Rev. Inst. Med. trop. São Paulo
30 (5):357-360, setembro-outubro, 1988
SPECIES A N D BIOTYPE DISTRIBUTION OF THERMOTOLERANT
CAMPYLOBACTERS IN A N I M A L RESERVOIRS IN SOUTHERN C H I L E "
Heriberto FERNANDEZ*2»
SUMMARY
The prevalence of thermotolerant Campylobacters in m a m m a l s and birds from Southern Chile was determined. Campylobacters were isolated from 4 6 . 3 % of the animals studied being C. jejuni biotipe 1 the most frequent (25.7%) followed by C.
coli (17.4%) and C. jejuni biotipe 2 ( 3 . 2 % ) .
KEY WORDS: Campylobacter; Campylobacteriosis; Reservoirs; Epidemiology.
INTRODUCTION
Thermotolerant Campylobacters -
Campy-lobacter jejuni and C. coli - are recognized as
important agents of acute diarrheal illness in
h u m a n s1 4 3 1. These microorganisms are widely
distributed all over the world having as natural reservoirs a great variety of animal species i n c l u d i n g c o m p a n i o n , d o m e s t i c a n d wild
animals'. ». 1 3.2 4.
H u m a n illness considered as z o o n o s i s3 0 can
be acquired by contact with a n i m a l s9-2° . 3 0, by
d r i n k i n g c o n t a m i n a t e d w a t e r with a n i m a l
faeces2- 2 7. 3 6 or by ingestion of contaminated
food of animal o r i g i n2.2 0.
In view of the lack of information about the n a t u r a l d i s t r i b u t i o n of t h e r m o t o l e r a n t Campylobacters in this region of the world, a study was undertaken to determine the preva-lence of C. jejuni and C. coli in different m a m m a l s a n d b i r d s f r o m s o u t h e r n C h i l e (province of Valdivia), southern latitude 39° 15' - 4 0 ° 1 5 ' .
MATERIALS A N D M E T H O D S
A total of 631 fecal samples obtained by rectal swabs from 150 dogs, 80 pigs, 80 cows,
40 cats, 100 hens, 75 sparrows, 60 geese and 46 pigeons were studied. None of the animals subjeted to this study showed clinical symptoms of diarrhea. Rectal samples were obtained using commercial sterile swabs (Linsan Laboratories, Chile) supplied with a 1 ml ampule of Cary Blair transport m e d i u m . After collection of the sample the ampules were crushed to moisten the swabs. Each sample was identified with an a p p r o p r i a t e code n u m b e r , and within 2 to 6 hours after collection were seeded on modified
Skirrow's medium2* comprising Brucella agar
(Difco), 5 % horse b l o o d , Skirrow's Antibiotic Supplement (Oxoid) plus 10 mg/1 of cephalotin ( L i l l y ) a n d F B P S u p p l e m e n t ( O x o i d ) . Inoculated plates were incubated at 43°C for 48 hours under microaerobic conditions utilizing the Gas generating box Campylobacter system (bio Merieux).
After the incubation period, the plates were examined for the presence of colonies presumed to be Campylobacter sp. The
identifi-cation was m a d e first presumptively1 7 using
catalase and oxidase reactions (both positive) and the morphological features observed in G r a m stain (curved S-shaped rods). Later, the
Campylobacter species and biotypes were
identified using the criteria p r o p o s e by
S K I R R O W & B E N J A M I N3 3.
(1) S u p p o r t e d by research grant S-84-1 from t h e Research F u n d of Universidad A u s t r a l d e Chile. (2) Institute of Clinical M i c r o b i o l o g y . Universidad A u s t r a l d e Chile. P . O . Box 567, Valdivia, C h i l e .
RESULTS
Campylobacters were isolated from 292 (46.3%) of t h e animals studied The h i g h e s t i s o l a t i o n r a t e was f o u n d in pigs ( 7 0 % ) , followed by hens (60%) and dogs ( 5 1 . 4 % ) . The lowest rate was found in pigeons ( 1 7 . 4 % ) .
C. jejuni biotype 1 was the most frequently
isolated (25.7%) and was found in all the animals studied. C. coli was isolated from all the animals but not from cats and pigeons, with an average frequency of 17.4%. C. jejuni biotype 2 was a c i found in hens, sparrows and pigeons and presented the lowest isolation rate ( 3 . 2 % ) . Table 1 shows the distribution of these Campylobacters among the animal reservoirs studied.
