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Salmonella on feces, hides and carcasses in beef slaughter facilities in Venezuela
International Journal of Food Microbiology 166 (2013) 226–230
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Salmonella on feces, hides and carcasses in beef slaughter facilities in Venezuela Claudia Narváez-Bravo a,⁎, Argenis Rodas-González c,1, Yrimar Fuenmayor b, Carolina Flores-Rondon c, Gabriela Carruyo b, Mireya Moreno b, Armindo Perozo-Mena d, Armando E. Hoet e a
Food Science Department, University of Manitoba, Winnipeg, MB R3T 2N2, Canada Infectious Transmission Diseases Department, School of Veterinary Science, Universidad del Zulia, Venezuela Industry and Animal Production Department, School of Veterinary Science, Universidad del Zulia, Venezuela d Bacteriology Laboratory, Medicine School, Universidad del Zulia, Venezuela e Department of Veterinary Preventive Medicine, Ohio State University, United States b c
a r t i c l e
i n f o
Article history: Received 19 April 2013 Received in revised form 8 July 2013 Accepted 9 July 2013 Available online 17 July 2013 Keywords: Beef Carcasses Feces Hides Salmonella Slaughter plant
a b s t r a c t This study determined Salmonella prevalence at different stages during the slaughtering in three beef slaughter plants (A, B and C) located in the western region of Venezuela (Zulia and Lara states). Each facility was visited three times at monthly intervals, from the months October through December of 2006. Samples were collected from hides (n = 80), fecal grabs (n = 80) and carcasses (n = 80) at the phases of pre-evisceration, afterevisceration and pre-cooler at three sampling sites on the animals (rump, flank and brisket). Salmonella prevalence was higher on hides (36.3%) than on feces (13.8%) (P b 0.05). Differences among slaughter plants for overall Salmonella prevalence were observed (P = 0.001; A: 3.5%, B: 11.1%, C: 4.4%). From the isolated strains, Salmonella enterica subspecies enterica ser. Saintpaul, Salmonella ser. Javiana and Salmonella ser. Weltevreden were identified. Cattle feces and hides might be considered as important sources of Salmonella for carcass contamination at different slaughter stages. The presence of potentially pathogenic Salmonella serotypes at the slaughtering stages is an evidence of the circulation of this pathogen in the food environment; its presence could increase consumers' risks of infection if proper food handling and preparation techniques are not followed. These data should serve as a baseline for future comparisons in Salmonella prevalence on beef carcasses to be used by the government and industry in order to establish preventive measures and to better address the risks of Salmonella contamination. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Developing countries are affected by a wide range of foodborne diseases. The World Health Organization (WHO) has estimated that 1.5 billion episodes of diarrhea occur every year in developing countries, resulting in 3 million deaths (Alper, 2003). In Latin America and the Caribbean, the Pan-American Institute for Food Protection and Zoonosis (INPPAZ) reported 5283 outbreaks of foodborne disease that affected 174,976 persons and caused 275 deaths between the years 1995 and 2001 (Franco et al., 2003). More recently data collected for Pan-American Health Organization (PAHO/WHO) on developing countries indicated that 9180 foodborne outbreaks were reported
⁎ Corresponding author at: Department of Food Science, Office number 238, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Tel./fax: +1 204 474 7630. E-mail address: [email protected] (C. Narváez-Bravo). 1 Current address: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.07.009
from the years 1993 to 2010 from 22 countries of the region, from these outbreaks 69% were caused by bacteria, 9.7% by viruses, 9.5% by marine toxins, 2.5% by chemical contaminants, 1.8% by parasites and 0.5% by vegetal toxins and among bacteria, Salmonella spp. was the most frequent agent (Pires et al., 2012), being responsible for 58.1% of the outbreaks and 66.2% of the cases (Franco et al., 2003). Among Salmonella serovars circulating in Latin America and the Caribbean, Campos et al. (2012) reported that Salmonella ser. Typhi, Salmonella ser. Typhimurium and Salmonella ser. Enteritidis were the more frequent serovar isolates from human infections in six countries of the region (Argentina, Brazil, Colombia, Costa Rica, Chile and Paraguay). In Venezuela, few studies have been conducted to date on the detection of Salmonella spp. in the beef production chain. Nava (2005) screened 15 dual purpose cattle farms (n = 1463), recovering Salmonella in all of them, with a prevalence that ranged between 1.1% and 55.7%. In beef products, Narváez-Bravo et al. (2005) reported high Salmonella prevalence in ingredients (45%) and during beef patty process (up to 66%), and seven Salmonella serotypes were reported (Scharzengrund, Braenderup, Sintorf, London, Anatum, Tennessee and Derby). Regrettably, publications addressing the prevalence of Salmonella in the beef cattle
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harvest process in Venezuela do not exist according to the authors' knowledge. Nevertheless, in developed countries, the prevalence of Salmonella shedding at feedlots is well documented, as are the dynamics of carcass contamination during the slaughter process, and the specific locations for distribution of pathogens on the carcass (Bell, 1997; Fegan et al., 2005). The ability to consistently identify patterns of contamination on carcasses in processing plants enables the implementation of interventions that target high contamination areas and results in further reduction of pathogens in the beef supply (Rekow et al., 2011). This type of information is lacking in Venezuela, and as a consequence, the establishment of pathogen reduction interventions is unusual at beef processing facilities even though, Venezuelan food regulations establish zero tolerance for Salmonella in beef (COVENIN, 1988). Also it is important to mention that the implementation of food safety programs, such as Hazard Analysis and Critical Control Point (HACCP) systems, is not required by law in this country. Therefore, it is important to generate scientific data that will lead to a further understanding of the dynamics of carcass contamination during the slaughter process that will help to develop mitigation strategies for pathogen reduction by the government and industry in order to better address the risks of Salmonella contamination. Furthermore, the objective of this research was to determine the Salmonella prevalence in feces, hides and carcasses during the slaughter processes in three distinct slaughter plants located in Venezuela.
2. Material and methods 2.1. Experimental design This study was carried out in three distinct abattoirs in the western region of Venezuela. The abattoirs were referred to as A, B and C, which kill an average of 900, 300 and 150 animals daily, respectively. The animals slaughtered in these three abattoirs originated in the main beef production regions of Venezuela and represented different breeds (crossbred Bos indicus × Bos taurus), sex classes (bulls, steers, heifers and cows) and ages (1–11 years). Their diets consisted mainly of grazed material. The slaughter plants were under official inspection to meet quality control, sanitation and hygiene standards during processing. Each slaughterhouse was sampled three times, at monthly intervals during the months of October, November and December of 2006. At the harvest facility, after each animal was stunned, bled and placed onto the rail system, a sample from each hide (n = 80) was obtained. Animals were randomly selected; samples were taken from the following sampling sites: rump, flank and brisket using sponges hydrated with 10 ml of buffered peptone water (BPW) (Difco® Laboratories, Sparks, MD). Approximately a 250 cm2 area was swabbed at each sampling site. Each of the sampled carcasses was tagged after hide removal, so that the samples were matched from the hide through all of the processing steps. Carcass sampling (n = 80) was performed at three different stages in the slaughtering process of each plant and at three different anatomical sites on the carcasses. The stages in the slaughtering chain were designated as pre-evisceration immediately after de-hiding, afterevisceration immediately after removal of the internal organs and pre-cooler after washing the carcass at the final rail. The sample sites on the carcasses were brisket, flank and rump, as listed in the FSIS's final rule (FSIS, 1996). Beef carcass samples were collected using sponges hydrated with 10 ml of BPW. During collection of the samples, care was taken to avoid cross contamination. On each of the carcass's sample sites, an area of 100 cm2 was swabbed using the sponge technique and disposable sterile templates. Intestinal feces samples were collected after evisceration from each tagged carcass (n = 80). The entire gastrointestinal tract was tagged and followed to the viscera room. Once there, the recto-colon portion of the intestine was cut and put individually into a labeled sterile bag.
