International Journal of Food Microbiology 170 (2014) 44–47

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Short communication

Microbial quality of frozen Nile crocodile (Crocodylus niloticus) meat samples from three selected farms in Zimbabwe Tsitsi B. Makanyanga a, Gideon Mutema a, Norman L. Mukarati a, Sylvester M. Chikerema a, Pious V. Makaya b, Shuvai Musari b, Gift Matope c,⁎ a b c

Department of Clinical Veterinary Studies, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe Central Veterinary Laboratory, P.O. Box CY 551, Causeway, Harare, Zimbabwe Department of Paraclinical Veterinary Studies, University of Zimbabwe, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe

a r t i c l e

i n f o

Article history: Received 21 March 2013 Received in revised form 15 August 2013 Accepted 26 October 2013 Available online 4 November 2013 Keywords: Aerobic plate counts E. coli Salmonella Crocodile farms

a b s t r a c t Microbial quality of frozen Nile crocodile (Crocodylus niloticus) meat from three farms in Zimbabwe was assessed based on 2051 samples collected for pre-export testing during 2006 to 2011. Data were perused by season and year in terms of aerobic plate (APC), coliform (CC), Escherichia coli (ECC) and Listeria monocytogenes (LMC) counts and the presence of Salmonella spp. The log10-transformed data were compared among the farms and seasons using the Kruskal–Wallis test. Microbial quality of the samples was graded based on the EC No. 2073.2005 criteria for beef. The mean APC and CC for the crocodile meat differed significantly (P = 0.000) among the farms with the highest APC (3.2 ± 0.05 log10 cfu/g) and the lowest (2.7 ± 0.05 log10 cfu/g) recorded from farms A and C, respectively. There were no significant differences (P N 0.05) in ECC and LMC among the farms, while Salmonella spp. were only isolated from one farm. Although the microbial quality of frozen crocodile meat from these farms was generally within acceptable limits, the isolation of E. coli and Salmonella spp. is of public health concern. Thus, implementing of measures to control the pasteurizing process and to minimize bacterial contamination of crocodile meat after pasteurization need to be carefully considered. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Commercial farming of the Nile crocodile (Crocodylus niloticus) in Zimbabwe dates back to the 1950s (Child, 1987) when they were primarily exploited for their skin which was exported to overseas markets. However, due to pressure from reduced skin prices following tremendous growth of the industry in the 1990's (Collins, 1995), crocodile meat became an alternative source of income and was later marketed widely as a delicacy especially in restaurants frequented by tourists (Huchzermeyer et al., 2008; Magnino et al., 2009). The export of frozen crocodile meat to overseas markets was commenced in 1988 (Madsen, 1993) and by the year 2007, over 250,000 kg was exported compared to approximately 400 kg in 1990 (Caldwell, 2010; Fergusson et al., 2004). The slaughter, processing, packaging and storage of the crocodile meat for export are monitored for compliance with the requirements for Crocodile abattoirs in Zimbabwe (Madsen, 1996) by the Veterinary Public Health (VPH) of the Department of Livestock and Veterinary Services. The animals are stunned by a ‘nap-stab’ which involves severing of the spinal cord by a knife driven in at the nap of the neck and pithing of both the spinal cord and the brain. To reduce bacterial contaminations, carcasses are scrubbed and rinsed in running water to ⁎ Corresponding author. Tel.: +263 772 954 390. E-mail address: [email protected] (G. Matope). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.10.020

