Growth capacity of thermotolerant campylobacters in culture media supplemented with pig and cow blood

HUMAN AND ANIMAL HEALTH

Growth capacity of thermotolerant campylobacters in culture media supplemented with pig and cow blood

Álvaro Tresierra-Ayala^I,II; Manuel Navas^I; Josué Flores^I; Ramsés Perea^I; Juan Huanaquiri^I; María Bendayán^I; Heriberto Fernández^III,*

^IDepartment of Microbiology; Universidad Nacional de la Amazonia Peruana; Pevas 5ta. Cuadra; Iquitos - Perú

^IIResearch Institute; Universidad Particular de Iquitos; Av. Quiñones Km, 2,5; Iquitos - Perú

^IIIInstitute of Clinical Microbiology; Universidad Austral de Chile; P. O. Box 567; Valdivia - Chile

ABSTRACT

In this work 60 thermotolerant Campylobacter strains (37 C. jejuni and 23 C. coli) isolated from the cows, pigs, chickens and ducks (15 strains of each type of animal) were used to establish their growth capacity on media containing cow or swine blood as potential substitutes of sheep or horse blood. The growth capacity was assessed by viable counts on cow and swine blood media, using the modified Miles and Misra method. Campylobacter strains showed better growth in the media supplemented with pig or sheep blood than with cow blood. Thus, the use of pig blood could be a supplement for Campylobacter culture medium, when there was no availability of sheep or horse blood.

Key words: Campylobacter, culture media, cow blood, swine blood

RESUMO

Na cidade de Iquitos (Região Amazônica do Peru), tanto o sangue de carneiro quanto o de eqüino, ambos recomendados como suplemento nos meios de cultura para Campylobacter, são difíceis de encontrar. No entanto, sangues de bovino e de suíno são de fácil disponibilidade. Por esta razão, 60 amostras de Campylobacter termotolerantes (37 C. jejuni e 23 C. coli) isoladas de bovinos, suínos, frangos e patos (15 amostras de cada animal) foram utilizadas para estabelecer sua capacidade de crescimento em meios de cultura contendo sangue de bovino ou de suíno como potenciais substitutos do sangue de carneiro ou de eqüino. A capacidade de crescimento foi estabelecida através da contagem de células viáveis utilizando o método de Miles e Misra modificado. Todas as amostras de Campylobacter mostraram melhor crescimento em meios suplementados com sangue de eqüino ou de suíno. Estes resultados permitem propor o uso de sangue de suíno como suplementos em meios de cultura para Campylobacter.

INTRODUCTION

The classical thermotolerant campylobacters (Campylobacter jejuni and C. coli) are zoonotic bacteria frequently associated with human diarrhoea in both developing and industrialized countries. Many animal species harbor these agents in their intestinal tract, being dogs, cats, cows, pigs and poultry their most important household reservoirs (Fernández, 1992; Tresierra-Ayala et al., 1995; Fernández and Pisón, 1996; Engberg, 2006; Fernández et al., 2008; Debruyne et al., 2008). Campylobacter species are strictly microaerophilic organisms that grow optimally in an atmosphere containing 5% oxygen. Such an environment may be obtained by (a) providing a specified microaerobic gas mixture, (b) using commercial atmosphere-generating systems, or (c) culturing the organisms in a normal atmosphere in a semisolid medium that naturally provides an oxygen gradient (Stern and Kazmi, 1989; Engberg, 2006).

George et al. (1978) reported that aerotolerance of many Campylobacter strains increases when the culture media were supplemented with 0.025% each of ferrous sulfate, sodium metabisulfite, and sodium pyruvate (FBP). They suggested that incorporating the FBP supplement into media enhanced the growth of the organisms and, consequently, its recovery from samples could be improved. On the other hand, Hoffman et al. (1979) suggested that FBP might act as an oxygen and oxygen products (peroxides and superoxides) scavenger that otherwise would produce a toxic effect on these organisms. Stern and Kazmi (1984) reported that supplements of ferrous sulfate, sodium pyruvate, charcoal and blood, acting as quenching or detoxifying agents, prevented the accumulation of toxic oxygen derivatives allowing the growth of the organisms. Most of the Campylobacter culture media described in the literature are also supplemented with sheep or horse blood.

A variety of animal bloods and banked human blood are used for preparing some microbiological culture media, especially for enrichment and studying some characteristics such as hemolytic properties. In many countries, defibrinated sheep blood is accepted as the most efficient blood supplement for routine work, being horse blood recommended as the second choice (Vera and Power, 1980).

In Iquitos city (in the Peruvian jungle region, Southern latitude 3º 45'), thermotolerant campylobacters have been considered an important cause of childhood diarrhea being isolated from 23% of the cases (Grados et al., 1988). However, the routine diagnosis of Campylobacter is not implemented due to the difficulties of the clinical microbiology laboratories in obtaining sheep and horse blood easily, associated to local animal husbandry practices. The aim of this work was to study the growth capacity of these bacteria using cow and pig blood instead sheep or horse blood.