D i s t r i b u t i o n of t h e r m o t o l e r a n t Campylobacters a m o n g various m a m m a l s and birds from s o u t h e r n Chile. C. j e j u n i b i o t y p e 1 C. jejuni b i o t y p e 2 C. coli T o t a l A n i m a l s s t u d i e d (n) N°. % N 9 % N ° % N ° % Dogs ( 1 5 0 ) 4 6 30.7 6 4.0 25 16.7 77 51.4 Pigs ( 8 0 ) 8 10.0 4 5.0 4 4 5 5 . 0 5 6 70.0 C o w s ( 80) 12 15.0 6 7.5 6 7.5 24 30.0 C a t s ( 4 0 ) 6 15.0 3 7.5 0 0.0 9 22.5 H e n s ( 1 0 0 ) 4 5 4 5 . 0 0 0.0 15 15.0 60 6 0 . 0 S p a r r o w s ( 7 5 ) 2 1 2 8 . 0 0 0.0 9 12.0 30 4 0 . 0 G e e s e ( 6 0 ) 16 26.7 1 1.7 11 18.3 28 46.7 Pigeons ( 4 6 ) 8 17.4 0 0.0 0 0.0 8 17.4 T o t a l ( 6 3 1 ) 1 6 2 2 5 . 7 20 3.2 1 1 0 17.4 2 9 2 4 6 . 3 DISCUSSION
Birds and m a m m a l s constitute important links in the e p i d e m i o l o g i c a l c h a i n of
Campylobacter i n f e c t i o n s2' 2 0. We s t u d i e d
several animal reservoirs that, according to their epidemiological relations with h u m a n s , can be divided in four g r o u p s :
1. c o m p a n i o n animals (dogs and cats); 2. food animals (cows and pigs); 3. poultry (hens and geese); 4. wild birds (sparrows and pigeons). Our results show that thermotolerant species of
Campylobacter were present in high frequency
in all of t h e m .
C. jejuni biotype 1 was isolated from all
the animals under study with an average frequency of 2 5 . 7 % . C. jejuni biotype 2, with a frequency of 3 . 2 % , was found in all the animals but not in hens, sparrows and pi?eons. With the exception of the cats and pigeons fecal samples, C. coli was isolated from the other animal feces with a frequency of 1 7 . 4 % . SK1RROW30" and S H A N K E R et a U s reported that C. jejuni biotype 1 was the most prevalent in fecal samples from hens and dogs. O u r results support these findings. However they found that C. jejuni biotype 2 was also frequent
in these two animal species. C. coli was not
isolated by S H A N K E R et al.2» from cloacal
samples of hens. In our study C. jejuni biotype 2 was found in low frequency (4%) in dogs but not in hens. On the other h a n d , the prevalence of C. coli was 16.7% in dogs and 15% in hens. This results are very simmilar to that obtained
by F E R N A N D E Z3 in fecal samples from dogs
and hens in São P a u l o - Brazil.
Some differences in the epidemiology of c a m p y l o b a c t e r i o s i s have been established a m o n g developed and developing countries such as the high percentage or normal carriers and a greater frequency of cases in the first two years of life observed in the latter
countries 4 5.
The fact that C. jejuni biotype 2 is less and C. coli m o r e frequent in hens and dogs in
B r a z i l 3 a n d s o u t h e r n C h i l e , c o u l d be
considered a peculiar epidemiological situation observed in developing countries. However, more data must be m a d e available from third world countries to establish definitively this statement.
The prevalence of Campylobacter in dogs (51.4%) was higher than that obtained by
M c O R I S T & B R O W N I N G '1' in A u s t r a l i a
( 9 . 5 % ) a n d J O R G E N S E N '2 in D e n m a r k
(11.1%). Nevertheless, it was simmilar to that
reported by S I M P S O N et a l .2 9 in stray dogs
(52.6%) in England. Remarkable differences exist in the incidence of intestinal carriage of
Campylobacter a m o n g stray and pet d o g s2.2 9
-A l t h o u g h all t h e d o g s s t u d i e d h a d a n individualized owner, many of them presented loiterer habits reflecting a poor standard of canine hygiene that predisposes to intestinal
colonization with Campylobacter3 0.
The prevalence of Campylobacter in cats (22.5%) was higher than that reported by
M c O R I S T & B R O W N I N G1 9 in Australia (3%)
and by P A T T O N et a l .2 2 in U . S . A . ( 1 4 % ) .
Because the cats studied presented the same habits observed in the dogs (owners references) we c a n use t h e s a m e c o n s i d e r a t i o n s as
S K I R R O W3 0 to explain the high isolation rates
of Campylobacter obtained in both animal s p e c i e s . T h e s e r e s u l t s c o n s t i t u t e a n o t h e r evidence of t h e i m p o r t a n c e of d o g s a n d c a t s as n a t u r a l r e s e r v o i r s of C a m p y l o
-bacter as p o t e n c i a l s o u r c e s for h u m a n
infections2-9.2°.2*. 3°.