remove excess dirt and then immersed in a bath of chlorinated (approximately 30 ppm) water for 10 min. To minimize further contamination of meat by intestinal contents, the head is enclosed in a plastic bag and the cloaca plugged before carcasses are skinned on clean stainless steel tables. The collected tails and filets are further decontaminated by dipping in chlorinated (30 ppm) water for 10 min, blanched at 82 °C for approximately 8 s and then vacuumpacked in plastic packs for blast freezing at − 20 °C. The microbial quality of export crocodile meat is monitored for compliance to the criteria prescribed by the VPH and the importing countries through bacteriological tests conducted monthly at a Government laboratory. Although a great deal of data has been accumulated over the years, the microbial quality of frozen crocodile meat from different farms country-wide has not been studied in detail. Analysis of such data may be useful in not only assessing changes in trends of the microbial quality of crocodile meat, but also monitoring for the risk of emergence of pathogenic food-borne bacteria. In view of the growing demand for Nile crocodile meat for human consumption, the production of safe, wholesome meat and products is imperative in order to minimize the risk of food-borne infections. There are fears that consumption of crocodile meat may be associated with the risk of transmission of zoonotic parasites; Trichinella zimbabwensis, Pentastomum spp. and Gnathostomum spp. (Magnino et al., 2009; Pozio et al., 2007), and bacteria; Listeria monocytogenes, Salmonella spp., Escherichia coli, Yersinia enterocolitica and Campylobacter spp.

T.B. Makanyanga et al. / International Journal of Food Microbiology 170 (2014) 44–47

(Madsen, 1993). Furthermore, meat may be rendered unfit for human consumption if contaminated by spoilage bacteria when processed under conditions of inadequate abattoir hygiene. On the other hand, the presence of E. coli in water or food of animal origin is used as a reliable indicator of fecal contamination. E. coli are common commensal microorganisms of the intestines of animals and humans while a few strains such as those producing verotoxins (VTEC), especially E. coli O:157: H7 are of zoonotic significance (Anon, 2010). Hence the recovery of E. coli from food of animal origin may be of public health concern. The objective of this study was to assess the microbial contamination and to detect Salmonella spp., Listeria monocytogenes and E. coli in frozen Nile crocodile (Crocodylus niloticus) meat obtained from three large scale commercial farms in Zimbabwe. 2. Materials and methods 2.1. Study design and sample collection The Nile crocodile meat samples were obtained from three selected farms in Zimbabwe between 2006 and 2011 for mandatory pre-export bacteriological testing. The collection of these meat samples from the abattoirs was conducted on one day a month, supervised by VPH personnel and not influenced by management of the farms. Essentially, samples were collected fresh on the day of slaughter immediately after packing using a systematic random sampling collecting one vacuum-packed meat sample (approximately 2 kg) for every 200 crocodiles slaughtered. Samples were cooled to 4 °C and transported in insulated boxes to the Central Veterinary Laboratory (CVL) in Harare by air at the end of the day's slaughter for bacteriological tests. At the laboratory, samples were immediately frozen at −20 °C and tested within 2 weeks of receipt. 2.2. Laboratory tests The crocodile meat samples were assessed for aerobic plate counts (APC) which were determined using the pour plate method conducted according to American Public Health Association (APHA) (1992). Briefly, following the arguments of Van der Merwe et al. (2013a) an excision method was used to obtain 1 g of sample, by excising approximately 1 cm3 using a sterile scalpel blade, which were then homogenized and used to make ten-fold serial dilutions in phosphate buffered saline. One mililiter of each appropriate dilution was added onto respective sterile petri dishes in duplicate and mixed with 20 ml of molten plate count agar (PCA) (Oxoid, Basingstoke, Hampshire, UK) cooled to approximately 45 °C. Plates were allowed to set at room temperature for 30 min and then incubated at 37 °C for 72 h as suggested for game meat (Van der Merwe et al., 2013a). Counts were done on plates that contained between 30 and 300 colony forming units. The coliform — (CC) and E. coli — (ECC) counts were performed using the surface spread technique (Cruickshank et al., 1975), on MacConkey agar (Oxoid) as described by Mhone et al. (2011) and plates incubated at 37 °C and 44 °C for 24 h, for coliforms and E. coli, respectively. The isolated E. coli colonies were further identified using standard biochemical tests (Barrow and Feltham, 1993). A randomly selected subset of the samples collected (n = 1524) was quantitatively assessed for contamination with Listeria monocytogenes (LMC) on Listeria selective agar (Oxoid) using the spread plate technique. The culture and isolation of Salmonella spp. were done using 25 g of meat samples which was homogenized in sterile phosphate buffered saline (PBS) in a stomacher (Seward Medical, USA). The homogenized samples (10% w/v) were pre-enriched in buffered peptone water (Oxoid), essentially using the method described by Schlundt and Munch (1993). The homogenized 25 g was transferred to 250 ml buffered peptone water and samples incubated aerobically at 37 °C for 18–24 h. Enrichment was carried out on Rappaport vassilliadis (RV) (Oxoid) by placing 0.1 ml sample into 10 ml RV broth and

