1 Research Associate, Project Directorate on Animal Disease Monitoring And Surveillance (PD_ADMAS), IAH & VB Campus, Hebbal, Bangalore – 560 024, INDIA. 2Department of Microbiology, College of Veterinary and Animal Sciences, Kerala Agricultural University, Mannuthy, Thrissur, Kerala, INDIA. *Corresponding author

Biochemical and restriction enzyme analysis of Corynebacterium pseudotuberculosis (goat) Mohan P, Jayaprakasan V Punnoose KT, Mini A 5:251-254, 2001. Corynebacterium pseudotuberculosis from goat caseous lymphadenitis samples were evaluated against standard reference strains by biochemical and restriction endonuclease analysis (REA) of chromosomal DNA.   The enzymes Eco R1, Bgl II, Pst1 and Bam H1 were used to characterize the organisms.  Both bacteria were similar except for one isolate which fermented lactose. REA of both organisms yielded different restriction patterns digested with Pst I but not with Eco RI, Bam HI or Bgl II. Both strains were nitrate negative. A 3.2-Kb fragment was absent in 4 enzymes whereas 6.6-Kb and 3.6-Kb DNA fragments were found in both strains. The results suggest  a high degree of homogeneity between field isolates and reference strains of C. pseudotuberculosis (goat).
 

Key words: Corynebacterium pseudotuberculosis, lymphadenitis - caprine - restriction endonuclease analysis.

REA of chromosomal DNA has been used to characterize C. pseudotuberculosis isolates. REA enzyme digest patterns of 14 cultures were used to designate and to confirm C. pseudotuberculosis using enzymes BamH1, Hind III and Eco RI to categorize group I nitrate non-reducing and group II nitrate-reducing organism. In the present study,  Eco R1, Bgl II, Pst1 and Bam H1, were used to characterize field isolates and standard reference strain of C. pseudotuberculosis.

Isolation: Caseous samples were collected with sterile swabs. Three local isolates and standard reference strain NCTC- 3450 of C. pseudotuberculosis were streaked on 10% sheep blood agar (Blood agar base was aseptically supplemented with 10% (v/v) defibrinated sheep blood) plates and incubated at 37oC for 72 h and observed for bacterial growth periodically. Hemolytic colonies suspected for C. pseudotuberculosis were examined by Gram staining and  sub-cultured on blood tellurite agar (Nutrient agar was supplemented with 10 percent (v/v) defibrinated sheep blood, 33mg percent potassium tellurite and 0.5g percent glucose). The isolates suspected for C. pseudotuberculosis from blood tellurite agar were taken to stock on blood tellurite agar slants.

Identification:  3 isolates were cultured on 10 percent sheep blood agar and incubated at 37o C for 48 to 72 h and observed for yellowish white, opaque convex colonies with matt surface. The isolates were also cultured on blood tellurite agar and incubated at 37o C for 48 to 72 h.

Biochemical tests: Identification of C. pseudotuberculosis was carried out using methods described by Muckle and Gyles, 1982,Garg et al., 1985 and Holt. et al.  (1994)

Bacterial DNA isolation: Cell harvest, lysis and DNA extractions were done following the procedures described by Groman et al. (1984).

Restriction enzyme digestion of DNA: Restriction enzyme digestion was carried out in 20ul volume. The following components were added to sterile microfuge tubes: DNA volume containing 2ug,  10x RE buffer 2ul, restriction enzymes 10 to 15 units and distilled water 20ul. The digestion mixture was kept at 37oC, overnight for complete digestion. RE was inactivated by heating at 65oC for 10 min before electrophoresis.

Agarose Gel Electrophoresis: Agarose gel electrophoresis was carried out in an Amersham Pharmacia Biotech 20x25 cm horizontal submarine electrophoresis unit. The digested DNA samples were mixed with one-fifth volume of gel loading buffer. For electrophoresis, 0.7% agarose in Tris-borate buffer containing ethidium bromide at a concentration of 5ug /ml was used. Lambda DNA Hind III digest was used as a DNA molecular weight marker.  After the addition of tracking dye each sample was loaded in the gel. Electrophoresis was carried out at 37V for 16 h until the bromophenol blue migrated more than two-third of the length of the gel. At the end of electrophoresis, the gel was visualized under ultra violet transilluminator and photograph was taken.

Isolates: Yellowish white, opaque, flat colonies of gram+ coco non-motile bacilli taken from 3 goats were designated F1, F2 and F3.  The isolates produced small uniformly black matt surfaced colonies on blood tellurite agar.