The fecal carriage of Campylobacter in pigs (70%) was very simmilar to that reported
by S T I C H T - G R O H3 4 in Germany (77%) and
L U E C H T E F E L D & W A N G " in U . S . A . ( 7 1 % ) , but lower to that published by R O S E F et aU5 in Norway (100%). The fact that C. coli forms part of the n o r m a l intestinal flora of
pigs3 explains the high isolation frequency of
this bacteria (55%) in the pigs subjected to our study.
Campylobacter was found in 3 0 % of the
cows under study. This result is lower than
reported by L U E C H T E F E L D & W A N G '6 in
U . S . A . (43%) but higher than the results obtained by S W E D H E M & K A I J S E R " in Sweden ( 1 9 % ) .
The isolation of thermotolerant species of
Campylobacter in high frequency from these
t w o a n i m a l s i n d i c a t e their i m p o r t a n c e as natural reservoirs and sources of these bacteria for m a n . H u m a n s can acquire the infection
by c o n t a c t with these a n i m a l s9. 2 0 or by
c o n s u m p t i o n of c o n t a m i n a t e d m e a t s a n d
milk2. i0.20,23.
In hens and geese the Campylobacter isolation rate was 6 0 % and 4 6 . 7 % respectively. High isolation rates of Campylobacter from b o t h , slaughtered and alive hens, is a c o m m o n
fact in many c o u n t r i e s2 6. Wild geese harbour
t h e s e m i c r o o r g a n i s m s f r e q u e n t l y in their
intestinal t r a c t1 5.
Poultry constitutes an important vehicle of
Campylobacter and the role of poultry meat in
the illness transmission is well established2.>°.1 1.
The isolation rate of Campylobacter in sparrows was 4 0 % . The corresponding values found by M A T S U S A K I et al.'» in Japan was
only 10% whereas, K A P P E R U D & R O S E F '3
did not isolated any Campylobacter from sparrows in Norway.
In pigeons we isolated only C. jejuni b i o t y p e 1 w i t h a f r e q u e n c y of 1 7 . 4 % M A T S U S A K I et al.'» and K A P P E R U D &
R O S E F '3 reported 7 % and 4 . 2 % of intestinal
c a r r i a g e of C a m p y l o b a c t e r in p i g e o n s
respectively. W E B E R et a l .3 7 in G e r m a n y ,
d e m o n s t r a t e d t h a t Campylobacter can be isolated with high frequency (54.9%) from carrier pigeons. Sparrows and pigeons are wild birds that can live near man and may be sources of contamination for water supplies and the environment as it has been previously
suggested 1-2 1.
Except for studies on pigs and h e n s6.7, this
work is the first study in Chile to ascertain the p r e v a l e n c e of t h e r m o t o l e r a n t C a m p y l o -bacters in animals. We think that more studies are necessary to understand the epidemiology of campylobacteriosis that seems to be a very complex p h e n o m e n o n in developing countries.
RESUMO
Espécies e biotipos de Campylobacter termotolerantes em reservatórios animais do
sul do Chile.
Foi determinada a prevalência de
Campy-lobacter termotolerantes em mamíferos e aves
do sul do Chile.
Campylobacter foi isolado em 4 6 , 3 % dos
animais estudados sendo C. jejuni biotipo 1 o mais freqüente (25,7%) seguido de C. coli (17,4%) e C. jejuni biotipo 2 ( 3 , 2 % ) . REFERENCES 1. B L A S E R , M . J . ; L A F O R C E , F . M . ; W I L S O N , N . A . & W A N G , W . L . L . — Reservoirs for h u m a n c a m p y l o b a c -teriosis. J. infect. D i s . , 14:665-669, 1980. 2. B L A S E R , M . J . ; T A Y L O R , D . N . & F E L D M A N , R . A . — Epidemiology of Campylobacter jejuni infections. Epidem. Rev., 5:157-176, 1983.
-lobacter: bacteriological, epidemiological and pathoge-nical aspects. São P a u l o , 1983. (Doctoral Thesis — School of Medicine).
4. F E R N A N D E Z , H . ; T O L E D O , M . R . F . ; F A G U N D E S N E T O , U . & T R A B U L S 1 , L . R . — Isolation ot Campy-lobacter jejuni in children with acute d i a r r h o e a and in healthy c o n t r o l s . Rev. méd. Chile, 122:238-241, 1984. 5. F E R N A N D E Z , H . ; T O L E D O , M . R . F . ; F A G U N D E S
N E T O , U. & T R A B U L S 1 , L . R . — Occurrence ot Campylobacter jejuni in diarrhoeic and n o n - d i a r r h o e i c children in São P a u l o , Brazil. Rev. Inst. Med. trop. S. Paulo, 27:102-104, 1985.
