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Abstract

Background and aim: Bangladesh,
being located in the humid tropical region is very rich in species diversity.
There are about 90 species of snakes in Bangladesh among which only one fourth
are venomous.  The
spectacled/binocellate cobras (Naja Naja)
are one of the most important venomous endangered reptiles that occur in
Bangladesh. The aim of the study was to isolate opportunistic Salmonella spp and E. coli
that are harmful for both human and animal health and also seen the
relationship of positive cases with the collected sample area, age of the
snakes and gender.

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Materials and methods: Total 26 samples of oral and
cloacal swabs were taken from binocellate cobras (Naja Naja) reared by the snake charmer’s of Mymensingh
and Sherpur district of Mymensingh division to isolate Slmonella
spp and E. coli by using
differential media.

Results: Salmonella
spp were isolated from 10 (n=26) oral and 10 (n=26) cloacal samples but no E coli was detected from any samples. No
significant variations were observed relating to bacterial isolation with area
and gender group. But Significant variation was found among the oral and
cloacal isolates (p=0.03) that are taken from more than 3 years of old snakes.

Conclusion: The
spectacled/binocellate cobras (Naja Naja)
harbor gram negative Samolella spp. To the author’s knowledge this is
the first survey of oral cavity and cloacal microflora of binocellate cobras in Bangladesh.

 

Key Word: Binocellate
cobras, Oral cavity, Cloaca, Salmonella
spp, E. coli

Introduction

Snakes are a group of
amazing reptiles which have fascinated human being since beginning of mankind. This elongated,
cylindrical, legless vertebrates belonging to the order serpentes under the
class reptilia. Its body is covered by scales and has no shoulder girdle and
limbs 1. Snakes are an important parameter of our ecosystem and have
tremendous importance in biopharmaceutical research, that been ignored for
proper recording possibly due to the danger and panic of handling of this
mysterious reptiles. There are about
3000 different snake species described on earth, of these, only about 350 are
regarded as poisonous and dangerous for humans.
 Most snakes are
non-poisonous and harmless to human being 2. Bangladesh, being located
in the humid tropical region is very rich in species diversity both for flora
and fauna. According to the report funded by UNDP, IUCN has reported that the
country has about 125 species of reptiles and 22 species of amphibians 3. There are about 90 species of snakes in Bangladesh
among which only one fourth are venomous. Poisonous
species of snakes with some kinds of poison or venom glands and poison fangs.
Also they must have the mechanism to pour or push the poison from the venom
gland through the fangs into the body of a prey or a victim 4. Amid of
venomous snakes cobras are most dangerous and commonly found in Bangladesh. Two species of cobra, the spectacled/binocellate cobra
(khoia or khodom paia gokhra), Naja
naja and monocled/monocellate
cobra (gokhra), Naja kaouthia occur in Bangladesh. Cobras
belonging to the family Elapidae and Naja naja were first described by
Swedish physician, zoologist, and botanist Carl Linnaeus in 1758 5. The cobra
is a moderately sized, heavy bodied species. This cobra species can easily be
identified by its relatively large and quite impressive hood, which it expands
when threatened. This species has a head which is elliptical, depressed, and
very slightly distinct from neck. The snout is short and rounded with large
nostrils. The eyes are medium in size and the pupils are round. The majority of
adult specimens range from 1 to 1.5 meters (3.3 to 4.9 ft) in length 6. Cobras are now a threatened species in Bangladesh. The
moncellate cobra is particularly vulnerable, and the binocellate cobra and the
king cobra are endangered snakes 7. It is estimated that about 1600 people
die every year of snakebite in the country. Cobra bites are associated with
neuromuscular paralysis; necrosis of soft tissues; and secondary bacterial
complication has also been reported. 

Recently studies using
reptiles have increased in the areas of infectious disease, comparative
anatomical physiology, the evaluation of phylogenic relations with birds and
other vertebrates, stem cell experiments, and therapeutic drug development 8,
9. As medicinal applications of venom
increase, its risk to human health should be examined. Even though snake health
can be affected by bacteria, there have not been many studies of the
distribution of bacteria in snakes or the influence of predominant bacteria in
snakes. Some bacteria not only affect snakes but also affect humans there have
not been many studies of the distribution of bacteria in snakes or the
influence of predominant bacteria in snakes 10. Venomous snakes may also
harbor a wide range of bacteria in their oral cavity able to complicate the
bite wounds 11 so the information on their microflora should not be
neglected. Secondary bacterial infections, such as subcutaneous abscess or
tetanus, are possible complications of snake bites, either venomous or
non-venomous snakes12, 13. In addition to bites, humans can be infected
during manipulation of animals or via infected equipment 14. The popularity
of snakes as pets and the models of biological and veterinary research
increased the risk for a public health due to the zoonotic potential of these
animals 15.