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incubated for a further 18–24 h at 37 °C. A loopful of the RV-enriched samples were then plated onto Xylose Lysine Desoxycholate agar (Oxoid) and incubated at 37 °C for another 18–24 h. The colonies suspected to be Salmonella spp. were further tested using the procedure described elsewhere (Makaya et al., 2012). 2.3. Statistical analysis Statistical analyses were carried out using STATA version SE 11.0 for windows (Stata corp. College Station, Texas, USA). Data for bacterial counts were tested for normality using the Shapiro–Francia test before and after logarithmic transformation. The log10-transformed data among the farms and seasons were further compared using the Kruskal–Wallis test. Four seasons were defined as follows: the hot–wet season (December to February), the cool wet season (March to May), cold–dry season (June to August) and the hot–dry season (September to November). Based on microbial levels, the crocodile meat samples were classified into “high-” and “low-microbial” contamination using cut off points of; 105 cfu/ml, 102 cfu/ml and 100 cfu/g for APC, CC and ECC, respectively. For Salmonella spp., they were graded on the basis of presence or absence of the bacteria in 25 g of sample, essentially as suggested by the Regulation (EC) No. 2073/2005 (Anon., 2005). Statistical significance of the categorized data was compared using simple proportion test and P-values b0.05 were regarded as significant. Further, based on the microbial quality for E. coli, L. monocytogenes and Salmonella spp., the meat samples were classified as ‘satisfactory’ (counts b 20 cfu/g), ‘acceptable’ (counts 20–102 cfu/g) and ‘potentially hazardous’ (counts N 102 cfu/g) (Anon., 2005). 3. Results and discussion The mean APC and CC for the crocodile meat differed significantly (P = 0.000) among the study farms with the highest APC (3.2 ± 0.05 log 10 cfu/g) and the lowest (2.7 ± 0.05 log 10 cfu/g) recorded from farms A and C, respectively (Table 1). There were no significant differences in the ECC (mean 1.1 ± 0.007 log10 cfu/g) among the farms. These data appeared to be lower than the 4.9 log10 cfu/g and 3.4 log10 cfu/g for APC and CC, respectively that were reported previously from some of these farms by Madsen (1993). Similarly crocodile meat from other regions has been reported to have APC of between 5–6 log10cfu/g (Rickard et al., 1995). Although, incubation temperatures of 25–30 °C generally result in significantly higher counts for APC compared to those obtained at 37 °C (Simmons et al., 2008), the use of incubation temperatures of 37 °C for APC in this study was prompted by the concern for isolating potential human pathogens as opposed to psychrotrophs which predominantly grow at lower temperatures. The use of incubation temperatures of 37 °C has also been recommended in other studies for beef (Eisel et al., 1997) and game meat (Van der Merwe et al., 2013a) where APC of 3–6.9 log10 cfu/g were obtained. We opted for the incision method instead of the EU approved swabbing method (Zweifel et al., 2005) because it was cheap, easy to standardize and was likely to include

Table 1 The logarithm10 of mean total viable bacterial counts (cfu/g) of crocodile meat (n = 2051) from selected farms in Zimbabwe. Farm

n

Mean log10 total bacterial counts ± S.E. (cfu/g) Aerobic plate counts

Farm A Farm B Farm C Total

712 753 586 2051

3.2 2.7 2.7 2.9

± ± ± ±

a

0.05 0.05b 0.05b 0.03

Coliforms 1.3 1.1 1.1 1.2

± ± ± ±

E. coli a

0.03 0.01b 0.02c 0.01

1.2 1.0 1.0 1.1

± ± ± ±

0.02a 0.003a 0.003a 0.007

Key: n = number; cfu = colony forming units. Values with different superscripts within columns are significantly different at P b 0.05.

