R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 0 ( 1 ) : 1 9 - 2 1 , J a n - M a r , 1 9 8 7
A STUDY OF BACTERIAL CONTAMINATION OF RATTLESNAKE VENOM
E . G a r c i a - L i m a a n d C . J . L a u r e
T h e a u t h o r s s t u d i e d t h e b a c t e r i a l c o n t a m i n a t i o n o f r a t t l e s n a k e v e n o m i s o l a t e d
f r o m s n a k e s in c a p t i v i t y a n d w i l d s n a k e s c a u g h t r e c e n t l y . T h e c a p t i v e s n a k e s s h o w e d a
r e l a t i v e l y h i g h i n c i d e n c e o f b a c t e r i a l c o n t a m i n a t i o n o f t h e i r v e n o m .
K ey words: Rattlesnake venom. Bacterial contamination.
Several investigators have studied cold-blooded animals in terms o f contam ination by different types o f
organisms. Hinshaw and M cN eil4 isolated S a l m o
n e l l a r u b i s l a w from 3 out o f 12 S c e l o p o r u s o c c i
d e n t a l specim ens. Zwart24 reported the iso
lation o f Salm onellae from 37.5% o f lizards. Le- N o c and Brygoo9 isolated 58 different types o f sal m onellae from 48 out o f 127 cham eleons examined. In studies on snakes captured in Pará and in lower A m azonia (Brazil), L ins1011 showed that 11.31% o f the animals exam ined were infected with salm onellae. Kourany et al.8 detected salm onellae in
27.3% o f lizards and 6.5% o f toads examined. M oreno et a l 14, in a study o f 6 13 cold-blooded animals raised in captivity, m ost o f which were snakes, detected in
271 animals several bacterial species, such as C i t r o
-b a c t e r , P r o t e u s v u l g a r i s , P r o t e u s m o r g a n i i and P r o
t e u s r e t t g e r i as w ell as several types o f salmonellae. This led the authors to em phasize the high incidence of contam ination with salm onellae among snakes.
D esp ite good physiological conditions o f the snakes several investigators have found high indices o f contamination. Thus, Furlanetto et al 3 detected high levels o f contam ination with enterobacteriaceae and other bacteria in an epizooty caused by a diphthe roid bacterium among snakes raised in an ani mal house. This bacillus caused a disease among these snakes that had not been reported before. The disease affected 70.0% o f snakes o f the genera
B o t h r o p s and C r o t a l u s w hose venom was going to be
u sed for the preparation o f antibothrops and anti- crotalus sera. The snakes were vaccinated with a W right-type vaccine prepared from the diphtheroid bacillus detected, which w as highly effective in low e ring the level o f infection from 70% to 0.4% .
Several investigators have attempted to esta blish a bacterial standard for the mouth flora o f snakes
Department o f Parasitology, Microbiology and Immunology and Department o f Biochemistry, Faculty o f Medicine o f Ribeirão Preto, University o f São Paulo. 14049 Ribeirão Preto, SP, Brazil.
Recebido para publicação em 2 5 /2 /1 9 8 6 .
and have reported the p rese n c e o f sev era l bacterial sp e c ie s ' 5 6 18 19 21 23_ S o eren sen et a l20 d escrib ed
“ single v iscera l drop” d ise a se affecting sn akes in
captivity. F u rla n etto e t a l.3 reported a type o f in fection o f the v en o m gland s o f B o t h r o p s l i n n e a e u s . 1758, and
C r o t a l u s w a g l e r , 1 8 2 4 , raised in the anim al h o u se.
M arcus13 detected “ mouth rot” in the upper and lower gums o f snakes, and W allach22 had also found inflammation o f the gums and dental arches o f snakes. This type o f inflammation caused large amounts o f yellow ish exudate w hich later induced secondary osteom yelitis. Klingelhoeffer7 had also reported two acute forms o f “ mouth rot” that led to the death o f affected snakes. Several other agents such as
A e r o m o n a s17 and P . l i q u e f a c i e n s , P a s t e u r e l l a and
P r o t e u s sp .19 were reported to be responsible for this
type o f disease.
In view o f the wide interest shown by many researchers in studying cold-blooded animals for in fections caused by bacteria and by other microor ganisms, w e decided to determine the bacterial con tamination present in the venom o f rattlesnakes, using both wild animals and animals raised in captivity.
M A T E R IA L S A N D M E T H O D S
The materials used were rattlesnake (C r o t a l u s
d u r i s s u s t e r r i f l c u s ) venom and blood-agar bacterial growth medium. The venom was obtained from re cently captured rattlesnakes (1 day) and from snakes held in captivity. V enom w as obtained from young and adult male and fem ale snakes restrained in the usual manner, by applying pressure to the venom glands. To avoid bacterial contam ination o f the sterilised venom flask, they were covered with plastic film during collection. O nly the snake’s fangs were allowed to perforate the plastic film, with the venom being deposited into the flask free from environmental contamination.