Studies on
oral of snakes are rare in Bangladesh. There is no information regarding
microflora and potential pathogens isolation from oral and cloaca of spectacled
cobra (Naja Naja) snakes. This present study was designated to isolate
opportunistic Salmonella spp and E. coli from oral and cloacal swab of
binocellate cobras (Naja Naja) that are rared by the snake charmer’s. The main
objective of this study was seen the relationship of positive cases with the
collected sample area, age of the snakes and gender.

Materials
and Methods

Study
area and duration

The study was conducted
over Mymensingh and Sherpur district of Mymensingh division. The time period of
study is march-2017 to june-2017 and it does involve from sample collection to
laboratory isolation of Salmonella and E. coli.

Study
population

The selected
population was those snakes which are reared by snake charmer for their earning
purpose and for recreational purpose. They mainly reared various type of local
snakes most of them are speckled cobra (Naja naja). The total snake population
is 15of different body weight, length and age.

Study
Design

This study was
done by using the following study design:

 

                                                                    
Live snake (Naja naja)

                                                                              

                                                   
   Sample collection (oral,
cloacal)

                                                                                                                                                                         

 

                                           
Transported by Stuart transport media                                                       

                                                               

                                                 
                Nutrient broth

 

                           XLD agar (Salmonella)                            MacConkey agar ( E.
coli)

 

            Biochemical test (Triple sugar iron)                         eosin
methylene blue agar(EMB)

                                                                                                                                                                               

                                                                                            
Biochemical test (indole test)

 

Collection
of sample

Swab sample were taken from oral cavity and cloacal
of live snakes. Oral swab are mainly taken from around the flank region and
upper jaw. The cloacal swabs are directly collected from cloaca by inserting
the cotton bar then suspending in 5 ml transport media within a falcon
tube.   At the mean time a developed questionnaire was filled upfor
each and every sample regarding with information of body weight, length, age,
management system, clinical state etc. Those samples were brought to the
laboratory for isolation of Salmonella and E. coli.

Pre-enrichment of sample at nutrient broth medium

The collected sample were than subculture at nutrient broth
which are prepared by dissolving 25gms powder in one litter distilled water and
dissolve the medium completely by heating then sterilize it by autoclaving at
10 lbs pressure (1150C) or alternatively at 15 lb pressure(1210C)for 15 minutes
or as per validated cycle. After sterilization pH of the medium should be
7.3±0.1.Normally each litter of nutrient broth medium contains 10gms peptone,
10gms beefextractand5gms sodium chloride. Beef extract and peptone provide then
necessary nitrogen compounds, carbon, vitamins and also some trace ingredients
to the non-fastidious organism and sodium chloride maintain osmotic equilibrium
of the medium. After giving subculture inoculation in nutrient broth, it was
incubated at 35-37°C for 24 hours.

Isolation of Salmonella

Isolation of Salmonella in xylose lysine deoxycholate (XLD)
agar

For the
isolation of Salmonella at first the subculture inoculums from nutrient broth
are streaked in the xylose lysine deoxycholate agar which is composed of
lactose 7.5gm, sucrose 7.5gm, sodium thiosulfate 6.8gm, l-lysine 5.0gm, sodium
chloride 5.0gm, xylose 3.75gm, yeast extract 3.0gm, sodium deoxycholate 2.5gm,
ferric ammonium citrate 0.8gm, phenol red 0.8gm, agar 15gm,  per liter of deionized water. XLD Agar is both
a selective and differential medium. It contains yeast extract as a source of
nutrients and vitamins. It utilizes sodium deoxycholate as the selective agent
and, therefore, is inhibitory to gram-positive micro-organisms. Xylose is
incorporated into the medium since it is fermented by practically all enterics
except for the Shigella and this property enables the differentiation of
Shigella species. Lysine is included to enable the Salmonella group to be
differentiated from the non pathogens since without lysine, salmonellae rapidly
would ferment the xylose and be indistinguishable from non-pathogenic species.
After the salmonellae exhaust the supply of xylose, the lysine is attacked via
the enzyme lysine decarboxylase, with reversion to an alkaline pH which mimics
the Shigella reaction.

Final identification of Salmonella through
biochemical (Triple Super Iron) test

Finally the presence of Salmonella organisms in sample was
confirmed by biochemical test in Triple Sugar Iron media. In Salmonella positive
case the slants of the media become red, button become yellow and gas was
formed, blackish appearance may indicate the production of hydrogen sulphide.

