An optimised recovery method for thermophilic Campylobacter from liver
© Moore; licensee BioMed Central Ltd. 2001
Received: 19 September 2001
Accepted: 30 November 2001
Published: 30 November 2001
The past three decades have witnessed the rise of Campylobacter enteritis in man from virtual obscurity to notoriety, with present isolation rates superseding those of other enteric pathogens such as Salmonella spp. and Shigella spp. in most developed countries. Although campylobacters are not completely new to applied bacteriology, they have evaded traditional isolation techniques used for the isolation of pure cultures, apart from single isolations that were free from competing organisms. Offals, in particular liver have been decribed as both a source of campylobacters, as well as a route of transmission of this organism to human. Therefore, the aim of this study was to develop an optimum method for the recovery of Campylobacter spp. from porcine liver.
Four isolation techniques (methods A-D) were compared in a small pilot study for their ability to successfully recover campylobacters from freshly eviscerated porcine liver. The optimum isolation method involved direct swabbing of the liver tissues followed by plating onto Preston Selective medium, which was superior to methods involving mechanical disruption to liver tissues, including direct plating and enrichment methods, with and without blood. Consequently, any isolation method that involves disruption of liver tissue e.g. homogenisation or stomaching, is not suitable for the detection of campylobacters from liver and hence it is recommended that employment of a direct swabbing technique without mechanical disruption of tissues in combination with selective plating to optimally recover campylobacters from freshly eviscerated liver.
Employment of a direct swabbing technique in combination with selective plating allow Campylobacter spp. to be optimally recovered from freshly eviscerated liver and therefore this technique is recommended when examining liver for the presence of this organism.
The past three decades have witnessed the rise of Campylobacter enteritis in man from virtual obscurity to notoriety, with present isolation rates superseding those of other enteric pathogens such as Salmonella spp. and Shigella spp. in most developed countries.
Unlike the salmonellae and other enteric pathogens, the majority (ca. 99%) of clinical reports concerning Campylobacter are sporadic and Campylobacter enteritis outbreaks are rare. The lack of well-developed typing schemes has hindered the epidemiological investigations seeking natural reservoirs of the organism and modes of transmission from these sources to man. Only about 15% of clinical isolates are identified to species level thus making epidemiological investigations extremely difficult to perform.
Campylobacters are not completely new to applied bacteriology. They have evaded traditional isolation techniques used for the isolation of pure cultures, apart from single isolations that were free from competing organisms. Until the development of a selective medium by Skirrow , these organisms were known mainly by veterinarians as animal pathogens which were responsible for a wide variety of disorders in cattle, sheep and pigs . Since the development of more sophisticated isolation techniques, the true disease potential of these organisms has become apparent and today campylobacteriosis is regarded as a zoonosis, which is capable of being transmitted to man by a wide range of domestic animals, their meat and offals.
Offals, in particular liver have been described as both a source of campylobacters [3–6], as well as a route of transmission of this organism to human . Therefore, the aim of this study was to develop an optimum method for the recovery of Campylobacter spp from porcine liver.
Results and discussion
Comparison of isolation methods (A-D) for campylobacters from freshly eviscerated porcine liver.
ISOLATION METHOD (% livers positive)
No. Herds examined
No. livers examined
A (Direct Isolation)
B (Enrichment with blood)
C (Enrichment without blood)
D (Swabbing technique)
Although there are numerous techniques for the isolation of Campylobacter spp. from meats and foodstuffs, there is presently no accepted standard reference method for the isolation of this organism. Several studies have addressed this in an attempt to define the most suitable isolation technique, so that all results are comparable [7–10], however one single protocol may not be ideal for all types of foodstuffs. Therefore, it is important to optimise the isolation technique for the particular foodstuff being examined.
In the present study, four isolation methods were compared to obtain an optimal isolation technique for porcine liver. As previous studies have shown that a low proportion (<10%) of liver may be infected with Campylobacter spp.[3, 4], an isolation method was sought which would allow rapid screening of a large number of livers, which was both specific and sensitive.
Only naturally contaminated livers were used in this study to assess the most effective isolation method, as employment of spiked specimens would not have truly reflected the sites of adherence, the natural adherence mechanism and the density of cells normally found in the wild-type state. Consequently, as it was difficult to obtain a large number of positive specimens, this small pilot study did show that the direct swabbing technique was the most sensitive isolation method employed (Table 1). Preston agar was selected as the selective plating medium, as this medium has been shown previously to be the optimum medium for the isolation of wild-type campylobacters from porcine liver . Campylobacters isolated from porcine liver were not considered to be sub-lethally stressed, as direct plating onto selective media did not cause a decrease in isolation rates. The swab method was shown to be sufficiently sensitive for the detection of Campylobacter spp., as isolation was on a presence/absence basis. The swab technique allowed the intrinsic contamination of liver to be assessed by selective sampling of deep tissue as opposed to surface tissue. Swabbing was also the most economical in terms of time and materials. Therefore swabbing onto selective media was considered to be the optimal method of recovery of campylobacters from liver.
Bolton and Robertson  and Bracewell et al. concluded that the incorporation of an enrichment stage was superior to direct plating. Although enrichment procedures may enhance isolation rates, Turnbull and Rose  showed that eight samples were positive on direct plates yet were negative by enrichment procedures. This might indicate that some of the enrichment methods employed were not fully reliable or may included a cell dispruption process such as homogenization or stomaching. Previously, it has been shown  that liver homogenates contain a heat-labile antagonistic factor, which is lethal to campylobcters even after six hours. As the liver is the organ of detoxification, any intracellular inhibitory peptides or proteins, which are released during homogenization, may act antimicrobially and hence inhibit the growth and proliferation of campylobacters. Therefore, it is proposed that any isolation method should therefore not include a disruption stage, such as homogenization or stomaching, which are normal stages in isolation protocols and thus an examination of liver homogenates in the study design was not included.
