X. METHODS IN CAMPYLORACTER
443
immunofluorescent method may distinguish between C . fetus and C.
bubulus (Bingol and Blobel, 1970). Following the results of Winter et al.
(1967), the fluorescent antibody technique or the cultural technique is
satisfactory for routine detection of carrier bulls. But the presence of C.
fetus cannot be ruled out by either test alone on the basis of a single negative
result.
1. Preparation of specimen
The preputial washing is filtered through a coarse paper (Whatman
No. 1) under slight negative pressure and the filtrate is centrifuged at
1000 x g for 15 min. Then the supernatant fluid is centrifuged for 30 min
at 12,000 x g at 4°C. Smears are made from sediments, fixed, and stained
with a fluorescent conjugate (Winter et al. 1967).
2. The fluorescent conjugate
Immunoglobulins against C. fetus subsp. fetus and subsp. intestinalis are
obtained by immunisation of rabbits (Bingol and Blobel, 1970). Sera possessing an agglutination titre of more than 1 : 1280 are pooled and the immunoglobulins are separated using a DEAE chromatography column
(Bingol et al., 1969). Then 0.04mg of fluorescein isothiocyanate per
milligram protein are used for labelling. The free dye is removed by
filtration on G25 Sephadex coarse (Philpott, 1966; Bingol et al., 1969);
the conjugate is kept at - 80°C.
G. Conservation of C. fetus
Tarrozi's liver broth is an exceptionally good substrate for short-term
preservation of micro-organisms in the laboratory (Ruckdeschel, 1973).
The cultures are incubated over four days at 37"C, closed with a rubber
stopper and stored at room temperature. In this way the micro-organisms
remain viable up to half a year. In the case of reisolation, 0.5 ml from the
shaken-up cultures is added to liquid culture and incubated as described.
For long-term storage, freeze-drying of cultures is the method of choice.
After growth for 36 h on liquid media, the culture is collected with a
pipette and suspended in sterile whole milk at a ratio of 1 : 2. This suspension is freeze-dried in 0.2 ml quantities and kept at 4°C. According to
the investigations of Jakovljevic (1972), cultures have been viable after eight
years. 0.5 ml of normal saline are necessary for rehydration of cultures;
they are then inoculated into liquid media. Mixture of cultures with sterile
horse serum 1 : 2 is also suitable for freeze-drying. Before dispatch of viable
strains, they are inoculated on chocolate media and incubated for two days
using the Fortner method.
443
immunofluorescent method may distinguish between C . fetus and C.
bubulus (Bingol and Blobel, 1970). Following the results of Winter et al.
(1967), the fluorescent antibody technique or the cultural technique is
satisfactory for routine detection of carrier bulls. But the presence of C.
fetus cannot be ruled out by either test alone on the basis of a single negative
result.
1. Preparation of specimen
The preputial washing is filtered through a coarse paper (Whatman
No. 1) under slight negative pressure and the filtrate is centrifuged at
1000 x g for 15 min. Then the supernatant fluid is centrifuged for 30 min
at 12,000 x g at 4°C. Smears are made from sediments, fixed, and stained
with a fluorescent conjugate (Winter et al. 1967).
2. The fluorescent conjugate
Immunoglobulins against C. fetus subsp. fetus and subsp. intestinalis are
obtained by immunisation of rabbits (Bingol and Blobel, 1970). Sera possessing an agglutination titre of more than 1 : 1280 are pooled and the immunoglobulins are separated using a DEAE chromatography column
(Bingol et al., 1969). Then 0.04mg of fluorescein isothiocyanate per
milligram protein are used for labelling. The free dye is removed by
filtration on G25 Sephadex coarse (Philpott, 1966; Bingol et al., 1969);
the conjugate is kept at - 80°C.
G. Conservation of C. fetus
Tarrozi's liver broth is an exceptionally good substrate for short-term
preservation of micro-organisms in the laboratory (Ruckdeschel, 1973).
The cultures are incubated over four days at 37"C, closed with a rubber
stopper and stored at room temperature. In this way the micro-organisms
remain viable up to half a year. In the case of reisolation, 0.5 ml from the
shaken-up cultures is added to liquid culture and incubated as described.
For long-term storage, freeze-drying of cultures is the method of choice.
After growth for 36 h on liquid media, the culture is collected with a
pipette and suspended in sterile whole milk at a ratio of 1 : 2. This suspension is freeze-dried in 0.2 ml quantities and kept at 4°C. According to
the investigations of Jakovljevic (1972), cultures have been viable after eight
years. 0.5 ml of normal saline are necessary for rehydration of cultures;
they are then inoculated into liquid media. Mixture of cultures with sterile
horse serum 1 : 2 is also suitable for freeze-drying. Before dispatch of viable
strains, they are inoculated on chocolate media and incubated for two days
using the Fortner method.