IMMUNO PROTECTIVE POTENTIAL OF PARTIALLY PURIFIED TOXOIDS OF Clostridium difficile IN MICE
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Date
2016-11
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Assam Agricultural University, Khanapara,Guwahati
Abstract
The present study was undertaken to characterize Clostridium difficile toxins, in respect to the influence of glucose and stages of growth (incubation period) on release of toxins, cytotoxic activities in Vero cells and the immune-protective potential of partially purified toxoids of C. difficile in mice. A total of 10 isolates of C. difficile from the repository of Department of Microbiology, College of Veterinary Science, Khanapara, Guwahati were revived and reconfirmed, based on morphological and staining characteristics, and molecular detection of gluD gene. Characterization of all the 10 isolates, in respect to certain virulence associated genes revealed presence of tcdA (toxin A) and tcdB (toxin B) genes in three strains of C. difficile each. Another three strains could reveal tcdA and tcdB together in the same isolate, while one strain was found to be negative for tcdA and tcdB gene.The protein concentration in the cell free supernatant of toxin A and toxin B positive isolate of C. difficile growth in nutrient media without addition of glucose was found to increase with advancement of growth phases and reached the highest conc. during the decline phase of 48 hr (5.24 µg/µl and 5.06 µg/µl, respectively). Similar trend of protein conc. was observed in the cell free supernatants of both the isolates, in presence of glucose in the nutrient media. However, the presence of glucose was found to suppress the protein conc. in the cell free supernatants of toxin A and toxin B of C. difficile (3.49 µg/µl and 3.99 µg/µl, respectively). The protein profile of toxin A positive C. difficile isolate, in presence of glucose could show 10 protein bands with mol. wt. ranging from 25 to 135 kDa, while the same isolates in absence of glucose in nutrient media revealed 16 protein bands within the range of 22.4 kDa and 100.0 kDa. Similarly, the isolate positive for toxin B revealed 8 protein bands of 35 to 135 kDa range in the cell free supernatant with addition of glucose, while the growth of the same isolate in nutrient media without glucose could exhibit 15 protein bands within the range of mol. wt. 20.0 to 135.0 kDa.
Toxin A, B and AB of C. difficile were extracted in thioglycolate media without addition of glucose at 48 hr of incubation and were partially purified by ammonium sulphate precipitation. The partially purified toxins were found to be cytotoxic for Vero cells at two dilution (1:10 and 1:100). Among the three toxins, toxin B was found to be more prominent cytotoxic activities than the other two toxins, A and AB. Complete detoxification was confirmed by testing the monolayer of Vero cells for no cytopathic effect.
The immune-protective efficacy of the three toxoid vaccine preparations were tested by immunization of groups of mice with challenge trial on 34th day of post immunization revealed variable protection level. The immunized groups of mice were found to have 100.0 percent protection against homologous challenge dose of 6.0x108CFU. However, the groups of mice, immunized with toxoid A and B could show 75.0 percent protection against challenge with 9.0x108CFU of homologous strains of C. difficile. On the other hand, the vaccine prepared from toxoid AB could confer only 25.0 percent protection in mice, following homologous challenge with 9.0x108CFU. The immunized affected mice, following challenge with 9.0x108CFU dose could show clinical symptoms, suggestive of intestinal disorder, with any mortality. All the affected immunized mice with clinical symptoms were found to recover by the end of the challenge study. The challenge trial with 6.0 x 108 and 9.0x108CFU / dose of homologous strain of C. difficile could produce 100.0 percent mortality in the mice of control group during 48 hr of post challenge observation. The affected mice of the control group revealed an initial development of clinical symptoms, suggesting intestinal infection during 24 hr of observation and all the clinically affected mice were died within 48 hr of challenge. Mortality in mice of control group due to inoculated strain of C. difficile was confirmed by re-isolation of the inoculated strains from the affected liver as well as haemorrhagic part of intestine and intestinal contents.
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