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All samples were transported in coolers containing ice packs, and were received and processed in the microbiology laboratory in the Veterinary Science Building at Zulia State University within 24 h of collection. 2.2. Salmonella detection For isolation of Salmonella from hide and carcass samples, 10 ml of BPW was added to each sponge bag, for a total volume of 20 ml. Each of the sponges was homogenized, by hand massage for 2 min, then 1 ml of homogenized sample was added to each 9 ml of Rappaport Vassiliadis broth (RV), (Himedia®) and Tetrathionate broth (TT) (Difco®). For fecal samples, sterile scissors were used to open the colon–rectum samples. Samples were processed according to methods previously described by Dargatz et al. (2000) and Narváez-Bravo et al. (2013). Briefly, 1 g of feces was weighed and added to each of the 9 ml RV and TT broths. After the addition of the samples to the enrichment broth, each tube was homogenized and incubated at 42 ± 0.5 °C for 24 h. After incubation, TT and RV enrichment for hides, carcasses and fecal samples were streaked onto XLT4 (Difco®) and Hecktoen Enteric (Himedia®) agar medium and incubated for 24 h at 37 ± 0.5 °C. Negative plates were incubated for an additional 24 h at 35 °C. All presumptive colonies (at least five characteristic colonies were tested for each media type plate, if available) were screened through the following biochemical tests: triple sugar iron (TSI) and LIA slants. All presumptive Salmonella, based on TSI and LIA outcome, were subjected to additional biochemical tests: urea, Voges– Proskauer (VP), methyl red (MR), indole, citrate, potassium cyanide, malonate, dulcitol, lysine, ornithine and arginine. All of the isolates with typical results for Salmonella, on the biochemical tests mentioned above, were tested for somatic antigens using polyvalent O antiserum (Difco®), following the manufacture recommendations. Some of the isolates were sent for serotyping at the Bacteriology Laboratory at the Medical School, Zulia State University. Once the purity of the submitted isolated material was tested, the strains underwent a complete set of biochemical tests (indole production, methyl red, Voges–Proskauer, Simmons Citrate, hydrogen sulfide on TSI, urea hydrolysis, phenylalanine deaminase, lysine decarboxylase, ornithine decarboxylase, arginine dehydrolase, motility, gelatin hydrolysis, grown in KCN, malonate utilization, D-glucose-acid, D-glucose-gas, fermentation of lactose, sucrose, Dmannitol, dulcitol, salicin, adonitol, myo-inositol, D-sorbitol, L-arabinosa, raffinose, L-rhamnose, maltose, D-mannose, D-xylose, threalose, Darabitol, glycerol, cellobiosa, mellibiose, esculin hydrolysis, acetate utilization, DNase, nitrate–nitrite and oxidase) and polyvalent somatic antisera. Once the confirmation step for generic Salmonella was completed, the serotypes were designated according to the Kauffmann– White scheme (Grimont and Weill, 2007) using Denka Seiken (Tokyo, Japan) Agglutinating antisera (somatic and flagellar) following manufacturer recommendations. Reference Salmonella strains (ATCC 123215 and 9842) were used as positive controls to evaluate the quality of the media and reagents used in this research. 2.3. Statistical analysis The data collected was analyzed using SAS (Cary, NC) version 9.2 (SAS, 2003). For each pathogen, a Chi-squared analysis (Fisher's exact test) was used, to test for differences among plants, slaughter processes and anatomical sites. 3. Results and discussion 3.1. Salmonella detection on hide and fecal samples The overall prevalence of Salmonella on hides and intestinal feces is shown in Table 1. Positive samples for Salmonella were greater on hides than on feces (P = 0.001, 36.7% vs. 13.8%; respectively). However,
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Table 1 Prevalence of positive samples for Salmonella on hides and intestinal feces. Sourcea
n
Positive, n (%)
Hide Fecal grab
80 80
29 (36.25) 11 (13.75)
a
Chi-square analysis indicated that the prevalence was different by source (P = 0.001).
when a comparison was made among the slaughter plants on hides and fecal samples (Fig. 1), there was no statistical difference (P = 0.8 and P = 0.1; respectively) with the exception of intestinal feces' prevalence observed in Plant A, as the animals from this plant showed lower Salmonella prevalence (P b 0.05) when compared with the other two plants. A wide variation of Salmonella prevalence on hides has been published by other researchers from 17.7% up to 94% (Bacon et al., 2002; Brichta-Harhay et al., 2008; Fegan et al., 2005; Reid et al., 2002). These differences could be due to differences in sampling, detection procedures, epidemiological factors, breeds, and diets, among others. Fegan et al. (2005) conducted similar research in Australia and reported Salmonella prevalence on hides of 68%; while in the research conducted by Brichta-Harhay et al. (2008), the prevalence ranged from 86% to 94%. Bacon et al. (2002) reported an overall incidence of Salmonella on cattle hides of 15.4%. Also, other research in South West England reported similar incidence of Salmonella on hides of 17.7% (Reid et al., 2002). In feces, the results of the current study were similar to those reported by Fegan et al. (2005), who reported a prevalence of 16% in cattle fecal samples collected after evisceration. Dargatz et al. (2000) in the United States, as part of the National Health Monitoring System, reported that approximately 1.4% (70 of 5049) of the total samples were positive for Salmonella. In Mexico, a higher prevalence at the intestinal feces (46.8%) and hides (92.4%) was reported (Narváez-Bravo et al., 2013). Comparing the prevalence obtained in the current study with the international data, it can be determined that the prevalence found in this research is of a low to intermediate range. Contrasting the percentage of positive Salmonella samples for hide and fecal samples, it was found that hide samples showed a higher (P b 0.05) percentage of positives. These results were consistent with other research where Salmonella prevalence on beef cattle's hides is higher than that on feces (Fegan et al., 2005). Overall Salmonella prevalence at intestinal feces showed that 13.8% of the animals were Salmonella carriers. Interestingly the intestinal feces prevalence for incoming animals in Plant A was very low (3.3%) when compared with Plan B (20.0%) and Plant C (20.0%). Surprisingly, the prevalence on hides was similar for all three abattoirs; we were expecting a lower prevalence on hide samples collected at Plant A. These results might suggest that cross contamination avoidance procedures at the
farm, during transportation for these particular animals shipped to Plant A, were probably deficient. Additionally, the slaughter plant received animals from a diverse group of farms, and although these animals were kept separate at the abattoir holding pens eventually the same holding pen was used for other animal lots over time, making possible the contamination from one processing day to another. Cross contamination at holding pen areas has been described before (Arthur et al., 2008; Buncic and Sofos, 2012; Carrasco et al., 2012). Small et al. (2003) reported that pathogens survived for N 1 week in the holding pen environment, and it was also reported that contamination of holding pens with pathogens could be carried over from one batch of animals to another and/or from one day to the next (Small et al., 2006; Small et al., 2003). However, to enable more detailed conclusions regarding contamination routes and origins of Salmonella at the abattoir, genomic DNA fingerprinting analysis must be performed (pulse field electrophoresis and/or multiple-locus variable number tandem repeat analysis) on the recovered Salmonella isolates. The microbial loads carried by incoming cattle are important, considering that the exterior of the hide is the primary source of contamination. In this regard, many