Im m ediately after collection, the venom sam ples were placed on a blood-agar plate with the aid o f a platinum wire. The plates were incubated for 24 hours at 3 7° C and examined. The colonies detected on the
G a r c i a - L i m a E , L a u r e C J . A s t u d y o s b a c t e r i a l c o n t a m i n a t i o n o r r a t t l e s n a k e v e n o m . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 0 : 1 9 - 2 1 , J a n - M a r , 1 9 8 7 .
plates were exam ined and the microorganisms iden tified.
R E S U L T S
A total o f 4 8 0 rattlesnakes were examined; 4 5 0 o f them belong to the animal house o f a private laboratory in the State o f São Paulo, and the remaining 30 to the animal house o f the Faculty o f M edicine o f Ribeirão Preto, University o f São Paulo. The first group is maintained under controlled temperature and humidity conditions, and the second in an open animal pit. The snakes from the private laboratory were divided into two groups: 3 6 0 snakes held in captivity and 90 being held in quarantine after being captured (1 day earlier). The snakes belonging to the animal house o f the F acuity o f M edicine have been captive for a long time. The animals belonging to the private laboratory originated from Bauru, State o f São Paulo.
V enom contamination w as detected in 42 snakes, as shown in Table 1. The incidence o f the microorganisms involved is shown in T able 2.
T a b l e 1 - D í s l n ò u í i o n a n d o r i g i n o f c o n t a m i n a t e d s n a k e s .
O r i g i n C a p t i v i t y Q u a r a n t i n e T o t a l
I n f e c t e d s n a k e s / T o t a i % I n f e c t e d s n a k e s / T o t a l % n u m b e r i n t h e g r o u p n u m b e r i n t h e g r o u p
B a u r u 3 5 / 3 6 0 9 .7 1 / 9 0 1.1 3 6
F a c u l t y o f M e d ic in e o f 6 / 3 0 R ib e ir ã o P r e to
20.0
— 6
T o t a l 4 1 / 3 9 0 1 0 .5 1/90 i.r 4 2 / 4 8 0
Table 2 - I n c i d e n c e o f m i c r o o r g a n i s m s a m o n g t h e
s n a k e s s t u d i e d .
M i c r o o r g a n i s m C a p t i v i t y Q u a r a n t i n e T o t a l
B a c i l l u s s u b t i l i s 7 - 7
E s c h e r i c h i a c o l i 2 - 2
M o n i l i a sp
P a r a c o l o b a c t r u m
3 3
a e r o g i n o i d e 1 1
P r o t e u s v u l g a r i s
P s e u d o m o n a s
7 1 8
a e r u g i n o s a 2 - 2
S h i g e l l a sp
S t a p h y l o c o c c u s
2 2
e p i d e r m i d e s
S t r e p t o c o c c u s
22 — 22
h a e m o l y t i c u s
S t r e p t o c o c c u s b e t a
4 __ 4
h a e m o l y t i c u s 2 - 2
Total 52 1 53
D IS C U S S IO N
Several investigators have studied the contami nation o f cold-blooded animals, specially snakes, with particular emphasis on mouth contamination. Infec tions caused by salm onellae were detected by H inshow and M c N e il,4 Z wart,24 L e-N oc and Brygoo9, L ins10 11 Kourany et al 8 and M oreno et al14. M oreno et al14 also detected other bacterial genera such as C i t r o b a c t e r and P r o t e u s . The high incidence o f con
tamination with salm onella among snakes m ay be due to their crawling and to their feeding habits. However, the high incidence o f contam ination o f low-body temperature animals with enterobacteriaceae is more difficult to explain, since these bacteria require higher temperatures to develop.
Conflicting results have been reported by the investigators who have studied the mouth flora o f snakes 1 5 6 18 19 21 23. A ll the mouth infections o f snakes lead to the death o f the animal.
In the present study, w hose objetive was simply
to study the contam ination o f venom , 8 .7 5 % o f the
captive snakes studied showed contamination as
opposed to 1.1 1 % o f so-called wild snakes captured
less than one day earlier. The wild snake was conta minated with a single bacterial genus, whereas several snakes held in captivity were contaminated with more than one genus o f bacteria. T hese results suggest that contam ination is due to handling which involves human contact and the consum ption o f contamined food and water2 512 15 16. The recently captured wild snake which was found to be contaminated may have been infected through handling, although it had been captive for only 2 4 hours. In view o f the above conside rations it is recommended that individuals bitten by snakes, in addition to being submitted to serum therapy, be also observed clinically for possible signs o f bacte remia.
