0115 966 7955 Today's Opening Times 10:00 - 20:00 (BST)
Place an Order
Instant price

Struggling with your work?

Get it right the first time & learn smarter today

Place an Order
Banner ad for Viper plagiarism checker

Leptospira Cultures Maintenance

Disclaimer: This work has been submitted by a student. This is not an example of the work written by our professional academic writers. You can view samples of our professional work here.

Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UK Essays.

Published: Mon, 04 Jun 2018

RESULTS

The Leptospira serovars maintained in the Department of Veterinary Microbiology were used in the present study. For maintenance, EMJH medium (Difco) with albumin supplement was employed and subcultured at seven day intervals and incubation was carried out at 28-30°C. In addition, the stock cultures were maintained in semi-solid medium with subculturing at one month interval.

IDENTIFICATION OF LEPTOSPIRES

Under dark field microscope, the live leptospiral organisms were found tightly coiled and actively motile. The motility observed was of both spinning and bending. In highly concentrated cultures, the organisms formed entangled masses. No contaminants were observed in most of the time when streaked on blood agar plates for purity checking of the cultures. In case of contamination they were purified by filtration.

RECOMBINANT PROTEIN PRODUCTION

Preparation of template DNA from Leptospira

The genomic DNA isolated from Leptospira interrogans serovar Australis had a DNA concentration ranging from 40- 60µg/ml. The purity of the extracted DNA was checked by measuring the ratio of absorbance (O.D of DNA preparation at 260 and 280 nm). The value of the ratio obtained was found to be in the range of 1.8 to 1.93, indicating that the preparations were almost free of proteins.(in mat and methods).

Amplification of lipl21 gene and lipl32 genes

The amplification of lipl21and lipl32 genes were carried out and observed amplicons of 507 bp and 767 bp, respectively. (Fig 1)

Cloning of the lipl21 and lipl32 gene

The colonies of E.coli Dh5∞ cells transformed with lipl21 and lipl32 genes was picked up separately and tested for the presence of the two genes. It was observed that lipl21 clones yielded an amplicon size of 507 bp and lipl32 with 767 bp. These confirmed clones were preserved for further studies (Fig 2)

Induction of recombinant protein

The above clones were subcultured in LB broth containing Ampicillin (100µg/ml) and expression was optimized with 2 mM IPTG for LipL21 and 1mM IPTG for LipL32. The induced recombinant cells were harvested after six hours and five hours for LipL21 and LipL32, respectively. Uninduced controls were set for each. The cells were then pelleted and lysed. The expression of recombinant 21 kDa (rLipL21) and 32 kDa outer membrane proteins (rLipL32) were confirmed in comparison with that of the uninduced cells where there was no significant protein expression (Fig 3)

Purification of recombinant lipl21 and lipl32 proteins

The rLipL21 and rLipL32 proteins were purified by Nickel chelating affinity chromatography without any contamination. The protein concentrations were estimated to be 0.69 mg/ml and 2.07mg/ml for rLipL21 and rLipL32, respectively.

Immunoreactivity of the proteins

The immunogenicity of both rLipL21 and rLipL32 proteins were tested using MAT positive canine sera and observed that both the proteins were reacting. Further the protein did not react when blotted with hyper immune serum raised against the different bacteria namely E.coli, Staphylococcus aureus and Pasteurella multocida.

DIAGNOSIS

Microscopic Agglutination Test

A total of 124 canine serum samples from Leptospira suspected dogs were tested using MAT and among this 22 (17.74 per cent) were found to be positive for leptospirosis. Serum samples having a titre of 1:400 and above were considered as positive. The infecting serovars identified with canine leptospirosis are depicted in Table 3

Enzyme Linked Immunosorbent Assay

Indirect ELISA was done in separate microtitre plates employing rLipL21 and rLipL32 as antigens and the results were compared with that of MAT.

Checker board analysis

Using checker board analysis the optimum concentration of antigen, antibody and conjugate were estimated. The optimum concentration of antigen was found to be 50 ng/well and 150 ng/well for rLipL21 and rLipL32, respectively. The rabbit anti-canine IgG HRP conjugate concentration estimated was 1:2500 and 1:2000 for rLipL21 and rLipL32, respectively. A 1:50 dilution of test serum was used as optimum working dilution in both ELISAs.

