Advance Publication

The Journal of Veterinary Medical Science

Accepted Date: 3 Jun 2018

J-STAGE Advance Published Date: 27 Jun 2018
 1 Note

2 Public Health

3 The importance of wild gecko as a source of human Salmonella infection

5 Khanh Thuan NGUYEN1, Mizuki HASEGAWA1, Thu Tam NGUYEN2, Thi Minh Tam VO1,

6 Thi Hong To TRAN1, Thi Lien Khai LY2, Takahide TANIGUCHI1 and Hideki

7 HAYASHIDANI1,*

1
9 Institute of Agriculture, Tokyo University of Agriculture and Technology, Japan

10 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509

2
11 Department of Veterinary Medicine, Can Tho University, Vietnam

12 Campus II, 3/2 street, Ninh Kieu District, Can Tho city, Vietnam

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14

15 *Corresponding author:

16 Dr. Hideki HAYASHIDANI

17 Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture

18 and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan

19 Tel: +81 42 367 5775

20 Fax: +81 42 367 5775

21 E-mail address: eisei@cc.tuat.ac.jp

22

23 Running Head: GECKO AS A SOURCE OF SALMONELLOSIS

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25 ABSTRACT

26 A total of 201 wild geckos from the region of Mekong Delta, Vietnam were collected

27 to determine the viable number and survival period of Salmonella in their feces. Of the 101

28 samples examined, 24 (23.8%) were Salmonella positive. These 24 geckos excreted

29 Salmonella in their feces in a range of 1 to 8.6 log CFU/g with a mean of 4.5 ± 3.2 log CFU/g.

30 Among the Salmonella serovars, Salmonella Weltevreden was the most predominant serovar

31 (37.5%). Moreover, Salmonella could survive for 6 weeks in gecko feces at room

32 temperature in Vietnam. These results indicate that the wild gecko seems to play an important

33 role as a reservoir for Salmonella and a source of Salmonella infection in humans in

34 Southeast Asian countries.

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36 Key words: a source of infection, feces, Salmonella, survival, wild gecko

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51 Salmonella is known to be one of the important human bacterial pathogens in both

52 developed and developing countries. Human salmonellosis was found in 0.6% to 7% of the

53 total human diarrhea cases in Southeast Asian countries such as Laos, Myanmar, and

54 Vietnam [3]. Wild geckos were commonly found in the residential areas of these countries

55 [12] and are widely distributed and seen in close contact with humans. Recently, some

56 researchers indicated that geckos could harbor Salmonella at a relatively high rate in

57 Southeast Asian countries [1, 5]. Humans could be infected with Salmonella via contact with

58 reptiles, and reptiles were considered to be a reservoir for Salmonella and a source of human

59 salmonellosis [4, 7, 9-11, 14, 15]. However, no report has been published on quantification

60 and survival analysis of Salmonella in gecko feces. Therefore, this study was carried out to

61 determine the number and persistence of Salmonella in gecko feces.

62 In this study, a total of 201 wild geckos (138 Hemidactylus frenatus and 63

63 Hemidactylus platyurus) were captured in the Mekong Delta region, located in the South of

64 Vietnam. Of 201 gecko fecal samples, 101 were examined for the number of Salmonella in

65 their feces. About 0.1 g of feces (rectum contents) in each sample was collected and

66 suspended in 9 times volume of phosphate buffer saline (PBS, pH 7.2). Following this, 0.1 ml

67 of the suspension diluted 10-fold with PBS was plated on mannitol lysine crystal violet

68 brilliant green agar (MLCB, Nissui, Tokyo, Japan) and deoxycholate hydrogen sulfide

69 lactose agar (DHL, Nissui, Tokyo, Japan). The number of Salmonella colonies was counted

70 on these selective media, after incubation at 37oC for 24 hr. If suspected Salmonella colonies

71 did not appear on the selective media, samples were enriched with Hajna tetrathionate broth

72 (Eiken, Tokyo, Japan) at 37oC for 24 hr. A loopful of enrichment broth was then streaked on

73 the selective agars. The suspected Salmonella isolates were examined for biochemical

74 characteristics, and serovars were identified by following the methods described by Tran et al.