Biochemical tests: The isolates were identical after biochemical tests except for one which fermented lactose. The isolates degraded urea and fermented glucose, maltose, mannose and starch but not mannitol, salicin, sucrose, trehalose and xylose. The bacteria were positive for catalase, aesculin hydrolysis, methyl red, gelatin liquefaction, arginine hydrolysis and citrate, but negative for oxidase, H2S production from Triple sugar iron agar, Voges proskaur and nitrate reduction.

DNA isolation from C. pseudotuberculosis culture:  Brain Heart Infusion broth supplemented with 0.1 percent Tween 80 (BHITW) supported growth for both. Growth was characterized by formation of a granular deposit which increased on incubation at 37C and reached 5 x 10⁶ cells per ml by 72 h. Penicillin G (1ug/ml) was added to the medium at 37 C for 2h. Lysis occurred when cells were grown in penicillin treated with lysozyme.  Penicillin treatment was retained both for the additional lysis and the fact that cells grown in penicillin were easier to suspend. Incubation was carried out at 55C since this appeared to increase the lysis of concentrated cell suspensions.
Cell debris and membrane-chromosome complexes were removed by centrifugation 15,000 rpm at 4oC for 30 minutes.  The OD value of 1.8 was obtained by the nucleic acid was clearly precipitated after extraction with cold phenol and chloroform- isoamyl alcohol by adding 2M sodium acetate and distilled ethanol and incubating at –200C for 30 minutes. A single clear DNA band was obtained, when the DNA was electrophoresed in 0.7 percent agarose in Tris borate buffer (TBB).

Restriction Endonuclease Analysis (REA): The DNA was extracted from the three isolates and RS of C. pseudotuberculosis and were digested by Pst I, Bgl II, Eco RI and Bam HI enzymes. The digestion mixtures were kept at 37oC overnight and halted at 65oC for 10 min. The mixtures were subjected to electrophoresis using Hind III digested l-DNA as molecular marker.   There were no observable differences between the three isolates and standard reference, when the DNA digested with four restriction enzymes.DNA of F1, F2 and F3 field isolates.

The Pst I, Bam HI, Eco RI, and Bgl II digested DNA from F1, F2, F3 and RS of C. pseudotuberculosis presented 22,13,18 and 16 DNA fragments respectively.   The 6.6 Kb and 3.6 Kb DNA fragments were present in all enzymes digested DNA samples from C. pseudotuberculosis. The 6.6Kb, 5.3 Kb, 4.2 Kb and 3.6 Kb DNA fragments were present in Bam HI, Eco RI and Bgl II enzymes digested DNA samples.  The lower molecular weight DNA fragments of 1.8 Kb followed by 2 Kb, 3.1 Kb and 3.6 Kb produced by Pst I, Eco RI, Bgl II and Bam HI enzymes respectively.  Among the restriction patterns, the DNA of F1, F2, F3 and RS when digested with Pst I produced different restriction pattern and the Eco RI and Bam HI and Bgl II produced similar restriction patterns.

REA was used by Songer et al (1988) to characterize C. pseudotuberculosis isolates.  The main difference in the REA patterns were between nitrate positive and negative isolates (Songer et al. 1988). Present results show that both strains tested were negative to nitrate reductase. There was no observable differences when DNA digested with Eco RV, Pst I, Bgl II, Eco RI and Bam HI enzymes. These findings agree with those of Songer et al (1988) and Sutherland et al (1993).

A significant difference between restriction patterns of nitrate reductase positive or negative isolates is a 3.2 Kb band in DNA from nitrate reducing organisms but it is absent from those not reducing nitrate. Differentiation into groups could not be done with all restriction enzymes but DNA digested with Eco RV and Pst I produced different restriction patterns.  Digestion with Eco RI, Hind III, or Bam HI did not produce any observable differences (Songer et al. 1988).

In the present study,  the 3 field isolates and reference strain were nitrate reductase negative and a 3.2 Kb band was absent in 5 enzyme digested DNA samples. Digestion of DNA of F1, F2, F3 and RS with Pst I produced different restriction patterns whereas digestion with Eco RI, Bam HI and Bgl II produced similar patterns confirming previous findings (Songer et al,(1988).

Near homogeneity of isolates of C. pseudotuberculosis biovar ovis in phenotypic and genotypic characteristics have been reported in earlier studies (Songer et al., 1988.  and Sutherland et al., 1993) where the isolates were reported to ferment carbohydrate and to be urease, catalase positive and to produce PLD. It is also reported that isolates from small ruminants are unable to reduce nitrate (Literak et al. 1999). The results suggest a high degree of homogeneity in field isolates and standard reference strain of C. pseudotuberculosis (goat).

The authors thank Dr. S. Sulochana, Dean of Veterinary College, for her timely help for the continuation of the scientific work.

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