Enteric colonisation following natural exposure to Campylobacter in pigs

Research in Veterinary Science 2000, 68, 75–78 doi:10.1053/rvsc.1999.0335, available online at http://www.idealibrary.com on

Enteric colonisation following natural exposure to Campylobacter in pigs C. R. YOUNG*, R. HARVEY, R. ANDERSON, D. NISBET, L. H. STANKER United States Department of Agriculture, Agricultural Research Service, Food and Feed Safety Research Unit, Food Animal Protection Research Laboratory, 2881 F&B Road, College Station, Texas 77845, USA SUMMARY A survey was conducted to establish the prevalence of Campylobacter in pigs from an integrated commercial hog farm. This study was carried out in four different groups of pigs: 1) adult gilts (50); 2) pregnant sows (9); 3) piglets at day-of-birth (73); 4) weaned piglets (20). Rectal and/or caecal samples were collected from each pig. Campylobacter was cultured and enumerated from such samples using Bolton enrichment broth and Campy-Cephex agar plates. Both biochemical and serological tests were used to determine Campylobacter species. Gilts had a 76 per cent incidence of Campylobacter with a mean of 76·3 per cent for C. jejuni, 21 per cent for C. coli and 2·6 per cent for C. lari. Pregnant sows had a 100 per cent incidence of Campylobacter with a mean of 87 per cent for C. jejuni and 13 per cent for C. coli. Newborn piglets had a 57·8 per cent incidence of Campylobacter, rising to 100 per cent by the time of weaning. Thus it appears that pigs, from the day of birth, are highly susceptible to colonisation by Campylobacter. © 2000 Harcourt Publishers Limited

CAMPYLOBACTER, a microaerophilic, Gram-negative bacterium, is a member of the epsilon division of bacteria. Campylobacter is recognised as one of the leading causes of human bacterial gastroenteritis (Gregory et al 1997). Campylobacter jejuni, Campylobacter coli and Campylobacter lari can be carried in the intestinal tract of animals and can thus contaminate foods of animal origin. These species of Campylobacter are recognised as causes of human diarrhoeal disease and hence their presence in food represents a possible health hazard. Epidemiological studies have revealed that chickens serve as important reservoirs for C. jejuni and are generally colonised by C. jejuni early in life (Berndston et al 1996, Shanker et al 1990). Infection with C. jejuni is commonly acquired from eating undercooked chicken and drinking unpasteurised milk or contaminated water (Tauxe 1992). Campylobacter has additionally been isolated from raw beef, pork, lamb, cooked meats and seafood (Fricker and Park 1989, Zanetti et al 1996). Pigs may be a natural reservoir of Campylobacter since Campylobacter has been frequently isolated from pork (Manser and Dalziel 1985, Stern et al 1985). It is generally accepted that the predominant species isolated from poultry is C. jejuni, whereas pigs are primarily colonised by C. coli (Manser and Dalziel 1985). However, isolation rates range considerably from between 46 to 95 per cent (Manser and Dalziel 1985, Svedhem and Kaijser 1981, Oosterom et al 1985). For example, a Danish study reported that 46 per cent of swine had detectable levels of Campylobacter, mostly C. coli, in the caecal content at slaughter (Nielsen et al 1997),

*Corresponding author Telephone: (409) 260–9397 Fax: (409) 260–9332 E-mail: [email protected]

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whereas other studies have found isolation rates between 47 per cent in healthy swine and 85 per cent of swine samples collected at the slaughterhouse (Weijtens et al 1993). Collectively, data indicates that pigs are an important reservoir of Campylobacter. It is possible that both pigs in finishing units and breeding farms are highly infected with Campylobacter. During a recent epidemiological survey of Campylobacter prevalence at slaughter in market age pigs, Campylobacter species were isolated from 70 to 100 per cent of the pigs, depending on the farm sampled (Harvey et al 1999). Isolations of C. coli ranged from 20 to 100 per cent (mean 60 per cent) and isolations of C. jejuni ranged from 0 to 76 per cent with a mean of 31·3 per cent (Harvey et al 1999). Furthermore, high numbers of C. coli or C. jejuni were isolated from these pigs, ranging from 103 to 107 colony forming units (cfu) per gram of caecal content. Because of this high incidence in market-age pigs, it was of interest to determine at what stage of production it is possible to isolate Campylobacter species from the porcine gastrointestinal tract. Additionally, the levels at which Campylobacter was present in such pigs were enumerated. The pigs used in these studies included gilts, pregnant sows and piglets from the day of birth until weaned at day 14.