Stern [7, 8] demonstrated that the swab technique was capable of recovering 32 Campylobacter cells per cm2 from lamb carcasses and this technique has been employed as an isolation technique by other workers . In the present study, direct plating of cells onto selective media by swabbing was considered to be a rapid and sensitive isolation technique for Campylobacter spp. in porcine liver.
In conclusion, employment of a direct swabbing technique in combination with selective plating allow Campylobacter spp. to be optimally recovered from freshly eviscerated liver and therefore this technique is recommended when examining liver for the presence of this organism.
Materials & Methods
30 samples were examined by each method after three visits over a two-month period to two EU-licensed pork processing plants in Northern Ireland (Plants A & B). Samples were taken from 17 herds of bacon pigs. Ten samples were taken at random on each plant visit constituting one batch. Campylobacter spp. were isolated from each sample by carrying out methods A-D in duplicate. Two replicate samples from all treatments (Methods A-D) were streaked onto Preston selective agar (Oxoid Ltd., England). Cultures were incubated as described (Figure 1). Presumptive positive colonies were streaked onto BA2 and incubated prior to characterization, employing several phenotypic tests as previously described . All livers from which Campylobacter spp. were isolated were recorded as positive. Statistical analyses were performed to compare recovery by methods A-D employing Microsoft Excel employing a paired student's t-test with a one-tailed distribution. Statistical significance was noted whenever the probability (P) was less than 0.05 (5%).
The authors wish to thank Mr. Michael Watts MRCVS for his help and assistance with the collection of liver specimens. JEM was supported by a Department of Agriculture for Northern Ireland (DANI) Postgraduate Studentship Award and also by the Department of Health & Social Services (Northern Ireland).
- Skirrow MB: Campylobacter enteritis: a "new" disease. Brit Med J. 1977, 2: 9-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Prescott JF, Bruin-Mosch CW: Carriage of Campylobacter jejuni in healthy and diarrheic animals. Am J Vet Res. 1981, 42: 164-165.PubMedGoogle Scholar
- Moore JE, Madden RH: Occurrence of thermophilic Campylobacter spp. in porcine liver in Northern Ireland. J Food Prot. 1998, 61: 409-413.PubMedGoogle Scholar
- Bolton FJ, Dawkins HC, Hutchinson DN: Biotypes and serotypes of thermophilic campylobacters isolated from cattle, sheep and pig offals and other red meats. J Hyg. 1987, 95: 1-6.View ArticleGoogle Scholar
- Soonattrakul W, Andersen BR, Bryner JH: Raw liver as a possible source of Vibrio fetus septicemia in man. Am J Med Sci. 1971, 261: 245-249.View ArticlePubMedGoogle Scholar
- Kramer JM, Frost JA, Bolton FJ, Wareing DR: Campylobacter contamination of raw meat and poultry at retail sale: identification of multiple types and comparison with isolates from human infection. J Food Prot. 2000, 63: 1654-1659.PubMedGoogle Scholar
- Stern NJ: Campylobacter fetus subsp. jejuni: Recovery methodology and isolation from lamb carcasses. J Food Sci. 1981, 46: 660-661.View ArticleGoogle Scholar
- Stern NJ: Recovery rate of Campylobacter fetus subsp. jejuni on eviscerated pork, lamb and beef carcasses. J Food Sci. 1981, 46: 1291-View ArticleGoogle Scholar
- Stern NJ: Methods for recovery of Campylobacter jejuni from foods. J Food Prot. 1982, 45: 1332-1337.Google Scholar
- Stern NJ, Line JE: Comparison of three methods for recovery of Campylobacter spp. from Broiler Carcasses. J Food Prot. 1992, 55: 663-666.Google Scholar
- Bolton FJ, Robertson L: A selective medium for isolating Campylobacter jejuni/coli. J Clin Path. 1982, 35: 462-467.PubMed CentralView ArticlePubMedGoogle Scholar
- Bracewell AJ, Reagan JO, Carpenter JA, Blankenship LC: Incidence of Campylobacter jejuni/coli on pork carcasses in the Northwest Georgia area. J Food Prot. 1985, 48: 808-810.Google Scholar
- Turnbull PCB, Rose P: Campylobacter jejuni and Salmonella in raw red meats: A public health laboratory service survey. J Hygiene. 1982, 88: 29-37.View ArticleGoogle Scholar
- Moore JE, Madden RH: Survival of Campylobacter coli in porcine liver. Food Microbiol. 2001, 18: 1-10. 10.1006/fmic.2000.0367.View ArticleGoogle Scholar
- Kwiatek K, Wojton B, Stern NJ: Prevalence and distribution of Campylobacter spp. on poultry and selected red meat carcasses in Poland. J Food Prot. 1990, 54: 127-130.Google Scholar
- Cary SG, Blair EB: New transport medium for shipment of clinical specimens. J Bact. 1964, 88: 96-98.PubMed CentralPubMedGoogle Scholar
- Bolton FJ, Holt AV, Hutchinson DN: Campylobacter biotyping scheme of epidemiological value. J Clin Path. 1984, 37: 677-681.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.