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T.B. Makanyanga et al. / International Journal of Food Microbiology 170 (2014) 44–47

Table 2 Classification of crocodile meat samples (n = 2051) from three crocodile farms in Zimbabwe based on common standards for coliforms and Escherichia coli in foods.a Variable

n

Samples with unacceptable (exceeding 105 cfu/g) total bacterial counts (%)

Total samples with coliforms exceeding 102 cfu/g (% of samples)

Total samples with E. coli exceeding 1 cfu/g (% of samples)

Farm A Farm B Farm C Total

712 753 586 2051

81 (11.4)a 43 (5.7)b 19 (3.2)c 143 (7.0)

94 (13.2)d 26 (3.5)e 27(4.6)e 147 (7.2)

91 (12.8)f 8 (1.1)g 4 (0.7)g 103 (5.0)

Frequencies with different superscripts within columns are significantly (s b 0.05) different. a Classification based on the Regulation (EC) No. 2073/2005 for livestock carcasses.

microorganisms that were deeply imbedded into the muscle tissues. Considering that good correlation between the two methods was reported previously (Van der Merwe et al., 2013b), the results obtained in this study are likely to be a true reflection of the microbial quality of the crocodile meat. Thus, the relatively good microbiological condition in terms of APC, CC and ECC observed in this study is probably the result of the meat being pasteurized (blanched) by immersion in hot water. Therefore, the microbiological condition could probably be improved if the pasteurizing process was better controlled and care was taken to avoid contamination of the meat after pasteurization. The differences in the levels of bacterial counts among farms cannot be explained by the available data, since there were no records on the numbers of animals slaughtered at ponds or abattoirs. However, given that two farms (B and C) that share the same abattoir had similar counts that were significantly lower than those from one farm (A) that uses a separate abattoir may be suggestive of differences in slaughter practices and/or abattoir processing hygiene. Our results indicated that the highest microbial contamination occurred during the cool-wet, followed by cold-dry and hot-wet seasons, respectively (data not shown). The cool-wet and cold-dry seasons coincide with the culling season, where the majority of crocodile slaughters are conducted. Hence, it is likely that the handling of large volumes of crocodile carcasses may impact negatively on farm and abattoir hygiene resulting in increased risk of carcass contamination. Further, the increased bacterial loads in the environment associated with the wet season would subsequently increase the chances of contamination of crocodile carcasses. This corroborated the findings of Fonkem et al. (2010) who reported season as having a significant effect on bacterial contamination of meat in other regions. Overall, there were 7.0% (143/2051), 7.2% and 5.0% of the crocodile meat samples that exceeded the acceptable limits for APC, CC and ECC, respectively, while Salmonella spp. were detected only in 0.05% (1/2051) of the samples from farm A (Table 2). Farm A had significantly higher (P b 0.05) proportions of samples that exceeded the recommended limits for APC and CC compared to both farms B and C. All of the samples (n = 1524) tested showed satisfactory counts (b20 cfu/g) for L. monocytogenes, while only 2.9% (60/2051) were classified as potentially hazardous for E. coli counts (data not shown). As noted by Madsen (1993), the presence of excessive APC and CC is generally used as product processing contamination indicators (Ashbolt et al., 2001). The recovery of E. coli is used as an indicator of fecal contamination and this may be of public health concern due to the possible presence of toxigenic strains, most notably E. coli O:157:H7 that cause hemolytic uremic syndrome (Kawano et al., 2008). However, given that most of the samples had APC, CC and ECC within the acceptable limits and that none were positive for L. monocytogenes was quite encouraging from a public health perspective. Previous work by Borch et al. (1996) and Bonardi et al. (2002) demonstrated that contamination of meat by L. monocytogenes does not occur primarily from the animal, but is mainly linked to the abattoir environment. Hence, L. monocytogenes is a useful indicator of the hygienic status of slaughterhouses (Bonardi et al., 2002). The observed low isolation of Salmonella spp. contrasted previous findings where prevalence of 15–30.9% was reported (Madsen, 1996; Madsen et al.,

