versión impresa ISSN 0301-732X
Arch. med. vet. v.33 n.1 Valdivia 2001
Survival rate of campylobacter coli strains in sterile buffalo and
Tasas de sobrevida de Campylobacter coli en leche de búfalo y de bovino
The survival rate of five Campylobacter coli strains, isolated from bovine faeces (3) and from buffalo faeces (2), was studied in sterile buffalo and bovine milk kept at 4°C under aerobic conditions. All strains lost their viability substantially more rapidly in buffalo milk than in bovine milk.
Palabras claves: Campylobacter coli, sobrevida, leche
Key words: Campylobacter coli, survival, milk
Campylobacter jejuni subsp. jejuni and C. coli are zoonotic bacteria frequently associated with human diarrhoea in both developing and industrialized countries. Many animal species harbour these agents in their intestinal tract; cows, pigs and poultry being the most important reservoirs (Skirrow & Blaser, 1992; Tresierra-Ayala et al., 1995; Fernández & Pisón, 1996).
Campylobacter enteritis is mainly a foodborne disease and human beings become infected by consumption of contaminated water or food of animal origin (Butzler & Oosterom, 1991). Some researchers (Barrell, 1981; Doyle & Roman, 1982; Koidis & Doyle, 1984; Tresierra-Ayala et al., 1999) have studied the survival rate of Campylobacter species in milk, but most of these studies have been done in bovine milk, considering only C. jejuni, although C. coli has a similar importance in public health (Tresierra-Ayala et al., 1996). In Iquitos (Eastern Peru), unpasteurized buffalo and bovine milk are frequently consumed and they are usually stored at 4°C for several days. For these reasons, it was decided to determine the survival rate of C. coli strains both in buffalo and bovine milk kept at 4°C under aerobic conditions.
MATERIAL AND METHODS
Five C. coli strains, 3 isolated from bovine and 2 from buffalo faeces were studied. Strains were cultured on blood agar plates according to Hoffman et al. (1979) consisting of (wt/vol): Brucella agar 4,3 g%; ferrous sulphate 0,05 g%, sodium metabisulfite 0,05 g%; sodium piruvate 0,05 g% and defibrinated horse blood 5 ml%. Plates were incubated at 42°C for 48 h under microaerophilic conditions. For survival experiments, bacterial suspensions of each strain were prepared in distilled water (109 CFU/ml) and 1ml aliquot added to 99 ml of sterile buffalo and bovine milk (pre-heated at 121°C for 15 min), for eliminating any influence of the accompanying flora or indigenous systems present in normal milk, and kept at 4°C, throughout the experiments, under aerobic conditions, without agitation. The pH of milk was monitored at the beginning and at the end of each experiment, ranging between 6.7 to 6.9 throughout the study.
Using a modified Miles and Misra method (Miles & Misra, 1938), viable counts were carried out at six-hour intervals up to 30 h or until no viable bacteria were detected. To achieve this, a 1-ml sample of the content of each assay was obtained, serially diluted in 0.1% peptone and 20 µl from each dilution plated out (in quintuplicate) onto blood agar plates and incubated at 42°C for 36 h under microaerophilic conditions. Finally, the results were corrected using the lineal regression method (Colton, 1974).
RESULTS AND DISCUSSION
A relative rapid decrease in viable counts of all strains was observed, especially in buffalo milk, in which viable bacteria were not detected after 30 h of incubation at 4°C. Strains 1, 4 and 5 did not survive after 24 h and viable cells of strains 2 and 3 were not detected after 30 h (table 1).
* lineal regression method (Colton, 1974)
On the other hand, the survival time of C. coli strains in bovine milk was more prolonged; strains 1, 3 and 4 did not survive after 96 h of incubation, whereas viable cells of strains 2 and 5 were not detected after 192 and 144 h, respectively (table 1).
The results of the present study have shown that, apparently, C. coli strains do not survive for extended times in sterile milk kept at 4°C under aerobic conditions, loosing their viability substantially more rapidly in buffalo milk than in bovine milk. Under the experimental conditions of this study, none of the 5 C. coli strains were recovered after 30 h of incubation in buffalo milk. This product is well known for its "heaviness" due to higher proportions of milk constituents such as fat, protein, lactose and minerals compared to bovine milk (Ganguli, 1980). We believe that chemical composition of buffalo milk may play an important role in the survival rate of Campylobacter strains in milk.