6. F I G U E R O A . G.; T O L E D O , M . S . ; T R O N C O S O , M . & S E P U L V E D A , C. — Isolation of Campylobacter fe-tus subsp. jejuni from broiler chicken". Rev. chil. Nutr., 10:87-98, 1982.
7. F I G U E R O A , G.; T R O N C O S O , M.; A L C A V D E , M . P . & S E P U L V E D A , C. — Isolation of Campylo-bacter fetus s u b s p . jejuni from local samples of pigs. Rev. chil. Tecnol. méd., 4:6-10, 1980.
8. F O X , J . G . — C a m p y l o b a c t e r i o s i s . A " n e w " disease in laboratory a n i m a l s . Lab. Anim. Sci., 32:625-637, 1982. 9. F O X , J . O . ; M O O R E , R. & A C K F R M A N N , J.I. — C a n i n e a n d feline campylobacteriosis: Epizootiology and clinical and public health features. J. Amer. vet. med. A s s . , 183:1420-1424, 1983.
10. H A R R I S , N . V . ; W E I S S , N . S . & N O L A N , C M . — l h e role of poultry and meats in the etiology of Campylobacter jejuni/coli enteritis. Amer. J. publ. Hltti., 76:407-411, 1986.
11. H O P K I N S , R. & S C O T T , A . S . - H a n d l i n g raw chic-ken as a source for sporadic Campylobacter jejuni in-fections. J. infect. D i s . , 148:770, 1983.
12. J O R G E N S E N , K. — Prevalence of Campylobacter fe-tus s u b s p . jejuni in Danish d o g s . Nord. Vet. - Med., 33:42-48, 1981.
13. K A P P E R U D . G. & R O S E F , O . — Avian wildlife re-servoirs of Campylobacter fetus s u b s p . jejuni, Yersinia s p p . , a n d Salmonella s p p . in N o r w a y . Appl. environ. Microbiol., 45:375-380, 1983.
14. L E V I N , S. & G O O D M A N , L. — Campylobacter in-fections t o d a y . J. Amer. med. A s s . , 253:1303, 1985. 15. L U E C H T E F E L D , N . A . W . ; B L A S E R , M . J . ; R E L -L E R , -L . B . & W A N G , W . -L . -L . — Isolation of Campy-lobacter fetus s u b s p . jejuni from m i g r a t o r y waterfowl. J. elin. Microbiol., 12:406-408, 1980.
16. L U E C H T E F E L D , N . W . & W A N G , W . L . L . — Ani-mal reservoirs of Campylobacter jejuni. In: N E W E L , D . G . , e d . Campylobacter: epidemiology, p a t h o g e n e -sis, biochemistry. L a n c a s t e r , M . T . P . Press, 1982. p . 249-251.
17. L U E C H T E F E L D , N . W . ; W A N G , W . L . L . ; B L A S E R , H . J . & R E L L E R , L . D . — Campylobacter fetus s u b s p . jejuni: b a c k g r o u n d and l a b o r a t o r y diagnosis. Lab. Med., 12:481-487, 1981.
18. M A T S U S A K I , S.; K A T A Y A M A , A. & 1 T A G A K I , K. - Prevalence of Campylobacter jejuni and C. coli in wild birds and domestic animals in Y a m a g u c h i , J a p a n . In: I N T E R N A T I O N A L W O R K S H O P O N Campylo-bacter I N F E C T I O N S , 3")., O t t a w a , C a n a d a , 1985. Abstract. Abstr. N? 160.
19. M c O R I S T , S. & B R O W N I N G , J . W . — C a r r i a g e of Campylobacter jejuni in health and diarrhoeic dogs and cats. Aust. vet. J . , 58:33-34, 1982.
