Trop Anim Health Prod (2011) 43:83–87 DOI 10.1007/s11250-010-9657-4
Occurrences of thermophilic Campylobacter in pigs slaughtered at Morogoro slaughter slabs, Tanzania Robinson H. Mdegela & Kibona Laurence & Petro Jacob & Hezron Emmanuel Nonga
Accepted: 17 March 2010 / Published online: 21 August 2010 # Springer Science+Business Media B.V. 2010
Abstract Occurrences of thermophlic Campylobacter in pigs and pig carcasses was investigated in a cross-sectional study that was carried out in three selected slaughter slabs in Morogoro municipality, Tanzania. Before sampling, the slab hygiene, slaughter, carcass dressing, and meat handling was assessed. Fecal samples were collected from 66 slaughter pigs at the kill floor. After slaughter, a 100-cm2 area on medial surface of the thigh muscles of dressed carcasses was sampled using sterile cotton swabs. Thereafter, the jejunal, cecal, and colon contents were also sampled. The samples were subjected to standard bacteriological examination using Skirrows protocol. In all slaughter slabs visited, it was found that pig slaughter, dressing, and meat handling was done on the ground under unhygienic condition. All the slaughter slab environment were dirty and had neither tap water or drainage systems. Thermophilic Campylobacter prevalence in slaughtered pig was 66.7% while contamination rate of dressed carcasses was 10.6%. Of the Campylobacter-positive carcasses, five (12.2%) were from the animals which were also positive to Campylobacter. The isolation rate of Campylobacter in the cecum was higher (34.8%) compared to the small intestines (28.8%) and colon (16.7%) although the difference was not statistically significant (P>0.05). Campylobacter jejuni was the most prevalent species as it constituted 74% of all isolates, while Campylobacter coli was isolated at 26%. This suggests possible risks of infection to people through consumption of contaminated pork or through contact with infected pigs. Cecum was found to be the major part of intestine highly colonized by Campylobacter. R. H. Mdegela : K. Laurence : P. Jacob : H. E. Nonga (*) Department of Veterinary Medicine and Public Health, Sokoine University of Agriculture, P.O. Box 3021, Morogoro, Tanzania e-mail:
[email protected] e-mail:
[email protected]
Keywords Thermophilic Campylobacter . Pigs . Slaughter . Morogoro . Tanzania
Introduction Thermophilic Campylobacter in particular Campylobacter jejuni and Campylobacter coli account for the majority of gastrointestinal diseases attributed to the genus Campylobacter. Campylobacter is of recent considered to be one of the main causes of diarrhoeal diseases in humans (Aarestrup and Engberg 2001). Campylobacter infection in human may emanate from ingestion of contaminated food, particularly undercooked poultry meat, unpasteurized milk, and improperly treated/untreated contaminated water (Shane 2000; Kassa et al. 2005). Other risk factors include contact with infected animals in particular pets and travel to Campylobacter endemic areas (Rao et al. 2002). Pork is also considered as among the important possible sources of Campylobacter infection in humans as a result of carcass contamination at slaughter (Fosse et al. 2009). Nevertheless, most farm animals may play a major role in the transmission of the disease since their intestinal system contains Campylobacter spp. According to some authors (Harvey et al. 1999; Moore and Madden 2001; Moore et al. 2002), the prevalence rates of Campylobacter spp. may vary significantly between pig farms. Similarly, the two most common species, C. coli and C. jejuni, may also be detected in pigs at different rates depending on geographical locations, Campylobacter spp. circulating in the local environment and management systems (Humphrey et al. 2007). Improving food safety by reducing the level of Campylobacter at the farm level, slaughter and meat processing level or at least gaining better understanding of the