On the other hand, all C. coli strains were recovered after storage at 4ºC for 48 h or more in bovine milk. Interestingly, one strain (C. coli N°2) was still recoverable at 144 h after inoculation (table 1). Barrell (1981) and Koidis & Doyle (1984) determined that some Campylobacter strains were still recovered from bovine milk after storage at 4°C for more than 120 h.
The greatest survival time of C. coli strains in milk, specially in bovine milk, indicates that these organisms remain viable for most of the time the milk is stored in the consumer's house. Being C. coli closely related to C. jejuni and also associated with enteric infections (Skirrow and Blaser, 1992; Fernández, 1992) and considering that the infective dose of C. jejuni for humans appears to be low (ca. 500 cells) as it was stated by Robinson (1981), the latter could be extrapolated to C. coli. Therefore, the presence of small numbers of Campylobacter cells in milk during storage in the consumer's house as well as the survival times of these bacteria, such as that found in this study, are other aspects to be considered for under-standing campylobacteriosis as a foodborne disease.
Se determinó, en leche de bovino y de búfalo autoclavadas y mantenidas a 4ºC bajo condiciones de aerobiosis, la sobrevida de cinco cepas de Campylobacter coli aisladas de fecas de bovino (3) y de búfalo (2). Todas las cepas perdieron su viabilidad más rápidamente en leche de búfalo que en leche bovina.
Barrell, R. 1981. The survival of Campy-lobacter coli/jejuni in unpasteurized milk. J Infect 3: 348-352. [ Links ]
Butzler, J. P., J. Oosterom. 1991. Campy-lobacter: pathogenicity and significance in foods. Int J Food Microbiol . 12: 1-8. [ Links ]
Colton, T. 1974. Statistics in Medicine. Little, Brown and Company, Massachusetts, pp. 372. [ Links ]
Doyley, M. P., D. J. Roman. 1982. Prevalence and survival of Campylobacter jejuni in unpasteurized milk. Appl . Environ Microbiol. 44: 1154-1158. [ Links ]
Fernández, H. 1992. Thermotolerant Campy-lobacter species associated with human diarrhea in Latin America. Ciênc. Cult. 44: 39-43. [ Links ]
Fernandez, H., V. PISON. 1996. Isolation of thermotolerant species of Campylobacter from commercial chicken livers. Int. J. Food Microbiol. 29: 75-80. [ Links ]
Ganguli, N. 1980. Buffalo as a candidate for milk production. International Dairy Federation, IDF Document 54. Brussels, Belgium, pp. 1-54. [ Links ]
Hoffman, P., N. Krieg, R. Smibert. 1979. Studies of the microaerophilic nature of Campylobacter fetus subsp. jejuni. I. Physiological aspects of enhanced aerotolerance. Can. J. Microbiol. 25: 1-7. [ Links ]
Koidis, P., M. Doyley. 1984. Procedure for Increased Recovery of Campylobacter jejuni from Inoculated Unpasteurized Milk. Appl. Environ Microbiol. 47: 455-460. [ Links ]
Miles, A., S. Misra. 1938. The estimation of the bactericidal power of the blood. J. Hyg. 38: 732-748. [ Links ]
Robinson, D. 1981. Infective dose of Campylobacter jejuni in milk. Br. Med. J. 282: 1584. [ Links ]
Skirrow, M., J. Blaser. 1992. Clinical and Epidemiologic Considerations. In: Nachamkin, I, Blaser, J, Tompkins, L, Campylobacter jejuni: Current status and future trends. American Society for Microbiology. Washington: pp 3-8. [ Links ]
Tresierra-Ayala, A., M. Bendayan, A. Bernuy, F. Espinoza, H. Fernandez. 1995. Carriage of the classical thermotolerant campylobacters in healthy domestic animals from eastern Perú. Rev. Inst. Med. Trop. São Paulo 37: 537-539. [ Links ]
Tresierra-Ayala, A., H. Fernandez, J. Zamora. 1996. Enterotoxigenic Capacity of Campylobacter coli Strains Isolated from Humans and Bovines. Mem. Inst. Oswaldo Cruz 91: 619-620. [ Links ]
Tresierra-Ayala, A., R. Ruiz, M. Bendayan, H. Fernandez. 1999. Survival times of Campylobacter coli in Sterilized Buffalo Milk. J. Vet. Med. B. 46:141-144. [ Links ]