Determination of cut off values

In IgG ELISA, the mean OD and standard deviation for the negative sera samples (n=44) was 0.49 and 0.11 for rLipL21 and 0.59 and 0.09 for rLipL32, respectively. The cut off value obtained was 0.82 for rLipL21 and 0.86 for rLipL32.

Test proper

The results of rLipL21 and rLipL32 based IgG ELISA are given in Table 4. Among 47 positive samples obtained by rLipL21 ELISA, only 20 found positive with MAT. In case of rLipL32 ELISA, 40 samples were recorded positive out of which 18 found positive with MAT.

Comparison of MAT and rLipL21IgG ELISA

The results of rLipL21 IgG ELISA were compared with that of MAT.

Among 124 canine sera examined, 47 (37.90 per cent) showed OD more than the cut off value i.e. 0.82 and were considered positive for leptospirosis by rLipL21 IgG ELISA. The sensitivity, specificity and accuracy of rLipL21 IgG ELISA as relative to MAT was calculated to be 90.90 per cent, 73.52 per cent and 76.61 per cent, respectively (Table 5).

On statistical analysis, it was found that there exists a significant difference between the two tests, ie, rLipL21 ELISA and MAT even at 1 per cent level of significance.

Comparison of MAT and rLipL32 IgG ELISA

The results of the IgG ELISA using rLipL32 as the antigen were compared with that of MAT.

Among 124 canine sera examined, 40 (32.25 per cent) showed OD more than the cut off value i.e. 0.86 and were considered positive for leptospirosis by IgG ELISA. The sensitivity, specificity and accuracy of rLipL32 IgG ELISA as relative to MAT was calculated to be 81.81per cent, 78.43 per cent and 79.03 per cent, respectively (Table 6).

Statistical analysis revealed that there exists a significant difference between the two tests, ie, rLipL32 ELISA and MAT at 1per cent level of significance.

Comparison of rLipL21 and rLipL32 IgG ELISA

On statistical analysis using Cochran’s Q test, at 1 per cent level of significance it was observed that there exists no significant difference between rLipL21 and rLipL32 ELISA, as the P value was found to be greater than 0.01.

Table 3 Infecting serovars identified with MAT

Australis

8 (36.36%)

Autumnalis

2 (9.1%)

Canicola

2 (9.1%)

Pomona

1 (4.54%)

Icterohaemorragiae

4 (18.18%)

Grippotyphosa

4 (18.18%)

Bataviae

0 (0%)

Hebdomadis

1 (4.54%)

Pyrogenes

0 (0%)

Table 4 Results of MAT, rLipL21 ELISA and rLipL32 ELISA

Sl.No.

MAT

rLipL21ELISA

rLipL32 ELISA

1

2

3

4

5

6

7

8

9

10

+

11

12

13

14

15

16

17

+

18

+

+

19

20

21

22

+

23

24

+

25

26

27

+

28

29

30

31

32

33

+

34

35

36

37

+

38

39

40

+

41

42

43

44

45

+

46

+

+

47

+

48

+

+

+

49

+

50

51

52

53

+

54

+

55

+

56

+

+

+

57

58

+

59

+

+

+

60

+

+

+

61

+

62

63

+

64

+

65

+

+

66

+

67

+

+

+

68

+

+

+

69

70

+

+

71

+

+

72

+

+

73

+

74

+

75

+

+

+

76

+

+

+

77

+

78

+

79

80

81

82

83

+

+

+

84

+

+

+

85

+

+

86

+

+

87

+

+

+

88

+

+

+

89

+

90

91

92

+

+

93

94

95

+

+

96

+

+

+

97

98

+

99

100

+

+

101

102

103

104

+

+

105

106

107

+

108

+

+

+

109

110

+

+

111

112

+

+

113

114

+

115

+

116

+

+

+

117

+

118

+

119

+

120

121

+

122

+

123

124

+

+

+

Table 5 Comparison between rLipL21 ELISA and MAT

 

MAT

rLipL21 ELISA

       
 

+

Total

+

20(a)

27(b)

47(a+b)

2(c)

75(d)

77(c+d)

Total

22(a+c)

102(b+d)

124(a+b+c+d)

Sensitivity = a/a+c = 90.90%

Specificity = d/b+d = 73.52%

Accuracy =a+d/a+b+c+d = 76.61%


To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Request Removal

If you are the original writer of this essay and no longer wish to have the essay published on the UK Essays website then please click on the link below to request removal:


More from UK Essays