Isolation of E. coli

Isolation of E. coli in MacConkey agar

On the
other hand for isolation of E. coli the initial pre-enriched nutrient broth inoculums
are streaked into the MacConkey agar which is used for the isolation of
gram-negative enteric bacteria and the differentiation of lactose fermenting
from lactose non-fermenting gram-negative bacteria. It has also become common
to use the media to differentiate bacteria by their abilities to ferment sugars
other than lactose. MacConkey agar is a selective and differential media used
for the isolation and differentiation of non-fastidious gram-negative rods,
particularly members of the family Enterobacteriaceae. It contains Peptone
17.0gm, Lactose 10.0gm, Sodium Chloride 5.0gm, Proteose Peptone 3.0gm, Bile
Salts 1.5gm, Neutral Red 30.0mg, Crystal Violet 1.0mg,Agar 13.5gm in per liter
of deionized water. E. coli growing on the media is differentiated by their
ability to ferment the sugar lactose. Fermentation of lactose cause the pH of
the media to drop and the resultant change in pH is detected by neutral red,
which is red in color at pH below 6.8. As the pH drops, neutral red is absorbed
by the bacteria, which appear as bright pink to red colonies on the agar.
Gram-negative bacteria that grow on MacConkey agar but do not ferment lactose
appear colorless on the medium and the agar surrounding the bacteria remains
relatively transparent. The suspected E. coli colonies then transferred to
nutrient broth medium and incubated at 35-37°C for 24 hours. In this case there
is no bright pint to red colour colonies. So it indicates that there is absence
of E. coli in the sample.

RESULTS

3.1. Proportion of Salmonella positive
case in relation to the study area

The study was
designed to show the number of Salmonella spp. and E. coli positive samples
taken from the Mymensingh and Sherpur district under Mymensing division. The
result was in respect of area of sampling states that, among the 10 samples 20%
cases found positive for Salmonella spp. both in oral and clocal swab
that are collected from Mymensingh with the p- value of 0.27.  On the other hand 8(50%) cases were found
positive for Salmonella spp. both in oral and clocal swab with the p
value of 0.27 in the samples of Sherpur district Figure2.

3.2.
Proportion of Salmonella
positive case in relation to age of population

Oral and
clocal samples taken from the snakes aged from (1-3) years and (>3-5) years
of age. Total 12 samples collected from snakes aged between (1-3) from which 4(33.33%) oral and 2(16.67%) cloacal samples were positive
for with the p-value of 0.619. Whereas 6(42.86%) oral and 8(57.14%) cloacal samples were found positive for with the statistically
significant p-value of 0.03; among 14 samples that are collected from the snakes aged from
(>3-5).   Results are graphically
represented below Figure 3.

3.3.
Proportion of Salmonella
positive case in relation to gender of population

Total samples
(n=26) were collected from both the sex (male: 8 and female: 18). The result was in respect of gender of
snake’s shows that; oral 2(25%) and cloacal 4(50%) samples from male were positive for Salmonella
spp with the p-value of 0.347. In female 8(44.4%) oral and 9(33.3%) cloacal sampes were found Salmonella spp positive with p-value
of 0.420. The
results are explained in the Figure 4.

Oral and cloacal samples collected from different
area, age and sex were also culture in the differential media for isolation and
identification of E. coli. But E. coli was negative in all the
samples.

Discussion

The binocellate cobras (naja naja)
are one of the most dangerous venomous snakes that are found in Bangladesh. In
Bangladesh binocellate snakes are now endangered 7. Few number of binocellate
cobra reared by the snake charmer’s of Mymenshing and Sherpur district.

The present study was planned to isolate the Salmonella spp. and E. coli from oral and cloacal sample that are taken from the snakes
of Mymenshing and Sherpur district of Mymenshing division. From total 26
snakes, bacteria were isolated from 10 oral and 10 cloacal swabs.In all the
positive samples Salmonella spp. was
isolated but E. coli were not found
in any case. This findings is not coincides with the result of Luka?, Horvatek Tomi? et al. 2017;
who isolated many gram negative organisms except Salmonella spp from the oral and cloacal sample of snake.

No significant differences between two districts in
the number or type of bacteria are noted. This gram negative opportunistic
microflora has already been described as a potential cause of disease in
animals and human 16.

Samples were collected from two different age groups
(1-3-5) years. Significant variation (p=0.03) were observed
among these two age group. All the samples both oral and cloacal are positive
for Salmonella that were collected from the snakes older than 3 years where 50%
samples found positive for Salmonella spp.
that are collected from younger than 3 years of old. No study was found
relating the age group of isolation of bacteria.

Salmonella spp.
were isolated from oral and cloacal swabs of both male 17(n=18) and female
6(n=8) samples. No significant variation was noted regarding the isolation of
bacteria among the male and female snakes. Authentic study was not found
relating to variation of the bacterial isolation with gender.

All the samples are collected from the snakes that
are reared by the snake charmer’s.  They
often handle their snake without caution. This may increase the danger of
contracting zoonotic disease responsible for bacterial infection 17. In addition to secondary
infection arising from bacteria coming from the snake’s mouth inoculated at the
time of a bite. Bacteria are also responsible for snake bite associated wound
infection 12.

Conclusion

In conclusion, the results of this
study indicate that binocellate cobras from Mymensingh and Sherpur
district of Mymensingh division harbor gram
negative opportunistic Samonella spp organism. This organism has already been
described as causes of infection in both reptiles and humans. To the author’s knowledge this is the
first survey of oral cavity and cloacal microflora of binocellate cobras (Naja
Naja) in Bangladesh.

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