20. M E G R A 1 J D , F. & L A T R I L L E . J. — Campylobacter jejuni en pathologie h u m a i n e . II. Diagnostic biologique
et e p i d e m i o l o g i c P a t h , et Biol., 29:305-314, 1981. 2 1 . P A L M E R , S.R.; G U L L Y , P . R . ; W H I T E , J . M . ; P E A R S O N , A . D . ; S U C K L I N G , W . G . ; J O N E S , D . M . ; R A W E S , J . C . L . & P E N N E R , J . L . — W a t e r -b o r n e o u t -b r e a k of C a m p y l o -b a c t e r gastroenteritis. Lan-cet, 1:287-290, 1983. 22. P A T T O N , C M . ; M I T C H E L L , S.W.; P O T T E R , M . E . & K A U F F M A N N , A . F . — C o m p a r i s o n of selective media for primary isolation of C a m p y l o b a c t e r fetus s u b s p . j e j u n i . J. cíin. M i c r o b i o l . , 13:326-330, 1981. 23. P O T T E R , M . E . ; B L A S E R , J . M . ; S I K E S , R.K.;
K A U F F M A N N , A . F . & W E L L S , J . G . — H u m a n C a m p y l o b a c t e r infection associated with certified raw milk. A m e r . J . E p i d e m . , 117:475-483, 1983.
24. P R E S C O T T , J . F . & M U N R O E , D . I . . — C a m p y l o b a c -ter jejuni in m a n and domestic a n i m a l s . .1. A m e r . vet. M e d . A s s . , 181:1524-1530, 1982.
25. R O S E F , O . ; G O N D R O S E N . B . ; K A P P E R U D , G. & U N D E R D A L , B. — Isolation and characterization of C a m p y l o b a c t e r jejuni and C a m p y l o b a c t e r coli from domestic and wild m a m m a l s in N o r w a y . A p p l . envi-r o n . M i c envi-r o b i o l . , 46:855-859, 1983.
26. S A C K , R . B . ; T 1 L T O N , R . C & W E I S S F E L D , A . S . — L a b o r a t o r y diagnosis of bacterial d i a r r h e a . A m e r . Soc. M i c r o b i o l . C u m i t e c h , 12:1-12, 1980.
27. S A C K S , J . J . ; L I E B , S.; B A L D Y , L . M . ; B E R T A , S.; P A T T O N , C M . ; W H I T E , M . C . ; B I G L E R , W . J . & WTTTE, J J . — Epidemic campylobacteriosis associa-ted with a c o m m u n i t y water supply. A m e r . J. p u b l . H l t h , 76:424-429, 1985.
28. S H A N K E R , S.; R O S E N F I E L D , J . A . ; D A V E Y G . R . & S O R R E L L , T . C . — C a m p y l o b a c t e r j e j u n i : incidence in processed broilers and biotype distribution in h u m a n and broiler isolates. A p p l . e n v i r o n . M i c r o b i o l . , 43:1219-1220, 1982. 29. S I M P S O N , J . W . ; B U R N I E , A . G . ; F E R G U S O N , S. & T E L L E R , W . A . B . — Isolation of t h e r m o p h i l i c C a m p y l o b a c t e r from t w o p o p u l a t i o n s of d o g s . Vet. Res. C o m m . , 5:63-66, 1981. 30. S K I R R O W , M . B . — C a m p y l o b a c t e r enteritis in dogs a n d cats: a " n e w " z o o n o s i s . Vet. Res. C o m m . , 5:13¬
19, 1981.
3 1 . S K I R R O W , M . B . — C a m p y l o b a c t e r enteritis: the first five years. J . H y g . ( L o n d . ) , 89:1975-1984, 1982. 32. S K I R R O W , M . B . — Epidemiological aspects of
C a m p y l o b a c t e r enteritis. In: N E W E L , D . G . , ed. -C a m p y l o b a c t e r I. L o n d o n , Public Health L a b o r a t o r y Service, 1982. p.67-69. 33. S K I R R O W , M . B . & B E N J A M I N , J. — Differentia-tion of e n t e r o p a t h o g e n i c C a m p y l o b a c t e r . J. clin. P a t h . , 33:1122,1980. 34. S T I C H - G R O H , V. — C a m p y l o b a c t e r in healthy slaughter pigs: a possible source of infection for m a n . Vet. R e c , 110:104, 1982.
35. S V E D H E M , A . & K A I J S E R , B. — C a m p y l o b a c t e r j e -j u n i from domestic a n i m a l s a n d pets: p r o b a b l e origin of h u m a n infection. J. Infect., 3:37-40, 1981. 36. V O G R , R . L . , S O U R S , H . E . ; B A R R E T , T . ; F E E D
-M A N , R . A . ; D I C K I N S O N R . J . & W I T H E R E L L , L. — C a m p y l o b a c t e r enteritis associated with c o n t a m i n a -ted water. A n n . intern. M e d . , 96:292-296, 1982. 37. W E B E R , V . A . ; L E M B K E , C . & K E T T N E R , A. —
Oc-currence of C a m p y l o b a c t e r jejuni in faecal samples of clinically healthy carrier pigeons. Berl. M u n c h , tie-rârztl. W s c h r . , 94:449-451, 198!.