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epidemiology of the disease organism is important in order to safeguard the public health (Steinhauserova et al. 2005). For its safety and suitability as food for humans, pork has to be produced from healthy pigs which have been properly slaughtered and inspected. At slaughter level, it is important to assure meat safety through reduction of all possibilities of carcass contamination with intestinal content since it is the major primary source of microbes in meat. Poor slaughter methods and unhygienic meat handling may constitute a potential risk of infections to humans (Steinhauserova et al. 2005). To prevent microbial contamination, which may affect consumer health and causes serious public health problems, appropriate slaughterhouse design, recommended slaughter methods, meat handling, and hygienic environment are necessary. There is generally little published information on Campylobacter infections in pigs in Tanzania; however, studies elsewhere have shown high prevalence in pigs managed in different management systems (Uaboi-Egbenni et al. 2008; Fosse et al. 2009). The prevalence study conducted in Ethiopia by Kassa et al. (2005) revealed thermophilic Campylobacter prevalence of up to 50% while Pezzotti et al. (2002) reported infection rate of 64% in Italy. A comparative study by Franco (1988) in pig carcasses showed that they were frequently contaminated with Campylobacter than cattle or sheep carcasses. Nevertheless, a study by Malakauskas et al. (2006) at pig slaughterhouse showed that 63.6% of the carcasses were contaminated by Campylobacter spp. Similarly, Aquino et al. (2002) isolated C. coli from 9% of pigs sent for slaughter. However, the rate of contamination from pig carcasses varies from 2.9% in Poland (Kwiatek et al. 1990) to 1.0% in Sweden (Lindblad et al. 2007). Despite the increased interest in Campylobacter studies in the world, there is a scarcity of information on prevalence of Campylobacter spp. in pigs and pork as possible sources of infections in humans in Tanzania. Simultaneously, pigs are increasingly becoming popular due to increased demand for pig meat in recent years in Tanzania. Therefore, the present study was undertaken to determine the prevalence of thermotolerant Campylobacter species in pigs slaughtered in selected slaughter slabs in Morogoro municipal and the possible carcass contamination consumed by the public within the municipality.
Material and methods Study design and areas This cross-sectional study was conducted in Morogoro municipality. Geographically, the municipality is situated at the latitude 5.7-10°S and longitude 35.6-39.5°E, with an
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elevation of 500-600 m above sea level and is about 200 km west of Dar es Salaam (Muhairwa et al. 2007). The samples for bacteriological culture were collected from pigs that were slaughtered at Chamwino, Bigwa, and Madizini slaughter slabs. Sample collection and handling On the day of slaughter slabs visit, questionnaires were administered to the butchers who were also owners of pigs slaughtered. Information collected included name of slaughter slab, source, age, sex, and general health status of pigs were recorded. In addition, the slaughter slab environment, slaughter, carcass dressing, and meat handling was assessed. Fecal swab samples were collected from the rectum of pigs at the kill floor just before slaughter. After carcass dressing, a 100-cm2 area on medial surface of the thigh muscles was sampled using sterile cotton swabs as described by Malakauskas et al. (2006). Intestinal contents were also collected for culture. By using a sterile pair of scissors, a small cut was made through the jejunal, cecal, and colon walls to expose the contents which were subsequently sampled by use of sterile cotton swabs. All the swab samples were placed in sterile universal bottles containing 10 ml of Preston broth (Oxoid Ltd, Basingstoke, UK) with Preston supplements (Oxoid Ltd, Basingstoke, UK) and stored in cool box with ice pack at 4°C. The samples were subsequently transported to the laboratory at the Faculty of Veterinary Medicine, Sokoine University of Agriculture for further processing using standard procedures. The samples were analyzed within 2 h from sampling period. Thermophilic Campylobacter isolation and identification The samples in the Preston broth were loaded in the microaerophilic candle jars (Coldstream Engineering Ltd, Arista, Sweden) with a lighting candle and incubated at 37°C for 24 h for enrichment as described by Skirrow and Benjamin (1980) and Atabay and Corry (1998). After incubation, the universal bottles with enriched samples were properly shaken and sub-cultured onto modified charcoal cefoperazone deoxycholate agar (Oxoid Ltd, Basingstoke, UK) for primary isolation of thermophilic Campylobacter. The inoculated Petri dishes were incubated at 42°C for 48 h under microaerophilic conditions and colonies resembling Campylobacter were sub-cultured onto blood agar (Oxoid Ltd, Basingstoke, UK) at 37°C under micro-aerophilic conditions for 24 h. Suspected thermophilic Campylobacter colonies on blood agar that were Gram negative, curved, or spiral rods and showed corkscrew-like motion; positive to catalase, oxidasem and nitrate reduction tests were further tested for hippurate hydrolysis, H2S production, and