studies have shown that cattle hide contamination is transferred to the carcasses during processing (Arthur et al., 2007b; Bell, 1997). Narváez-Bravo et al. (2013) found a positive correlation in the presence of Salmonella in intestinal feces from asymptomatic animal carriers and subsequent contamination of hides and carcasses. The authors established, through risk factor analysis, that positive animals for Salmonella in intestinal feces (carriers) had an eight times higher likelihood of testing positive on hides, were almost three times more likely to test positive on carcasses at pre-evisceration, and were two times more likely to test positive on carcasses at pre-cooler. Carrasco et al. (2012) highlight the importance of cross contamination and recontamination of food by Salmonella and the importance of these events to identify high-risk practices during food processing and preparation. Our results show the need of evaluated best practices at farm level and to establish food safety interventions that help in the reduction of Salmonella loads on hides before the slaughter process take place. Some beef slaughter plants have implemented hide wash cabinets or hide decontamination treatments after slaughter and before skinning, in order to reduce hide contamination; as a consequence the risk of cross contamination later on in the process could be further reduced (Arthur et al., 2007a; Buncic and Sofos, 2012). 3.2. Salmonella detection during the slaughter process The overall Salmonella prevalence on carcasses for each plant is summarized in Fig. 2. Regardless of the anatomic location and slaughter process involved, differences among slaughter plants for overall positive carcass samples were observed (P = 0.001), where Plant B (11.1%)
45.0
Salmonella %
35.0
40.0
36.7
12
NS
*
33.3
25.0 20.0
20.0
20.0
15.0 10.0 5.0
11.1
10
NS
30.0
Salmonella %
40.0
8 6
4.4 4
3.4
3.3
2
0.0 Plant A Hide
Plant B Plant C Intestinal Feces
Fig. 1. Salmonella prevalence detected on hides and intestinal feces at the different slaughter plants: A, B and C. Chi-square analysis indicated that the prevalence of positive samples for Salmonella on hide and intestinal feces was different by plant (*: P b 0.05; NS: P N 0.05).
0
Plant A
Plant B
Plant C
Fig. 2. Overall Salmonella prevalence detected on carcass samples by plants. Chi-square analysis indicated that the prevalence of positive samples for Salmonella between plants was different (P = 0.001).
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showed a higher number of positive samples, followed by Plant C (4.4%) and Plant A (3.5%). The overall Salmonella prevalence at slaughter stages and sampling sites is presented in Table 2. No differences were detected among sample sites (P N 0.05) at the different stages of the process (from 0.8% up to 3.4%). Regardless of the sample site, each slaughter plant did not show any differences and showed the same level of Salmonella contamination from 4.6% up to 7.2% (P = 0.4). The lack of reduction of contamination during the different slaughter stages could be attributable to the absence of pathogen reduction interventions in the three slaughter plants. In Mexico and the United States, the Salmonella prevalence at the slaughter process reported is higher than the prevalence reported by this study. Narváez-Bravo et al. (2010) reported a prevalence at preevisceration of 49%, at pre-cooler of 24.8% and at the cooler (after 24 h of dry chilling) of 6.0% in a Mexican beef slaughterhouse. While in four geographically distant regions of the United States, Brichta-Harhay et al. (2008) reported a prevalence at pre-evisceration of 50.2%. These authors found a higher reduction (reaching 0.1 to 1%) after the samples received the full complement of all processing interventions and after being chilled for no more than 2 h. No differences within each plant were observed at slaughter stages or sampling site (P N 0.05). The hygiene of cattle during slaughter has an important impact on the incidence of pathogens such as Salmonella on the carcasses, and in the final product. Positive carcasses for Salmonella at pre-evisceration could increase the chances of testing positive for Salmonella at the pre-cooler by 5.95 times, and carcasses positive at the pre-cooler have an 8-fold increased chance of a positive carcass after 24 h of dry chilling (Narváez-Bravo et al., 2013). In this study, the presence of Salmonella on hides and carcasses during the slaughter process was examined and the results suggest that there was a potential transfer of Salmonella from hides and feces to different anatomical sites of the carcass. This data provides important information about how well these slaughter plants follow SOP and GMP programs. It also allows the identification of the slaughter stages with higher risks of carcass contamination due to Salmonella and/or other bacteria. When slaughter procedures and sanitary programs are working properly, the ideal is that the load of pathogens that initially enter the plant with the live animals decreases at each step of the process, which should prevent an increase of pathogenic loads in the final products. In the case of Plant C, where the contamination increased at the pre-cooler stage, it suggests that there are weaknesses in processing, and that they must be corrected through reinforcing the SOP and GMP programs, through the implementation of microbial interventions. None of the abattoirs researched for this study had pathogen reduction interventions or decontamination treatments in place. These interventions/decontamination treatments are applied during slaughter and dressing in some countries to reduce the prevalence and number of pathogens and other microorganism in carcasses (Buncic and Sofos, 2012). It is important to mention that we were not allowed to collect samples on chilled carcasses. We only can infer that in regard to Table 2 Overall Salmonella prevalence for processing stages and sampling sites of the carcass. Slaughter process, n (%) a Anatomical site
Pre-evisceration n = 237
After-evisceration n = 237
Pre-cooler n = 237
Total
Brisket Flank Rump Total b
2 (0.84) 5 (2.11) 6 (2.53) 13 (5.49)
7 (2.97) 8 (3.39) 2 (0.84) 17 (7.17)
2 (0.84) 7 (2.95) 2 (0.84) 11 (4.64)
11 (4.65) 20 (8.45) 10 (4.21)
a Chi-square analysis indicated that the prevalence was not different by sampling site within pre-evisceration, after-evisceration and pre-cooler process (P = 0.4, P = 0.1, P = 0.1; respectively). b Chi-square analysis indicated that the overall prevalence was not different between harvest process (P = 0.4; regardless anatomical sites).
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regulatory requirements, carcasses showing a positive sampling site after chilling could be seized under Venezuelan regulation requirements. Under these regulations Salmonella standards for raw beef stipulate zero tolerance for Salmonella; additionally HACCP implementation is not a regulatory requirement by Venezuelan government. Regarding interventions during the slaughter process, the three slaughter plants investigated in this study only had a carcass washing step applying tempered water (physical decontamination) at the pre-cooler stage, before entry into the cooler. Nevertheless, carcass washing per se could further spread the microbial contamination to uncontaminated areas of the carcass if there was not a previous removal of the contaminated area by knife trimming (Buncic and Sofos, 2012). These results indicate that the feces and hides of animals carrying Salmonella are major sources of contamination. However, the spread of this pathogen during the process can be considered low and may be attributed to the relatively low number of incoming positive animals (hide or fecal samples) to the slaughter process, as well as the implementation of SOP and GMP programs by participating companies. It should be noted that carcasses, after chilling, should be evaluated in order to assess adherence of the abattoirs to Salmonella zero tolerance policies.