75 [13].

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 76 Of 101 gecko samples, 24 (23.8%) were Salmonella positive. Among these positive

77 samples, 14 geckos excreted Salmonella ≥ 4 log CFU/g in their feces. The highest number of

78 Salmonella in gecko feces was 8.6 log CFU/g, and the mean was 4.5 ± 3.2 log CFU/g (Fig. 1).

79 These results indicated that wild geckos excreted a high number of Salmonella in their feces.

80 Moreover, of 24 Salmonella isolates, S. Weltevreden was the most predominant serovar

81 (37.5%), followed by S. Worthington (12.5%), S. Lexington (8.3%), S. Albany (4.2%), and S.

82 Bellevue (4.2%) (Table 1). S. Weltevreden is known to be the predominant serovar in human

83 salmonellosis in Southeast Asian countries such as Thailand, Malaysia, and Philippines [2, 6].

84 Ly et al. [8] also reported that S. Weltevreden was isolated from human patients with diarrhea

85 in the region of Mekong Delta, Vietnam. Therefore, wild geckos seem to be the potential

86 source of Salmonella infection, especially S. Weltevreden serovar, and spread Salmonella in

87 the environment of these countries.

88 Of 201 gecko fecal samples, 101 were used for quantification analysis of Salmonella

89 in gecko feces and 100 were divided into 2 groups. Fecal samples in each group were mixed

90 and put into sterilized Erlenmeyer flasks (300 ml). These fecal mixtures were kept at the

91 room temperature (25-30oC) of Vietnam for 10 weeks. About 1 g of fecal sample was taken

92 from each mixture once a week for 10 weeks after storage. Isolation and identification of

93 Salmonella from fecal samples were also done following the same methods as described

94 above. In this experiment, Salmonella was isolated from fecal mixtures of both groups for 6

95 weeks after storage. However, no Salmonella was detected from fecal samples after 7 weeks.

96 These results indicate that Salmonella can survive for a long time in gecko feces in normal

97 environmental conditions in Vietnam. However, the mechanism involved in the survival of

98 Salmonella for a long time in gecko feces in the environment is still unclear.

99 The present study indicated that the wild gecko seems to play an important role as a

100 reservoir for Salmonella and a source of Salmonella infection in humans in Southeast Asian

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101 countries. Further research needs to be performed to evaluate the epidemiology of Salmonella

102 in wild geckos of this region.

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104 ACKNOWLEDGMENTS

105 This study was partially supported by a Health Sciences Research Grant from the

106 Ministry of Health, Labor and Welfare, Japan.

107

108 REFERENCES

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151 15. Whitten, T., Bender, J.B., Smith, K., Leano, F. and Scheftel, J. 2015. Reptile-associated

152 salmonellosis in Minnesota, 1996-2011. Zoonoses Public Health 62: 199-208.

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154

155 FIGURE LEGEND

156 Fig. 1: The number of Salmonella excreted in gecko feces (log CFU/g). The number of

157 Salmonella in gecko feces < 2 (log CFU/g) was calculated as 1 (log CFU/g).

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177 Fig. 1

178 12
179 Mean No. of Salmonella: 4.5 ± 3.2 log CFU/g
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180
No. of Salmonella positive samples

181 8

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<2 2～3 3～4 4～5 5～6 6～7 7～8 8～
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No. of Salmonella excreted in feces (log CFU/g)
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203 Table 1. Prevalence of Salmonella serovars

204 isolated from gecko feces (n = 24)

No. of 205
Serovar
isolates (%)
206
S. Weltevreden 9 (37.5)

S. Worthington 3 207
(12.5)
S. Lexington 2 (8.3)
208

S. Albany 1 (4.2)
209
S. Bellevue 1 (4.2)
210
Biovar IIIb 1 (4.2)
211
Biovar IV 3 (12.5)
Untyped 4 (16.7)
Total 24 (100.0)