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susceptibility to nalidixic acid and cephalothin. These parameters formed the basis for the identification of C. jejuni, C. coli, or Campylobacter lari as described by On (1996). For the hippurate hydrolysis test, those organisms yielding a positive test were considered C. jejuni, while those organisms that showed a negative reaction were considered C. coli or C. lari. For hydrogen sulfide (H2S) production test, all the tested Campylobacter strains were found to be negative for the test. Susceptibility tests to nalidixic acid (30 μg; Oxoid, UK) and cephalothin (30 μg; Oxoid, UK) were performed for all isolates of thermophilic Campylobacter spp. in accordance with the criteria set by the National Committee for Clinical Laboratory Standards using the disk diffusion method (NCCLS 2002). The isolates were classified as sensitive and/or resistant according to the standardized tables supplied by the (NCCLS 2002). Campylobacter strains that were sensitive to nalidixic acid but resistant to cephalothin were considered C. jejuni and C. coli, while strains that were resistant to both drugs were considered C. lari. Statistical analysis The Epi info version 2000 (approved by CDC, Atlanta, GA, US) was used for statistical analysis. Using statcalc, proportions of categorical variables were computed and further compared using chi-square test at critical probability of P0.05).
Discussion Findings from this study show that there is high Campylobacter infection rate (66.7%) in pigs slaughtered in Morogoro municipality. Our results are comparable to the findings by Kassa et al. (2005) in Ethiopia, Pezzotti et al. (2002) in Italy, and Harvey et al. (1999) in Texas, USA. Thus, in live pigs, presence of Campylobacter could stem from the farm level and cross-contamination during transportation. However, isolation of C. jejuni and C. coli from the intestinal contents of slaughter healthy pigs and their carcasses is of public health importance since are known to cause enteritis and other diseases in humans (Rao et al. 2002; Heymann 2004). In this study, among the thermophilic Campylobacter isolated from pigs, C. jejuni accounted for 74% of all the isolates. The observed prevalence of C. jejuni in pigs in comparison with other thermophilic Campylobacter appears to be comparable to the findings of Harvey et al. (1999). However, other investigators reported higher prevalance of C. coli than other thermophilic Campylobacter in pigs (Kassa et al. 2005; Malakauskas et al. 2006). Other workers also reported a higher carriage rate of C. coli than C. jejuni among healthy pigs, up to the stage of concluding that C. coli is a normal flora of these animals (Rosef et al. 1983; Franco 1988; Aquino et al. 2002). The difference in species isolations may depend on common Campylobacter spp. circulating in the local environment, sampling techniques employed, seasonality, identification methods employed, and management of pigs where the study was conducted. Furthermore, the difference in species isolated in slaughter animals may also possibly be contributed by differences of the slaughter environment and the conditions. In the present study, C. jejuni was
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Table 1 Thermophilic Campylobacter isolates from slaughter pig, intestines and carcasses in Morogoro municipality Source of sample
Animals Thigh muscles Intestines
Region/area sampled
Rectum Medial surface Small intestine Cecum Colon
Number of samples
66 66 66 66 66