3.3. Salmonella serotyping Eight hundred and seventy one samples were collected across the study and Salmonella were recovered from 81 samples. Through isolate serotyping, Salmonella ser. Saintpaul and Salmonella ser. Javiana were found on hides. In fecal samples, Salmonella ser. Javiana and Salmonella ser. Weltevreden var. 15+ (obsolete name Lanka) were found. Carcass samples at pre-evisceration, after-evisceration and pre-cooler tested positive for Salmonella ser. Saintpaul and Salmonella ser. Javiana. Due to economic constraints not all of the isolates were sent for serotyping. The different serotypes found in this research have been linked to human diseases in Venezuela and other countries (Araque, 2009; CDC, 2010; Kim, 2010; Toro et al., 2010). In Venezuela, there are not enough reports describing what Salmonella serotypes are causing human infections, nor is there any reliable epidemiological data. The reason is the limitations in the Venezuelan foodborne disease surveillance system that makes it difficult to properly study foodborne outbreaks and to identify pathogens related to the outbreaks (Toro et al., 2010); additionally, individuals that may present gastrointestinal illnesses, in the majority of the cases, do not seek medical care and therefore foodborne illness is probably underestimated. In the United States, in 2009, there were a total of 17,883 laboratoryconfirmed cases of infection in FoodNet surveillance areas, where 6371 (90.5%) Salmonella isolates were serotyped, and 10 serotypes accounted for 73.1% of infections. Among the top five serotypes reported were Enteritidis (19.2%); Typhimurium (16.1%), Newport (12.1%), Javiana (8.5%) and Heidelberg (3.6%), even though Salmonella ser. Saintpaul was not in the five top it accounted for 2.5% of infections (CDC, 2010). While in Latin America and the Caribbean, Salmonella ser. Typhi, Salmonella ser. Typhimurium and Salmonella ser. Enteritidis are the serovars more frequently reported (Campos et al., 2012). Information on what Salmonella serotypes are circulating and in what food sources is essential to prioritize food safety interventions and to implement appropriate control measures during food manufacture and preparation (Campos et al., 2012). The presence of potentially pathogenic Salmonella serotypes at the different stages is an evidence of the circulation of pathogens in the food environment. It could increase the risks of infection to consumers when Salmonella survival is allowed under cross contamination and recontamination conditions such as when there are inadequate temperatures during food preparation that allow for the survival of
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the pathogen through undercooking or food handling scenarios when food safety procedures are not followed (Carrasco et al., 2012). 4. Conclusions Comparing the prevalence obtained in the current study with the international data, the prevalence found in this research can be considered to range from low to intermediate. The data demonstrates that the feces and hides of these animals were the major sources of Salmonella beef carcass contamination. The data not only demonstrates the variation in the incoming Salmonella loads on cattle hides and feces, but also illustrates the differences among plants and their abilities to prevent cross contamination between hide and fecal material, during dressing and the evisceration process, as well as the need to implement best practices at the farms and food safety programs such as HACCP/pathogen reduction interventions in these slaughter plants in order to assure a proper control or reduction of pathogen dissemination through the slaughter process and to comply with zero tolerance policy for Salmonella. More research in this area is needed in order to determine if seasonality or other factors affect the Salmonella loads in beef cattle, and thus determine the real impact this pathogen represents to the Venezuelan beef industry. Acknowledgment This research was possible through the support of the CONDES (Consejo de Desarrollo Cientifico y Humanistico de la Universidad del Zulia) and the microbiology laboratory of the Faculty of Veterinary Medicine, University of Zulia. A special thanks to all of the slaughter plants which collaborated and supported the research team during sample collection. References Alper, J., 2003. Date gaps need bridging to assess infectious gastrointestinal diseases. ASM News 69, 65–68. Araque, M., 2009. Nontyphoid Salmonella gastroenteritis in pediatric patients from urban areas in the city of Mérida, Venezuela. Journal of Infection in Developing Countries 3, 28–34. Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2007a. Effects of a minimal hide wash cabinet on the levels and prevalence of Escherichia coli O157:H7 and Salmonella on the hides of beef cattle at slaughter. Journal of Food Protection 70, 1076–1079. Arthur, T.M., Bosilevae, J.M., Brichta-Harhay, D.M., Guerini, M.N., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2007b. Transportation and lairage environment effects on prevalence, numbers and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. Journal of Food Protection 70, 280–286. Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Kalchayanand, N., King, D.A., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2008. Source tracking of Escherichia coli O157:H7 and Salmonella contamination in the lairage environment at commercial U.S. beef processing plants and identification of an effective intervention. Journal of Food Protection 71, 1752–1760. Bacon, R.T., Sofos, J.N., Belk, K.E., Hyatt, D.R., Smith, G.C., 2002. Prevalence and antibiotic susceptibility of Salmonella isolated from beef animal hides and carcasses. Journal of Food Protection 65, 284–290. Bell, R.G., 1997. Distribution and sources of microbial contamination on beef carcasses. Journal of Applied Microbiology 82, 292–300. Brichta-Harhay, D.M., Guerini, M.N., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2008. Salmonella and Escherichia coli O157:H7 contamination on hides and carcasses of cull cattle presented for slaughter in the United States: an evaluation of prevalence and bacterial loads by
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(Eds.), International Handbook of Foodborne Pathogens. Marcel Dekker, Inc., New York, pp. 733–743. FSIS, 1996. Pathogen reduction; Hazard Analysis and Critical Control Point (HACCP) systems; final rule. In: Agriculture D.o. (Ed.), Federal Register, pp. 38806–38989. Grimont, P.A.D., Weill, F.X., 2007. Antigenic Formulae of the Salmonella Serovars WHO, 9th ed. WHO Collaborating Centre for Reference and Research on Salmonella Paris. Kim, S., 2010. Salmonella serovars from foodborne and waterborne diseases in Korea, 1998–2007: total isolates decreasing versus rare serovars emerging. Journal of Korean Medical Science 25, 1693–1699. Narváez-Bravo, C., Parra, K., Huerta-Leidenz, N., Rodas-González, A., Arenas de Moreno, L., 2005. Isolation of Salmonella and pathogenic Escherichia coli during processing of hamburger patty in a small plant of Maracaibo Venezuela. Revista Cientifica. FCV-LUZ 15, 551–559. Narváez-Bravo, C., Miller, M.F., Echeverry, A., Pond, K., Brashears, M.M., 2010. Salmonella and E. coli O157:H7 prevalence in cattle and on carcasses in a vertically integrated feedlot and harvest plant in Mexico. Proceedings 97th Annual Meeting of the International Association for Food Protection, Anaheim, California. Narváez-Bravo, C., Miller, M., Jackson, T., Jackson, S., Rodas-González, A., Pond, K., Echeverry, A., Brashears, M.M., 2013. Salmonella and E. coli O157:H7 prevalence in cattle and on carcasses in a vertically integrated feedlot and harvest plant in Mexico. Journal of Food Protection 76, 786–795. Nava, R., 2005. Aislamiento de Salmonella spp. en heces bovinas y determinación de los patrones de resistencia a los antimocrobianos. (Thesis) Facultad de Ciencias Veterinarias. Universidad del Zulia, Maracaibo, Venezuela 46. Pires, S.M., Vieira, A.R., Perez, E., Lo Fo Wong, D., Hald, T., 2012. Attributing human foodborne illness to food sources and water in Latin America and the Caribbean using data from outbreak investigations. International Journal of Food Microbiology 152, 129–138. Reid, C.A., Small, A., Avery, S.M., Buncic, S., 2002. Presence of food-borne pathogens on cattle hides. Food Control 13, 411–415. Rekow, C.L., Brashears, M.M., Brooks, J.C., Loneragan, G.H., Gragg, S.E., Miller, M.F., 2011. Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir. Meat Science 87, 361–365. SAS, 2003. SAS/STAT User's Guide: Statistics. SAS Institute Inc., Cary, NC, USA. Small, A., Reid, C.A., Buncic, S., 2003. Conditions in lairages at abattoirs for ruminants in southwest England and in vitro survival of Escherichia coli O157, Salmonella kedougou, and Campylobacter jejuni on lairage-related substrates. Journal of Food Protection 66, 1570–1575. Small, A., James, C., James, S., Davies, R., Liebana, E., Howell, M., Hutchison, M., Buncic, S., 2006. Presence of Salmonella in the red meat abattoir lairage after routine cleansing and disinfection and on carcasses. 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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Salmonella on feces, hides and carcasses in beef slaughter facilities in Venezuela Claudia Narváez-Bravo a,⁎, Argenis Rodas-González c,1, Yrimar Fuenmayor b, Carolina Flores-Rondon c, Gabriela Carruyo b, Mireya Moreno b, Armindo Perozo-Mena d, Armando E. Hoet e a
Food Science Department, University of Manitoba, Winnipeg, MB R3T 2N2, Canada Infectious Transmission Diseases Department, School of Veterinary Science, Universidad del Zulia, Venezuela Industry and Animal Production Department, School of Veterinary Science, Universidad del Zulia, Venezuela d Bacteriology Laboratory, Medicine School, Universidad del Zulia, Venezuela e Department of Veterinary Preventive Medicine, Ohio State University, United States b c
a r t i c l e
i n f o
Article history: Received 19 April 2013 Received in revised form 8 July 2013 Accepted 9 July 2013 Available online 17 July 2013 Keywords: Beef Carcasses Feces Hides Salmonella Slaughter plant
a b s t r a c t This study determined Salmonella prevalence at different stages during the slaughtering in three beef slaughter plants (A, B and C) located in the western region of Venezuela (Zulia and Lara states). Each facility was visited three times at monthly intervals, from the months October through December of 2006. Samples were collected from hides (n = 80), fecal grabs (n = 80) and carcasses (n = 80) at the phases of pre-evisceration, afterevisceration and pre-cooler at three sampling sites on the animals (rump, flank and brisket). Salmonella prevalence was higher on hides (36.3%) than on feces (13.8%) (P b 0.05). Differences among slaughter plants for overall Salmonella prevalence were observed (P = 0.001; A: 3.5%, B: 11.1%, C: 4.4%). From the isolated strains, Salmonella enterica subspecies enterica ser. Saintpaul, Salmonella ser. Javiana and Salmonella ser. Weltevreden were identified. Cattle feces and hides might be considered as important sources of Salmonella for carcass contamination at different slaughter stages. The presence of potentially pathogenic Salmonella serotypes at the slaughtering stages is an evidence of the circulation of this pathogen in the food environment; its presence could increase consumers' risks of infection if proper food handling and preparation techniques are not followed. These data should serve as a baseline for future comparisons in Salmonella prevalence on beef carcasses to be used by the government and industry in order to establish preventive measures and to better address the risks of Salmonella contamination. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Developing countries are affected by a wide range of foodborne diseases. The World Health Organization (WHO) has estimated that 1.5 billion episodes of diarrhea occur every year in developing countries, resulting in 3 million deaths (Alper, 2003). In Latin America and the Caribbean, the Pan-American Institute for Food Protection and Zoonosis (INPPAZ) reported 5283 outbreaks of foodborne disease that affected 174,976 persons and caused 275 deaths between the years 1995 and 2001 (Franco et al., 2003). More recently data collected for Pan-American Health Organization (PAHO/WHO) on developing countries indicated that 9180 foodborne outbreaks were reported
⁎ Corresponding author at: Department of Food Science, Office number 238, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Tel./fax: +1 204 474 7630. E-mail address: [email protected] (C. Narváez-Bravo). 1 Current address: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada. 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.07.009
from the years 1993 to 2010 from 22 countries of the region, from these outbreaks 69% were caused by bacteria, 9.7% by viruses, 9.5% by marine toxins, 2.5% by chemical contaminants, 1.8% by parasites and 0.5% by vegetal toxins and among bacteria, Salmonella spp. was the most frequent agent (Pires et al., 2012), being responsible for 58.1% of the outbreaks and 66.2% of the cases (Franco et al., 2003). Among Salmonella serovars circulating in Latin America and the Caribbean, Campos et al. (2012) reported that Salmonella ser. Typhi, Salmonella ser. Typhimurium and Salmonella ser. Enteritidis were the more frequent serovar isolates from human infections in six countries of the region (Argentina, Brazil, Colombia, Costa Rica, Chile and Paraguay). In Venezuela, few studies have been conducted to date on the detection of Salmonella spp. in the beef production chain. Nava (2005) screened 15 dual purpose cattle farms (n = 1463), recovering Salmonella in all of them, with a prevalence that ranged between 1.1% and 55.7%. In beef products, Narváez-Bravo et al. (2005) reported high Salmonella prevalence in ingredients (45%) and during beef patty process (up to 66%), and seven Salmonella serotypes were reported (Scharzengrund, Braenderup, Sintorf, London, Anatum, Tennessee and Derby). Regrettably, publications addressing the prevalence of Salmonella in the beef cattle
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harvest process in Venezuela do not exist according to the authors' knowledge. Nevertheless, in developed countries, the prevalence of Salmonella shedding at feedlots is well documented, as are the dynamics of carcass contamination during the slaughter process, and the specific locations for distribution of pathogens on the carcass (Bell, 1997; Fegan et al., 2005). The ability to consistently identify patterns of contamination on carcasses in processing plants enables the implementation of interventions that target high contamination areas and results in further reduction of pathogens in the beef supply (Rekow et al., 2011). This type of information is lacking in Venezuela, and as a consequence, the establishment of pathogen reduction interventions is unusual at beef processing facilities even though, Venezuelan food regulations establish zero tolerance for Salmonella in beef (COVENIN, 1988). Also it is important to mention that the implementation of food safety programs, such as Hazard Analysis and Critical Control Point (HACCP) systems, is not required by law in this country. Therefore, it is important to generate scientific data that will lead to a further understanding of the dynamics of carcass contamination during the slaughter process that will help to develop mitigation strategies for pathogen reduction by the government and industry in order to better address the risks of Salmonella contamination. Furthermore, the objective of this research was to determine the Salmonella prevalence in feces, hides and carcasses during the slaughter processes in three distinct slaughter plants located in Venezuela.