R E S U M O
O s a u t o r e s e s t u d a r a m a c o n t a m i n a ç ã o b a c t e
-r i a n a d o v e n e n o d ë c a s c a v é i s m a n t i d a s e m c a t i v e i -r o e
d a s r e c e n t e m e n t e c a p t u r a d a s . V e r i f i c a r a m q u e o s
v e n e n o s d o s a n i m a i s c a t i v o s a p r e s e n t a r a m a l t a i n
c i d ê n c i a d e c o n t a m i n a ç ã o e o s t i d o s c o m o r e c e n
t e m e n t e c a p t u r a d o s e s t a v a m c o m b a i x a c o n t a m i
n a ç ã o a p a r e n t e .
Palavras chaves: Veneno de cascavel. Conta m inação bacteriana.
R E F E R E N C E S
1. Aronson JD. Spontaneous tuberculosis in snakes. Archi ves o f Pathology 8:159, 1929.
2. Dobbs JS. Reptilian disease recognition and treatment. Ralph Curtis Books, Hollywood, 1973.
3. Furlanetto RS, Belluomini HE, Lizuka H, Rosa RR. Epizootia provocada por bacilo difteróide em serpentes
G a r c i a - L i m a E , L a u r e C J . A s t u d y o s b a c t e r i a l c o n t a m i n a t i o n o r r a t t l e s n a k e v e n o m . R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c i n a T r o p i c a l 2 0 : 1 9 - 2 1 , J a n - M a r , 1 9 8 7 .
mantidas em biotério. Revista de Microbiologia 10:139-143, 1979.
4. Hinshaw WR, M cNeil E. Lizards carriers o f salmonella and paracolon bacteria. Journal o f Bacteriology
53:715-718, 1947.
5. Jaksztien KP, Petzold HG. Durch Salmonella Infektion bedingte Schweirigkeiten bei der Anfzucht Schlangen und ihre Behandlung. VI. Ager Terrk 6:79-80, 1969. 6. Kauffeld CF. Newer treatment o f mouth rot in snakes.
Herpetologica 9:132, 1953.
7. Klingelhoeffer W. Terrarien-kunde. Vol. 1 ,2 , 3, u. 4. Alfred Kemen, Stuttgart, 1955.
8. Kourany M, Myers CW, Schneider C R Panamanian amphibians and reptiles and carriers o f salmonella. American Journal of Tropical Medicine and Hygiene
19:632-638, 1970.
9. Le-Noc P, Brygoo ER. Les salmonelles des camaléons de Madagascar. Archives de l’Institut Pasteur
30:93-104, 1962.
10. Lins ZC. Ocorrência do gênero salmonella em animais silvestres capturados na floresta amazônica. Anais de Microbiologia 12: 19-29, 1964.
11. Lins ZC . Studies on enteric bacteria in the lower Amazon region. II. Salmonella types isolated from wild reptiles in Pará State, Brazil. Revista de Microbiologia 2: 165-169, 1971.
12. Marcus LC. Infectious diseases o f reptiles. Journal o f the American Veterinary Medicine Association 159:1626-1631, 1971.
13. Marcus LC. Diseases o f snakes and turtles. N ew York Medicine Society Bulletin 8:3-12, 1971.
14. Moreno G, Lopes CAM , Belluomini H E, Pessoa GVA, Biasi P, Andrade JCR. Enterobactérias isoladas de anfíbios e répteis. Revista do Instituto de Medicina Tropical 15:122-126, 1973.
15. Murphy LB, ArmstrongBL. Maintenance o f rattlesnakes in captivity. Special Publication o f the Museum o f National History, 1978.
16. Murphy LB. A brief outline of suggested treatments for diseases of captive reptiles. Published by the Society o f the Study o f Amphibians and Reptiles and the Kansas Herpetological Society, 1981.
17. Page LA. Experimental ulcerative stomatitis in King snakes. Cornell Veterinary 51:259-266, 1961. 18. Patrick R, Werkman CH. N otes on the bacterial flora of
the s'nakes. Proceedings o f the Iowa Academy of Science 37:330, 1930.
19. Reichenbach-Kline, Elkan ME. The principal diseases of lower vertebrates. Academic Press, N ew York, 1965. 20. Soerensen B, Amaral PA , Belluomini H E, Saliba M A, Correa H S, Hoge AR. Gota única visceral em serpentes.
C r o t a l u s d u r i s s u s t e r r i f i c u s (Laurenti, 1768). Arquivos do Instituto Biológico 20:271-275, 1962.
21. Wagner CE. Mouth rot, a common disease of snakes. Bulletin of the Natural History Society 5:20-23, 1934. 22. Wallach JD. Medical care o f reptiles. Journal o f the American Veterinary Medicine Association 155: 1017-1041, 1969.
23. Williams FE, Freeman J, Kennedy E. The bacterial flora o f the mouth o f Australian venomous snakes in captivity. Medical Journal o f Australian 11: 190, 1934. 24. Zwart D. N otes on salmonella infections in animals.
Ghana Research Veterinary Science 2:150-169, 1961.