isolated from adult pigs of either sex sampled from different locations within Morogoro municipality. Previous similar studies in Morogoro also found a higher prevalence of C. jejuni in chickens, humans, and craws than C. coli (Jiwa et al. 1994; Mdegela et al. 2006). This shows that C. jejuni is the common species that may be circulating from either pig to humans, other animals and vice versa. Moreover, C. jejuni is recognized as the most important causal agent of human Campylobacter enteritis, whereas C. coli is less commonly involved in human infections (Skirrow and Benjamin 1982). In a study by Mdegela et al. (2006) reported that C. jejuni accounted for 96.6% of the cases of human campylobacteriosis in Tanzania. The finding of C. jejuni on the pig feces, intestinal contents, and carcasses is important as the majority of human cases of campylobacteriosis are caused by C. jejuni (Kapperud et al., 2003). The current study further observed that Campylobacter carcass contamination rate was 10.6% which is comparable to findings by Aquino et al. (2002). Nevertheless, Steinhauserova et al. (2005) and Malakauskas et al. (2006) reported a higher pig carcass contamination rate which ranged from 34-63.6%. In contrast, low carcass contamination rate was reported in Poland (Kwiatek et al. 1990), Belgium (Ghafir et al. 2007), and Sweden (Lindblad et al. 2007). Differences in Campylobacter isolation rates from carcasses may be influenced by sampling and analysis methods, slaughter and dressing methods, abattoir hygiene, infection rate in the slaughter animals, and sampling plan. The low contamination rate recorded in this study may be to a large extent affected by the area swabbed. In the present study, the area was quite small (100-cm2) which probably limited the possibility of recovering the Campylobacter from the wider contaminated carcass areas. Indeed, during this survey, the carcasses were in many times exposed to unclean surfaces, equipment, and personnel which gave a higher possibility for Campylobacter contaminations. Furthermore, evisceration process was not properly done such that spillover of the intestinal contents to the carcasses was common. In that regards, carcass contamination would certainly occur at any stage during slaughter and carcass dressing. Indeed, live animals are
Number of isolate (%)
44 7 19 23 11
(66.7) (10.6) (28.8) (34.8) (16.7)
Number of Campylobacter isolated (%) C. jejuni
C. coli
36 6 12 16 7
8 1 7 7 4
(81.8) (85.7) (18.2) (24.2) (10.6)
(18.2) (14.3) (10.6) (10.6) (6.1)
regarded as an important source of Campylobacter contamination in slaughterhouses due to carcass contamination by bacteria from the gastrointestinal tract during the course of slaughtering. Since the prevalence of Campylobacter in live pigs was high coupled with poor slaughter methods under unhygienic conditions, certainly high carcass contamination was expected. If the slaughter and carcass dressing processes were carried out perfectly under the hygienic environment, pigs with Campylobacter in their intestines would theoretically not contain Campylobacter on their carcasses or in the meat after slaughter. Cecum was found to be the part of intestinal tract which had the highest rate (34.8%) of Campylobacter infection. Similar other studies also reported higher isolation rate of Campylobacter in the cecum (Steinhauserova et al. 2005). Berry et al. (1988) reported that the best site of intestinal colonization by Campylobacter is the cecum. For the Campylobacter to establish and maintain colonization, a complex interaction of unknown mechanisms between the host and the organism comes into play (Stern 1994). Motility, chemotaxis, and flagella are known to be important factors for attachment and colonization of intestinal epithelium (Berry et al. 1988; Ketley 1997). Once Campylobacter are introduced in an animal, the main first site for colonization is the cecum and the organisms are concentrated in the mucus layer in the crypts of the villi where they undergo multiplications (Berry et al. 1988). Furthermore, cecum may serve as the best place since it has minimum oxygen concentration which favors Campylobacter survival. Even during very early stages of infection in animals, it is possible to recover Campylobacter in the cecal contents but rarely with rectal swabs and other parts of the intestines. From results of this investigation, it is concluded that pigs slaughtered at Morogoro municipal slaughter slabs poses health risks to meat consumers within the municipality. Cecum was found to be the major part of intestines highly colonized by Campylobacter. More work is required to establish the magnitude of zoonotic enteric campylobacteriosis in humans and epidemiological role of pig and other animals in the study area.
Trop Anim Health Prod (2011) 43:83–87 Acknowledgments The authors wish to acknowledge cooperation received from butchers. Mr. Ndaki L., Mr. Mkuchu P., and Mr. Makingi G. of the Department of Veterinary Medicine and Public Health are thanked for their technical assistance. This work was funded by the Higher Student Loan Body of Tanzania.
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