MATERIALS AND METHODS Pigs The study was carried out using pigs from a single commercial hog farm that used co-mingled multiple-site production. Gilts were offspring of Yorkshire sows x American Landrace boars. Gilts were raised in covered, open-sided, concrete-floored barns with conventional feed and water equipment. © 2000 Harcourt Publishers Ltd

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TABLE 1: Enteric colonization following natural exposure to Campylobacter in pigs Group of pigs

Gilts (n=50) Pregnant sows (n=9) Newborn piglets (day-of-birth) (n=73) Weaned piglets (n=20)

Number of pigs infected with Campylobacter speciesa (percentage)

C. coli

C. jejuni

C. lari

log10 cfub Campylobacter mean (S.D.)

8 (21) 1 (11) 50 (68·3)

29 (76·3) 8 (89) 23 (31·7)

1 (2·6) 0 0

5·08 (0·83) 5·31 (1·04) 4·84 (1·60)

4 (18)

16 (82)

0

7·25 (0·70)

a

Campylobacter species identification was carried out using both biochemical and serological techniques. Campylobacter were enumerated by colony counting of logarithmic dilutions of samples on Campy-Cephex agar plates.

b

Piglets were offspring of Yorkshire x American Landrace sows and either Duroc or Hampshire boars. Each nursery site consisted of two barns and pigs were weaned at 14 to 17 days. Experimental design Three experiments were conducted. First, an examination of the incidence of caecal Campylobacter in 50 six-monthold gilts; secondly, determination of the incidence of Campylobacter in nine sows at parturition; thirdly, assessment of the incidence of Campylobacter in piglets from the day-of-birth until weaning. Fifty adult gilts, chosen at random as representative of the commercial hog farm, were sampled at slaughter. At the slaughter plant, viscera were collected from the processing line and approximately 25 g caecal contents were collected aseptically into sterile plastic bags. The sample bags were placed on ice prior to microbiological evaluation in the laboratory. Nine pregnant sows were sampled at, or within 24 hours of, parturition. Rectal swabs were obtained from each sow and placed on ice prior to microbiological evaluation in the laboratory. Rectal swabs were taken from 73 day-of-birth piglets, from the above nine pregnant sows within 24 hours of birth. Samples were placed into sterile plastic tubes on ice prior to microbiological evaluation in the laboratory. Twenty weaned piglets (day 14 to day 17) were euthanised and caecal contents aseptically collected into sterile plastic bags. All samples were kept on ice prior to microbiological evaluation in the laboratory. Isolation techniques For Campylobacter detection, 0·1 g of caecal or rectal material was inoculated directly into 10 ml Bolton broth (Hunt et al 1988). The broth was incubated for 4 hours at 37°C, followed by 20 hours at 42°C. Then, 20 ml of broth was removed and streaked directly onto Campy-Cephex (Stern et al 1992) selective agar. Plates were then incubated at 42°C, for 48 hours, in a microaerobic atmosphere of 10 per cent carbon-dioxide, 5 per cent oxygen and 85 per cent nitrogen. Campy-Cephex agar is a moderately selective medium on which Campylobacter forms readily recognisable pink translucent colonies. Colonies that displayed typical Campylobacter morphology (up to three per plate) were serologically tested using a commercial latex agglutination kit and positive samples differentiated by testing for indoxyl acetate and hippurate metabolism.