2. Material and methods 2.1. Experimental design This study was carried out in three distinct abattoirs in the western region of Venezuela. The abattoirs were referred to as A, B and C, which kill an average of 900, 300 and 150 animals daily, respectively. The animals slaughtered in these three abattoirs originated in the main beef production regions of Venezuela and represented different breeds (crossbred Bos indicus × Bos taurus), sex classes (bulls, steers, heifers and cows) and ages (1–11 years). Their diets consisted mainly of grazed material. The slaughter plants were under official inspection to meet quality control, sanitation and hygiene standards during processing. Each slaughterhouse was sampled three times, at monthly intervals during the months of October, November and December of 2006. At the harvest facility, after each animal was stunned, bled and placed onto the rail system, a sample from each hide (n = 80) was obtained. Animals were randomly selected; samples were taken from the following sampling sites: rump, flank and brisket using sponges hydrated with 10 ml of buffered peptone water (BPW) (Difco® Laboratories, Sparks, MD). Approximately a 250 cm2 area was swabbed at each sampling site. Each of the sampled carcasses was tagged after hide removal, so that the samples were matched from the hide through all of the processing steps. Carcass sampling (n = 80) was performed at three different stages in the slaughtering process of each plant and at three different anatomical sites on the carcasses. The stages in the slaughtering chain were designated as pre-evisceration immediately after de-hiding, afterevisceration immediately after removal of the internal organs and pre-cooler after washing the carcass at the final rail. The sample sites on the carcasses were brisket, flank and rump, as listed in the FSIS's final rule (FSIS, 1996). Beef carcass samples were collected using sponges hydrated with 10 ml of BPW. During collection of the samples, care was taken to avoid cross contamination. On each of the carcass's sample sites, an area of 100 cm2 was swabbed using the sponge technique and disposable sterile templates. Intestinal feces samples were collected after evisceration from each tagged carcass (n = 80). The entire gastrointestinal tract was tagged and followed to the viscera room. Once there, the recto-colon portion of the intestine was cut and put individually into a labeled sterile bag.
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All samples were transported in coolers containing ice packs, and were received and processed in the microbiology laboratory in the Veterinary Science Building at Zulia State University within 24 h of collection. 2.2. Salmonella detection For isolation of Salmonella from hide and carcass samples, 10 ml of BPW was added to each sponge bag, for a total volume of 20 ml. Each of the sponges was homogenized, by hand massage for 2 min, then 1 ml of homogenized sample was added to each 9 ml of Rappaport Vassiliadis broth (RV), (Himedia®) and Tetrathionate broth (TT) (Difco®). For fecal samples, sterile scissors were used to open the colon–rectum samples. Samples were processed according to methods previously described by Dargatz et al. (2000) and Narváez-Bravo et al. (2013). Briefly, 1 g of feces was weighed and added to each of the 9 ml RV and TT broths. After the addition of the samples to the enrichment broth, each tube was homogenized and incubated at 42 ± 0.5 °C for 24 h. After incubation, TT and RV enrichment for hides, carcasses and fecal samples were streaked onto XLT4 (Difco®) and Hecktoen Enteric (Himedia®) agar medium and incubated for 24 h at 37 ± 0.5 °C. Negative plates were incubated for an additional 24 h at 35 °C. All presumptive colonies (at least five characteristic colonies were tested for each media type plate, if available) were screened through the following biochemical tests: triple sugar iron (TSI) and LIA slants. All presumptive Salmonella, based on TSI and LIA outcome, were subjected to additional biochemical tests: urea, Voges– Proskauer (VP), methyl red (MR), indole, citrate, potassium cyanide, malonate, dulcitol, lysine, ornithine and arginine. All of the isolates with typical results for Salmonella, on the biochemical tests mentioned above, were tested for somatic antigens using polyvalent O antiserum (Difco®), following the manufacture recommendations. Some of the isolates were sent for serotyping at the Bacteriology Laboratory at the Medical School, Zulia State University. Once the purity of the submitted isolated material was tested, the strains underwent a complete set of biochemical tests (indole production, methyl red, Voges–Proskauer, Simmons Citrate, hydrogen sulfide on TSI, urea hydrolysis, phenylalanine deaminase, lysine decarboxylase, ornithine decarboxylase, arginine dehydrolase, motility, gelatin hydrolysis, grown in KCN, malonate utilization, D-glucose-acid, D-glucose-gas, fermentation of lactose, sucrose, Dmannitol, dulcitol, salicin, adonitol, myo-inositol, D-sorbitol, L-arabinosa, raffinose, L-rhamnose, maltose, D-mannose, D-xylose, threalose, Darabitol, glycerol, cellobiosa, mellibiose, esculin hydrolysis, acetate utilization, DNase, nitrate–nitrite and oxidase) and polyvalent somatic antisera. Once the confirmation step for generic Salmonella was completed, the serotypes were designated according to the Kauffmann– White scheme (Grimont and Weill, 2007) using Denka Seiken (Tokyo, Japan) Agglutinating antisera (somatic and flagellar) following manufacturer recommendations. Reference Salmonella strains (ATCC 123215 and 9842) were used as positive controls to evaluate the quality of the media and reagents used in this research. 2.3. Statistical analysis The data collected was analyzed using SAS (Cary, NC) version 9.2 (SAS, 2003). For each pathogen, a Chi-squared analysis (Fisher's exact test) was used, to test for differences among plants, slaughter processes and anatomical sites. 3. Results and discussion 3.1. Salmonella detection on hide and fecal samples The overall prevalence of Salmonella on hides and intestinal feces is shown in Table 1. Positive samples for Salmonella were greater on hides than on feces (P = 0.001, 36.7% vs. 13.8%; respectively). However,
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Table 1 Prevalence of positive samples for Salmonella on hides and intestinal feces. Sourcea
n
Positive, n (%)
Hide Fecal grab
80 80
29 (36.25) 11 (13.75)
a
Chi-square analysis indicated that the prevalence was different by source (P = 0.001).