MATERIAL AND METHODS

Sixty thermotolerant Campylobacter strains (37 C. jejuni and 23 C. coli) were used in this study. They were isolated from the cloacal or rectal samples obtained from the healthy domestic animals (chickens, ducks, cows and pigs) from different peri-urban zones in Iquitos city. Once obtained, all the samples were immediatly placed into the transport and enrichment medium proposed by Fernández (1992), consisting of (w/v%): Brucella broth (Difco) 2.8 g; agar (Difco) 0.15 g; ferrous sulphate (Merck) 0.05 g, sodium metabisulfite (Merck) 0.05 g; sodium pyruvate (Merck) 0.05 g, trimethoprim (Sigma) 1 mg; rifampicin (Sigma) 1.5 mg; colistin (Sigma) 1000 IU; amphotericine (Squibb) 1 mg and defibrinated horse blood 3 ml. After that, they were streaked onto modified Skirrow plates (Fernández et al. 1994) consisting of (w/v%): Brucella agar (Difco) 4.3 g; ferrous sulphate (Merck) 0.05 g, sodium metabisulfite (Merck) 0.05 g; sodium piruvate (Merck) 0.05 g, vancomicin (Sigma) 1 mg; trimethoprim (Sigma) 0.5 mg; polimixin B 250 IU; cephalotin (Sigma) 1 mg; amphotericine (Squibb) 0.1 mg and defibrinated horse blood 5 ml. The plates were incubated at 42ºC for 48 h in an atmosphere of 5% O₂ -10% CO₂ and 85% N₂. Suspected colonies were identified to species level by their morphological characteristics, susceptibility to cephalothin and nalidixic acid, growth at 26, 37 or 42ºC and their biochemical profiles using the API Campy system (bioMérieux).

The growth capacity in different bloods containing media (sheep, pig and cow blood) was assessed by viable counts of each strain using the modified Miles and Misra method (Tresierra-Ayala et al., 1999). In brief, bacterial suspensions of each strain were prepared in distilled water (3 x 10⁸ CFU/ml); log10 serially diluted in 0.1% peptone water and 20µl from each dilution were seeded, in quintuplicate, onto blood agar plates. After 36h of incubation at 42ºC for 36h under microaerophilic conditions, viable counts were determined.

The results were compared by the analysis of variance (ANOVA), provided by SPSS program.

RESULTS AND DISCUSSION

All the thermotolerant Campylobacter strains under study showed growth capacity in culture media supplemented with cow blood and pig blood, as well as in the media supplemented with sheep blood used as control. However, the analysis of the results showed that the growth on the culture medium with pig blood was higher than the growth observed on the culture medium supplemented with cow blood, being this difference statistically significant (p <0.05). On the other hand, the growth on the culture medium supplemented with pig blood was similar to that obtained on the culture medium supplemented with sheep blood (p >0.05). This trend was similar, regardless of the bacterial species studied and the animal source of the strains (Tables 1 and 2).

Therefore, it could be concluded that Campylobacter strains showed better growth in the presence of pig or sheep blood than with cow blood. Thus, of pig blood could be used as a supplement for culturing Campylobacter, especially when there was no availability of sheep or horse blood. This suggestion was in agreement with Anand et al. (2000), who expressed that in many developing countries, pigs, goats, or both were more readily available than sheep or horses and constituted an alternative source of blood for bacteriological purposes. Bolton et al. (1984) and Corry et al. (2001) suggested that blood as a supplement of the bacterial growth media prevented the accumulation of toxic oxygen derivatives (peroxides and superoxides) and allowed the growth of the organisms because these substances acted as quenching or detoxifying agents.

Erythrocytes possess superoxide dismutase and catalase enzymes (Chauhan et al., 1982), some with more enzymatic activity than others, so this could influence on the detoxifying capacity of blood. Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies (Florin-Christensen et al., 2001) that could have influence on their physiological activities. On the other hand, copper deficiencies in bovines give rise to an alteration in the antioxidant capacity (Sukalsky et al., 1997). Probably, due to those facts, the erythrocytes of cow blood could have a lower enzymatic activity than the sheep or pig erythrocytes.

From the resukts, it could be concluded that pig blood could be a suitable alternative to sheep blood for use in Campylobacter media in regions where sheep and horse blood were not readily available.

ACKNOWLEDGEMENTS

This work was supported by the Grants DID-UACH S-2007-37 and DID-UACH SE-1-2009.

Received: March 17, 2009; Revised: October 26, 2009; Accepted: April 13, 2010.

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Publication Dates

• Publication in this collection
  03 Nov 2010
• Date of issue
  Oct 2010

History

• Accepted
  13 Apr 2010
• Reviewed
  26 Oct 2009
• Received
  17 Mar 2009