For Campylobacter enumeration, 0·1 g of caecal or rectal material were diluted in 9·9 ml phosphate buffered saline (PBS). The samples were serially diluted and 0·1 ml of each dilution (range 10–2 through 10–5) was spread on the surface of Campy-Cephex agar plates. Plates were incubated, colonies identified and counted as described above. Colonies typical of Campylobacter were picked (maximum of three per plate) and differentiated with Campylobacter antibodies as described above. All of the colonies selected tested positive for Campylobacter by that agglutination test. Campylobacter species identification Colonies displaying typical Campylobacter morphology were serologically tested using a commercial latex agglutination kit (Integrated Diagnostics, Inc., Baltimore, MD 21227). Positive samples were differentiated for species by testing for indoxyl acetate (Sigma-Aldrich, St. Louis, MO 63118) and hippurate hydrolysis (Difco, Detroit, MI). Data analysis Plate count data were converted to the logarithmic form and are presented as the geometric means and standard deviations of the enumerated Campylobacter (Schlotzhauer and Littell 1987).

RESULTS The average prevalence of Campylobacter in gilts was 76 per cent. For Campylobacter-positive pigs the incidence of C. jejuni was 76·3 per cent, 21 per cent for C. coli and 2·6 per cent for C. lari. The number of Campylobacter in gilts ranged from log10 cfu/g 3·95–6·32 with a mean log10 cfu/g 5·08±0·83 (mean ± standard deviation) (Table 1). Campylobacter was isolated from all the pregnant sows tested with C. jejuni being isolated from 89 per cent and C. coli from 11 per cent. At the time of parturition, Campylobacter in pregnant sows ranged from log10 cfu/g 3·30–6·30 with a mean of log10 cfu/g 5·31 (1·04) (mean ± standard deviation) (Table 1). Newborn piglets had an average incidence of Campylobacter of 57·8 per cent within 24 hours of birth. For positive newborn piglets, the mean incidence of C. jejuni was 31·7 per cent and 68·3 per cent for C. coli. The number of Campylobacter in newborn piglets ranged from log10 cfu/g of 3·00–8·00, with a mean of log10 cfu/g of 4·84 (1·60) (mean ± standard deviation (Table 1).

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Prevalence of Campylobacter in pigs

Weaned piglets, at day 14, had an 85 per cent incidence of Campylobacter. For positive piglets, the mean incidence of C. jejuni was 82 per cent and 18 per cent for C. coli. The number of Campylobacter ranged from log10 cfu/g 5·15–8·00, with a mean log10 cfu/g 7·25 (0·70) (mean ± standard deviation) (Table 1).

DISCUSSION The aim of this study was to improve our knowledge of the epidemiology of Campylobacter in pigs. This should enable us to understand better at what stage of the production cycle attempts should be made to reduce or eliminate Campylobacter colonisation of the gastrointestinal tract of the pig. Examples of possible intervention strategies to produce Campylobacter-free porcine meat are probiotic cultures or vaccines. In this study, C. jejuni, C. coli and C. lari were isolated from pigs on a commercial hog farm. The involvement of C. jejuni and C. coli in human gastroenteritis is well documented (Nielsen et al 1997), however, the pathogenicity of C. lari was largely unknown until six cases of human illness associated with C. lari were reported (Tauxe et al 1985). The isolation rate of Campylobacter varied from 57·8 per cent for day-of-birth piglets to 100 per cent for weaned piglets and pregnant sows. These findings are in general agreement with other results indicating prevalence of Campylobacter at 79·3 per cent amongst piglets (Adesiyun 1993). In an enclosed barn, it is possible that if one animal carries Campylobacter it might be very quickly transferred from one pig to another, and hence these high prevalence rates of Campylobacter in pigs. The high prevalence rates reported in this study for day-of-birth and weaned piglets may be due to direct transmission from the infected sows. This hypothesis of direct transmission of Campylobacter from sows to piglets, although not tested here, could be addressed by subtyping isolates using techniques such as pulsed-field gel electrophoresis (Bourke et al 1996). Unexpectedly, in this study large numbers of C. jejuni were isolated from both newborn piglets (31·7 per cent), weaned piglets (82 per cent), pregnant sows (87 per cent) and gilts (76·3 per cent). This detection of such high numbers of C. jejuni from either caecal or rectal samples was somewhat unexpected since it has previously been reported that the major Campylobacter species of pigs is C. coli (Manser and Dalziel 1985). However, there are additional studies indicating high prevalence rates of C. jejuni in pigs, ranging from 24 per cent for healthy pigs at slaughter (Sticht-Groh 1982) to 59 per cent of pig carcasses at slaughter (Hudson and Roberts 1981, Finlay et al 1986). It is possible that the prevalence of the respective Campylobacter species might differ considerably between farms, breeding companies and countries. Also, the prevalence of Campylobacters in the rodent population on the farm might be important epidemiologically. The differences in isolation rates reported in this and other groups may be related to the subjective methods, such as the intensity of the violet color reaction in the hippurate test, used to distinguish between C. coli and C. jejuni (Bär and Fricke 1987). The apparent very high numbers of the dominant Campylobacter strain in the samples could indicate that