when a comparison was made among the slaughter plants on hides and fecal samples (Fig. 1), there was no statistical difference (P = 0.8 and P = 0.1; respectively) with the exception of intestinal feces' prevalence observed in Plant A, as the animals from this plant showed lower Salmonella prevalence (P b 0.05) when compared with the other two plants. A wide variation of Salmonella prevalence on hides has been published by other researchers from 17.7% up to 94% (Bacon et al., 2002; Brichta-Harhay et al., 2008; Fegan et al., 2005; Reid et al., 2002). These differences could be due to differences in sampling, detection procedures, epidemiological factors, breeds, and diets, among others. Fegan et al. (2005) conducted similar research in Australia and reported Salmonella prevalence on hides of 68%; while in the research conducted by Brichta-Harhay et al. (2008), the prevalence ranged from 86% to 94%. Bacon et al. (2002) reported an overall incidence of Salmonella on cattle hides of 15.4%. Also, other research in South West England reported similar incidence of Salmonella on hides of 17.7% (Reid et al., 2002). In feces, the results of the current study were similar to those reported by Fegan et al. (2005), who reported a prevalence of 16% in cattle fecal samples collected after evisceration. Dargatz et al. (2000) in the United States, as part of the National Health Monitoring System, reported that approximately 1.4% (70 of 5049) of the total samples were positive for Salmonella. In Mexico, a higher prevalence at the intestinal feces (46.8%) and hides (92.4%) was reported (Narváez-Bravo et al., 2013). Comparing the prevalence obtained in the current study with the international data, it can be determined that the prevalence found in this research is of a low to intermediate range. Contrasting the percentage of positive Salmonella samples for hide and fecal samples, it was found that hide samples showed a higher (P b 0.05) percentage of positives. These results were consistent with other research where Salmonella prevalence on beef cattle's hides is higher than that on feces (Fegan et al., 2005). Overall Salmonella prevalence at intestinal feces showed that 13.8% of the animals were Salmonella carriers. Interestingly the intestinal feces prevalence for incoming animals in Plant A was very low (3.3%) when compared with Plan B (20.0%) and Plant C (20.0%). Surprisingly, the prevalence on hides was similar for all three abattoirs; we were expecting a lower prevalence on hide samples collected at Plant A. These results might suggest that cross contamination avoidance procedures at the
farm, during transportation for these particular animals shipped to Plant A, were probably deficient. Additionally, the slaughter plant received animals from a diverse group of farms, and although these animals were kept separate at the abattoir holding pens eventually the same holding pen was used for other animal lots over time, making possible the contamination from one processing day to another. Cross contamination at holding pen areas has been described before (Arthur et al., 2008; Buncic and Sofos, 2012; Carrasco et al., 2012). Small et al. (2003) reported that pathogens survived for N 1 week in the holding pen environment, and it was also reported that contamination of holding pens with pathogens could be carried over from one batch of animals to another and/or from one day to the next (Small et al., 2006; Small et al., 2003). However, to enable more detailed conclusions regarding contamination routes and origins of Salmonella at the abattoir, genomic DNA fingerprinting analysis must be performed (pulse field electrophoresis and/or multiple-locus variable number tandem repeat analysis) on the recovered Salmonella isolates. The microbial loads carried by incoming cattle are important, considering that the exterior of the hide is the primary source of contamination. In this regard, many studies have shown that cattle hide contamination is transferred to the carcasses during processing (Arthur et al., 2007b; Bell, 1997). Narváez-Bravo et al. (2013) found a positive correlation in the presence of Salmonella in intestinal feces from asymptomatic animal carriers and subsequent contamination of hides and carcasses. The authors established, through risk factor analysis, that positive animals for Salmonella in intestinal feces (carriers) had an eight times higher likelihood of testing positive on hides, were almost three times more likely to test positive on carcasses at pre-evisceration, and were two times more likely to test positive on carcasses at pre-cooler. Carrasco et al. (2012) highlight the importance of cross contamination and recontamination of food by Salmonella and the importance of these events to identify high-risk practices during food processing and preparation. Our results show the need of evaluated best practices at farm level and to establish food safety interventions that help in the reduction of Salmonella loads on hides before the slaughter process take place. Some beef slaughter plants have implemented hide wash cabinets or hide decontamination treatments after slaughter and before skinning, in order to reduce hide contamination; as a consequence the risk of cross contamination later on in the process could be further reduced (Arthur et al., 2007a; Buncic and Sofos, 2012). 3.2. Salmonella detection during the slaughter process The overall Salmonella prevalence on carcasses for each plant is summarized in Fig. 2. Regardless of the anatomic location and slaughter process involved, differences among slaughter plants for overall positive carcass samples were observed (P = 0.001), where Plant B (11.1%)
45.0
Salmonella %
35.0
40.0
36.7
12
NS
*
33.3
25.0 20.0
20.0
20.0
15.0 10.0 5.0
11.1
10
NS
30.0
Salmonella %
40.0
8 6
4.4 4
3.4
3.3
2
0.0 Plant A Hide
Plant B Plant C Intestinal Feces
Fig. 1. Salmonella prevalence detected on hides and intestinal feces at the different slaughter plants: A, B and C. Chi-square analysis indicated that the prevalence of positive samples for Salmonella on hide and intestinal feces was different by plant (*: P b 0.05; NS: P N 0.05).
0
Plant A
Plant B
Plant C
Fig. 2. Overall Salmonella prevalence detected on carcass samples by plants. Chi-square analysis indicated that the prevalence of positive samples for Salmonella between plants was different (P = 0.001).
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showed a higher number of positive samples, followed by Plant C (4.4%) and Plant A (3.5%). The overall Salmonella prevalence at slaughter stages and sampling sites is presented in Table 2. No differences were detected among sample sites (P N 0.05) at the different stages of the process (from 0.8% up to 3.4%). Regardless of the sample site, each slaughter plant did not show any differences and showed the same level of Salmonella contamination from 4.6% up to 7.2% (P = 0.4). The lack of reduction of contamination during the different slaughter stages could be attributable to the absence of pathogen reduction interventions in the three slaughter plants. In Mexico and the United States, the Salmonella prevalence at the slaughter process reported is higher than the prevalence reported by this study. Narváez-Bravo et al. (2010) reported a prevalence at preevisceration of 49%, at pre-cooler of 24.8% and at the cooler (after 24 h of dry chilling) of 6.0% in a Mexican beef slaughterhouse. While in four geographically distant regions of the United States, Brichta-Harhay et al. (2008) reported a prevalence at pre-evisceration of 50.2%. These authors found a higher reduction (reaching 0.1 to 1%) after the samples received the full complement of all processing interventions and after being chilled for no more than 2 h. No differences within each plant were observed at slaughter stages or sampling site (P N 0.05). The hygiene of cattle during slaughter has an important impact on the incidence of pathogens such as Salmonella on the carcasses, and in the final product. Positive carcasses for Salmonella at pre-evisceration could increase the chances of testing positive for Salmonella at the pre-cooler by 5.95 times, and carcasses positive at the pre-cooler have an 8-fold increased chance of a positive carcass after 24 h of dry chilling (Narváez-Bravo et al., 2013). In this study, the presence of Salmonella on hides and carcasses during the slaughter process was examined and the results suggest that there was a potential transfer of Salmonella from hides and feces to different anatomical sites of the carcass. This data provides important information about how well these slaughter plants follow SOP and GMP programs. It also allows the identification of the slaughter stages with higher risks of carcass contamination due to Salmonella and/or other bacteria. When slaughter procedures and sanitary programs are working properly, the ideal is that the load of pathogens that initially enter the plant with the live animals decreases at each step of the process, which should prevent an increase of pathogenic loads in the final products. In the case of Plant C, where the contamination increased at the pre-cooler stage, it suggests that there are weaknesses in processing, and that they must be corrected through reinforcing the SOP and GMP programs, through the implementation of microbial interventions. None of the abattoirs researched for this study had pathogen reduction interventions or decontamination treatments in place. These interventions/decontamination treatments are applied during slaughter and dressing in some countries to reduce the prevalence and number of pathogens and other microorganism in carcasses (Buncic and Sofos, 2012). It is important to mention that we were not allowed to collect samples on chilled carcasses. We only can infer that in regard to Table 2 Overall Salmonella prevalence for processing stages and sampling sites of the carcass. Slaughter process, n (%) a Anatomical site
Pre-evisceration n = 237
After-evisceration n = 237
Pre-cooler n = 237
Total
Brisket Flank Rump Total b
2 (0.84) 5 (2.11) 6 (2.53) 13 (5.49)
7 (2.97) 8 (3.39) 2 (0.84) 17 (7.17)
2 (0.84) 7 (2.95) 2 (0.84) 11 (4.64)
11 (4.65) 20 (8.45) 10 (4.21)
a Chi-square analysis indicated that the prevalence was not different by sampling site within pre-evisceration, after-evisceration and pre-cooler process (P = 0.4, P = 0.1, P = 0.1; respectively). b Chi-square analysis indicated that the overall prevalence was not different between harvest process (P = 0.4; regardless anatomical sites).
