artificial peptic juice.

Effect of meat processing on isolated cystozoites
Cystozoites isolated from fresh meat were suspended in PBS and exposed to conditions imitating meat processing.They were examined after 5,10,15,20,25,30,45,60, and 90 minutes of exposure to 55°C or 70 °C.Isolated cystozoites were kept at -18°C for 48 hours and then examined.The effect of the nitrite curing mixture was evaluated after 72 hours of exposure.

Verification of vitality of cystozoites by assays in definitive hosts
To verify our classification of cystozoites, assays were carried out with four whelps aged two months and three kittens aged three months.Two pups and two kittens were fed meat from which isolated cystozoites displayed no fluorescence.Two pups and one kitten were fed meat from which isolated cystozoites emitted fluorescence.Parasitological examinations of their faeces were carried out daily over the period of 20 days.

Results
The evaluation of vitality of Sarcocystis sp.cystozoites is demonstrated in Table 1.Living cystozoites emitted no fluorescence, while there was an intensive fluorescence of nuclei in dead cystozoites.Fragments of cystozoites only could be isolated from frozen, cooked, or pickled meat samples.As far as there was a cell nucleus within the fragment, it emitted intensive fluorescence.
The process of isolation and purification had rio effect on cystozoites.Their exposure to peptic juice for 120 minutes resulted in no alteration in vitality.
The effect of meat processing was illustrated by experiments with isolated cysto-zoites.Exposure to the temperature of -18 C for 48 hours affected the destruction of cystozoites.Likewise destroyed were cystozoites treated by the nitrite curing mixture.The effect of high temperature on isolated cystozoites is shown in Table 2.  Whelps and kittens fed meat from which isolated cystozoites emitted no fluorescence, began elimination by day 11.Animals fed meat from which isolated cystozoites emitted fluorescence, eliminated no sporocysts.

Discussion
The results of our evaluating vitality of sarcocysts by means of the DAP! fluorescence test are in agreement with the results achieved by biological assays carried out by Gestrich andHeydorn(1974), Payer (1975), Golubkov (1978), and Leek and Payer (1978).
Morphological and biological similarities of sarcocysts and Toxoplasma gondii suggest comparison with analogical assays on vitality and infectivity of toxoplasma cysts (Jacobs et aI., 1960;Sommer et aI., 1965;Dubey 1974).The vitality of toxoplasma cysts and sarcocysts in fresh, chilled, frozen and cooked meat is similar, and so is the survial of bradyzoites of Toxoplasma gondii and cystozoites of Sarcocystis sp. in peptic juice (Sharma and Dubey, 1981).
Determining vitality of cystozoites in beef after different processing is of considerable hygienic and epizootiological importance.
that nonspecific fluorescence was not observed.The only fragments of cystozoites with broken structure of cell components emitted weak fluorescence.The intensity of fluorescence depends on the concentration of DAP!.While testing various concentrations of D API the concentration recommended by N i emann et al. (1981) (1 : 100000) was found optimum.

Table 2
Effect of temperature on vitality of isolated S"rcocysts sp.cystozoites from beef