other Campylobacter subtypes were only present in very low numbers. To determine if this were the case would involve screening a large number of colonies per plate. Since the current study involved 152 pigs, such a screening of colonies would require an enormous amount of work. An alternative way of answering this question would be to carry out 165 RNA analysis or pulsed field gel electrophoresis of the single colonies. It is possible that in the future we may address this question using the latter approach. The experiments reported here show that the concentration of Campylobacter isolated from pigs, within the production cycle, is high. For example, the log10 cfu g–1 for gilts was 5·08 (0·83), for pregnant sows 5·31 (1·04), whereas the log10 cfu g–1 for weaned piglets was 7·25 (0·70). Our findings in adult pigs are in agreement with previous findings showing a mean log number of 3·6 cfu g–1 of Campylobacter in the caecal contents of pigs (Oosterom et al 1985) which is in itself in accordance with findings obtained in ileal samples (Weijtens et al 1993). Previous epidemiological studies have shown that pigs already have intestinal Campylobacter by the age of 11 weeks, and that nearly all pigs remain carriers until slaughter (Weijtens et al 1993). Furthermore, it has been suggested, although not demonstrated, that piglets may be infected with Campylobacter at a young age on the breeding farm (Weijtens et al 1993). Our data clearly demonstrate that piglets are infected with either C. jejuni or C. coli very early in life, probably within the first few hours of birth. Additionally, the number of Campylobacter was higher in 14-day-old weaned piglets [log10 cfu g–1 7·25 (0·70)] than in the pregnant sows [log10 cfu g–1 5·31 (1·04)] or gilts [(log10 cfu g–1 5·08 (0·83)]. The reason for this finding is unclear, but is supported by epidemiological studies in dogs and pigs showing that the level of Campylobacter in the faeces decreases as the animals get older (Weijtens et al 1993; Matsusaki et al 1986, Weijtens et al 1997). Overall, this high incidence of Campylobacter in young animals probably indicates that a primary site of Campylobacter transmission is the nursery. The important finding in this study is that piglets all probably become colonised with Campylobacter within a few hours of birth on the breeding farms. Consequently, the prevalence and level of infection of pigs with Campylobacter will be most effectively controlled by intervention strategies on the breeding farms. Experiments are in progress in our laboratory to address such intervention strategies in the breeding farms to control Campylobacter colonisation of pigs. REFERENCES ADESIYUN, A. A. (1993) Prevalence of Listeria spp., Campylobacter spp., Salmonella spp., Yersinia spp., and toxigenic Escherichia coli on meat and seafoods in Trinidad. Food Microbiology 10, 395–403. BÄR, W., & FRICKE, G. (1987) Rapid and improved gas-liquid chromatography technique for detection of hippurate hydrolysis by Campylobacter jejuni and Campylobacter coli. Journal of Clinical Microbiology 25, 1776–1778. BERNDSTON, E. M. L., DANIELSSON-THAM, & ENGVALL, A. (1996) Campylobacter incidence on a chicken farm and the spread of Campylobacter during the slaughter process. International Journal of Food Microbiology 32, 35–47. BOURKE, B., SHERMAN, P. M., WOODWARD, D., LIOR, H., & CHAN, V. L. (1996) Pulsed-field gel electrophoresis indicates genotypic heterogeneity among Campylobacter upsaliensis strains. FEMS Microbiology Letters 143, 57–61. FINLAY, R. C., MANN, E. D., & HORNING, J. L. (1986) Prevalence of Salmonella and Campylobacter contamination in swine carcasses. Canadian Veterinary Journal 27, 185–187.

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