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regulatory requirements, carcasses showing a positive sampling site after chilling could be seized under Venezuelan regulation requirements. Under these regulations Salmonella standards for raw beef stipulate zero tolerance for Salmonella; additionally HACCP implementation is not a regulatory requirement by Venezuelan government. Regarding interventions during the slaughter process, the three slaughter plants investigated in this study only had a carcass washing step applying tempered water (physical decontamination) at the pre-cooler stage, before entry into the cooler. Nevertheless, carcass washing per se could further spread the microbial contamination to uncontaminated areas of the carcass if there was not a previous removal of the contaminated area by knife trimming (Buncic and Sofos, 2012). These results indicate that the feces and hides of animals carrying Salmonella are major sources of contamination. However, the spread of this pathogen during the process can be considered low and may be attributed to the relatively low number of incoming positive animals (hide or fecal samples) to the slaughter process, as well as the implementation of SOP and GMP programs by participating companies. It should be noted that carcasses, after chilling, should be evaluated in order to assess adherence of the abattoirs to Salmonella zero tolerance policies.
3.3. Salmonella serotyping Eight hundred and seventy one samples were collected across the study and Salmonella were recovered from 81 samples. Through isolate serotyping, Salmonella ser. Saintpaul and Salmonella ser. Javiana were found on hides. In fecal samples, Salmonella ser. Javiana and Salmonella ser. Weltevreden var. 15+ (obsolete name Lanka) were found. Carcass samples at pre-evisceration, after-evisceration and pre-cooler tested positive for Salmonella ser. Saintpaul and Salmonella ser. Javiana. Due to economic constraints not all of the isolates were sent for serotyping. The different serotypes found in this research have been linked to human diseases in Venezuela and other countries (Araque, 2009; CDC, 2010; Kim, 2010; Toro et al., 2010). In Venezuela, there are not enough reports describing what Salmonella serotypes are causing human infections, nor is there any reliable epidemiological data. The reason is the limitations in the Venezuelan foodborne disease surveillance system that makes it difficult to properly study foodborne outbreaks and to identify pathogens related to the outbreaks (Toro et al., 2010); additionally, individuals that may present gastrointestinal illnesses, in the majority of the cases, do not seek medical care and therefore foodborne illness is probably underestimated. In the United States, in 2009, there were a total of 17,883 laboratoryconfirmed cases of infection in FoodNet surveillance areas, where 6371 (90.5%) Salmonella isolates were serotyped, and 10 serotypes accounted for 73.1% of infections. Among the top five serotypes reported were Enteritidis (19.2%); Typhimurium (16.1%), Newport (12.1%), Javiana (8.5%) and Heidelberg (3.6%), even though Salmonella ser. Saintpaul was not in the five top it accounted for 2.5% of infections (CDC, 2010). While in Latin America and the Caribbean, Salmonella ser. Typhi, Salmonella ser. Typhimurium and Salmonella ser. Enteritidis are the serovars more frequently reported (Campos et al., 2012). Information on what Salmonella serotypes are circulating and in what food sources is essential to prioritize food safety interventions and to implement appropriate control measures during food manufacture and preparation (Campos et al., 2012). The presence of potentially pathogenic Salmonella serotypes at the different stages is an evidence of the circulation of pathogens in the food environment. It could increase the risks of infection to consumers when Salmonella survival is allowed under cross contamination and recontamination conditions such as when there are inadequate temperatures during food preparation that allow for the survival of
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the pathogen through undercooking or food handling scenarios when food safety procedures are not followed (Carrasco et al., 2012). 4. Conclusions Comparing the prevalence obtained in the current study with the international data, the prevalence found in this research can be considered to range from low to intermediate. The data demonstrates that the feces and hides of these animals were the major sources of Salmonella beef carcass contamination. The data not only demonstrates the variation in the incoming Salmonella loads on cattle hides and feces, but also illustrates the differences among plants and their abilities to prevent cross contamination between hide and fecal material, during dressing and the evisceration process, as well as the need to implement best practices at the farms and food safety programs such as HACCP/pathogen reduction interventions in these slaughter plants in order to assure a proper control or reduction of pathogen dissemination through the slaughter process and to comply with zero tolerance policy for Salmonella. More research in this area is needed in order to determine if seasonality or other factors affect the Salmonella loads in beef cattle, and thus determine the real impact this pathogen represents to the Venezuelan beef industry. Acknowledgment This research was possible through the support of the CONDES (Consejo de Desarrollo Cientifico y Humanistico de la Universidad del Zulia) and the microbiology laboratory of the Faculty of Veterinary Medicine, University of Zulia. A special thanks to all of the slaughter plants which collaborated and supported the research team during sample collection. References Alper, J., 2003. Date gaps need bridging to assess infectious gastrointestinal diseases. ASM News 69, 65–68. Araque, M., 2009. Nontyphoid Salmonella gastroenteritis in pediatric patients from urban areas in the city of Mérida, Venezuela. Journal of Infection in Developing Countries 3, 28–34. Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2007a. Effects of a minimal hide wash cabinet on the levels and prevalence of Escherichia coli O157:H7 and Salmonella on the hides of beef cattle at slaughter. Journal of Food Protection 70, 1076–1079. Arthur, T.M., Bosilevae, J.M., Brichta-Harhay, D.M., Guerini, M.N., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2007b. Transportation and lairage environment effects on prevalence, numbers and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. Journal of Food Protection 70, 280–286. Arthur, T.M., Bosilevac, J.M., Brichta-Harhay, D.M., Kalchayanand, N., King, D.A., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2008. Source tracking of Escherichia coli O157:H7 and Salmonella contamination in the lairage environment at commercial U.S. beef processing plants and identification of an effective intervention. Journal of Food Protection 71, 1752–1760. Bacon, R.T., Sofos, J.N., Belk, K.E., Hyatt, D.R., Smith, G.C., 2002. Prevalence and antibiotic susceptibility of Salmonella isolated from beef animal hides and carcasses. Journal of Food Protection 65, 284–290. Bell, R.G., 1997. Distribution and sources of microbial contamination on beef carcasses. Journal of Applied Microbiology 82, 292–300. Brichta-Harhay, D.M., Guerini, M.N., Arthur, T.M., Bosilevac, J.M., Kalchayanand, N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., 2008. Salmonella and Escherichia coli O157:H7 contamination on hides and carcasses of cull cattle presented for slaughter in the United States: an evaluation of prevalence and bacterial loads by
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