1998; Obwolo and Zwart, 1993). It is noteworthy that these Salmonella spp. were predominantly isolated from intestinal contents and not from meat and skin surfaces (Madsen et al., 1998). Salmonella spp. are normally carried in the intestinal tracts of reptiles and shed intermittently when animals are stressed (Austin and Wilkins, 1998). Intestinal carriage rates of Salmonella spp. exceeding 50% in asymptomatic animals (Geue and Loschner, 2002) have been reported and together with high crocodile pond water concentration of Salmonella spp. (Madsen, 1994) they may act as possible sources of contamination for the meat. The low recovery of Salmonella spp. may be due to disinfection through chlorination (Purdie, 1999) and pasteurization of carcasses. Dipping in chlorine has been reported to be effective in destroying Salmonella spp. (Madsen et al., 1992) although unhygienic handling of meat afterwards may result in recontamination. Salmonella spp. are important zoonotic pathogens in the risk analysis of crocodile meat and hence strict measures should be put in place to minimize the risk of human exposure (AQIS, 2000). It is concluded that notwithstanding the differences of APC and CC of frozen crocodile meat among the study farms, the microbiological quality in terms of the these process indicator microorganisms in addition to ECC, LMC and Salmonella presence was good. However, the isolation of E. coli and Salmonella spp. from some of the investigated meat samples is a cause for concern from a public health point of view. Thus, strict control and monitoring of the pasteurizing process and implementation of measures to avoid contamination of the meat after pasteurization need to be carefully considered. Further studies to determine the preponderance of verocytotoxin producing E. coli (VTEC) strains from crocodile meat will be beneficial in ascertaining their zoonotic potential. Acknowledgments We are grateful to members of staff from the Central Veterinary Laboratories for their technical assistance. References American Public Health Association (APHA), 1992. Compendium of Methods for Microbiological Examination of Foods, In: Vanderzant, C., Splittstoesser, D.F. (Eds.), 3rd ed. American Public Health Association Inc., Washington D.C. Anon, 2010. The Prevention of Verocytotoxigenic E. coli (VTEC) Infection: A Shared Responsibility, Report of the Scientific Committee of the Food Safety Authorityof Ireland, 2nd ed. Food Safety Authority of Ireland, Dublin, Ireland. Anon, 2005. Commission Regulation (EC) No. 2073/2005 of 15 November, 2005 on Microbiological Criteria of Foodstuffs. European Parliament, Brussels, Belgium. AQIS, 2000. The AIQS Import Risk Analysis Process Handbook, Australian Quarantine and Inspection Service — Department of Primary Industries and Energy. National Capital printing, Canberra. Ashbolt, N.J., Grabow, W.O.K., Snozzi, M., 2001. Indicators of microbial water quality. World Health Organisation (WHO) Water Quality Guidelines, Standards and Health. In: Fewtrell, L., Bartram, J. (Eds.), IWA Publishing, London, UK. Austin, C.C., Wilkins, M.J., 1998. Reptile-associated salmonellosis. J. Am. Vet. Med. Assoc. 212 (6), 866–867. Barrow, G.I., Feltham, R.K.A., 1993. Cowan and Steel's Manual for the Identification of Medical Bacteria, third ed. Cambridge University Press, Cambridge, UK. Bonardi, S., Brindani, F., Maggi, E., 2002. Isolation of Listeria monocytogenes and Listeria spp. from pigs at slaughter in Italy. , 22. Annali Facolta' di Medicina Veterinaria—Universita'degli Studi di Parma 205–210. Borch, E., Nesbakken, T., Christensen, H., 1996. Hazard identification in swine slaughter with respect to foodborne bacteria. Int. J. Food Microbiol. 30, 9–25.

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