Effective antimicrobial intervention strategies to reduce Shiga toxin–producing Escherichia coli (STEC) risks associated with veal are needed. This study evaluated the efficacy of lactic acid (4.5%, pH 2.0), Citrilow (pH 1.2), and Beefxide (2.25%, pH 2.3) for reducing STEC surrogates on prerigor and chilled bob veal carcasses and monitored the effects of these interventions on chilled carcass color. Dehided bob veal carcasses were inoculated with a five-strain cocktail of rifampin-resistant, surrogate E. coli bacteria. E. coli surrogates were enumerated after inoculation, after water wash, after prechill carcass antimicrobial spray application, after chilling for 24 h, and after postchill carcass antimicrobial spray application; carcass color was measured throughout the process. A standard carcass water wash (~50°C) reduced the STEC surrogate population by 0.9 log CFU/cm2 (P ≤ 0.05). All three antimicrobial sprays applied to prerigor carcasses delivered an additional ~0.5-log reduction (P ≤ 0.05) of the surrogates. Chilling of carcasses for 24 h reduced (P ≤ 0.05) the surrogate population by an additional ~0.4 log cycles. The postchill application of the antimicrobial sprays provided no further reductions. Carcass L*, a*, and b* color values were not different (P > 0.05) among carcass treatments. Generally, the types and concentrations of the antimicrobial sprays evaluated herein did not negatively impact visual or instrumental color of chilled veal carcasses. This study demonstrates that warm water washing, followed by a prechill spray treatment with a low-pH chemical intervention, can effectively reduce STEC risks associated with veal carcasses; this provides processors a validated control point in slaughter operations.
There are approximately 1,400 veal calf producers in the United States, primarily based in Indiana, Michigan, New York, Ohio, Pennsylvania, and Wisconsin (42). In 2013, it was reported that 725,020 veal calves were slaughtered in the United States, resulting in a market value between $283 million and $366 million (33). There are three different types of commercial veal calves, designated by how they are raised and fed, as well as by the color and texture of the harvested meat. Special-fed veal calves, which are fed a nutritionally balanced diet containing iron and 40 other essential nutrients such as amino acids, carbohydrates, fats, minerals, and vitamins (29), are normally raised until they are 18 to 22 weeks old and weigh around 205 to 225 kg. Meat from special-fed veal calves is usually pink in color, with a firm velvety texture, and represents approximately 85% of the veal consumed in the United States (42). Bob veal calves are initially fed milk from the mother until they are sold to a meat processor for harvesting, which is usually by 3 weeks of age (29). These calves weigh about 68 kg, and their meat is light grayish-pink with a soft texture. They represent 10 to 15% of the veal industry and usually are used for veal sausages, veal patties, and other value-added veal products (42). Grain-fed veal calves are initially fed milk and are then transitioned to grain, hay, and formula. Grain-fed veal calves are usually harvested at 5 to 6 months of age at 205 to 270 kg, and the resulting meat is darker in color and has increased marbling. Grain-fed veal calves represent a comparatively smaller portion of the total veal market in the United States and are primarily raised in southern states (42).
Shiga toxin–producing E. coli (STEC) and Salmonella can be recovered from veal products and have resulted in outbreaks and recalls. In 2011, raw veal liver was recalled in Canada due to possible E. coli O157:H7 contamination. Illnesses were reported in several provinces, leading the Canadian Food Inspection Agency to issue the recall and warnings, but no information was provided regarding the number of illnesses (14, 21). In 2012 in Canada, ground beef and veal were recalled because of possible E. coli O157:H7 contamination; no illnesses were reported (15). In 2013, an outbreak in Canada occurred due to E. coli O157:H7 contamination associated with beef and veal tartare (13). The outbreak resulted in seven illnesses, two hospitalizations, and one case of hemolytic uremic syndrome. A small (350 kg) raw veal recall in Ohio occurred in 2009 due to E. coli O157:H7 contamination, again with no illnesses reported (12). In 2013, there were two Class I veal recalls in the United States associated with STEC contamination. A California firm recalled 572 kg of veal trimmings due to possible E. coli O157:H7 contamination (30), and a New York firm recalled 5,715 kg of boneless veal product due to possible E. coli O157:H7 and non-O157 contamination (31). Two more Class I recalls occurred in 2015 in the United States, associated with veal products contaminated with STEC. In the first, 1,134 kg of boneless veal trim products was recalled due to possible E. coli O157:H7 contamination (39). The second involved recalling 17 months of veal trimmings due to possible E. coli O157:H7 and non-O157 STEC contamination (40). None of the Class I recalls that occurred in the United States between 2013 and 2015 resulted in any reported illnesses.
STEC bacteria are estimated to cause 265,000 illnesses, 3,600 hospitalizations, and 30 deaths annually in the United States (8). For the past 20 years, since it was declared an adulterant in ground beef, E. coli O157:H7 has been the main STEC serotype focused upon by processors, researchers, and regulatory agencies (27). This focus on E. coli O157:H7 is due to the number of disease outbreaks linked to this serotype, its strong association with hemolytic uremic syndrome and thrombotic thrombocytopenic purpura (3, 17), and its negative impact on the beef industry due to the resultant recalls. Six other STEC serotypes (O26, O45, O103, O111, O121, and O145) have emerged as causes of similar foodborne illnesses both domestically and abroad (3). These six non-O157 STEC serotypes cause 20 to 50% of all STEC infections in the United States (16), and they have been declared to be adulterants in ground and nonintact raw beef, including veal (28).
The U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) has found a higher risk associated with veal-derived raw ground beef components (RGBC) compared to beef RGBC. In 2012, FSIS published data showing beef and veal RGBC contamination prevalence for all seven regulated serotypes of STEC in raw ground and nonintact beef products. These data established a lower STEC recovery rate in beef RGBC (41 of 4,901, 0.84%) compared to veal RGBC (25 of 311, 8.04%) (34); and the trend continued in 2013, with a lower STEC recovery rate in beef RGBC (27 of 6,162, 0.44%) than in veal RGBC (12 of 244, 4.92%) (35). USDA-FSIS has also implemented a nationwide beef and veal carcass microbiological baseline data collection program to estimate STEC presence and levels in raw beef and veal (32). Data made available from the first 6 months of this program further substantiate that veal carcasses pose a greater risk of non-O157 STEC contamination compared to beef carcasses. These results showed 27.08% of veal carcasses to be positive for non-O157 STEC after hide removal, compared to 4.52% of beef carcasses (36). With the caveat that far fewer veal carcasses have been tested, collectively, the results to date for both RGBC and carcasses indicate that it is much more likely for STEC to be recovered from veal than from beef samples.
In general, abattoirs that process veal cattle or beef cattle utilize similar practices; however, inherent differences in the size, age, immune status, and husbandry practices for these two types of cattle most likely contribute to differences in the types and levels of indigenous microbes found both in and on these animals at the time of harvest. Thus, it may be necessary to develop new interventions and/or to repurpose interventions proven effective for beef cattle to lower the prevalence and concentration of STEC on veal carcasses. Although commercial veal processors are implementing carcass intervention strategies to varying degrees to mitigate these STEC issues, little research has been reported to validate common antimicrobial technologies approved for use in beef processing for their effectiveness in decontaminating veal carcasses. The objectives of this study were to validate the efficacy of three common USDA-FSIS approved antimicrobial sprays (i.e., lactic acid, Beefxide, and Citrilow) for reducing populations of nonpathogenic STEC surrogates on both prerigor and chilled bob veal carcasses and to monitor for any associated veal carcass color changes throughout the slaughter and chilling processes.
MATERIALS AND METHODS
Design of study.
Bob veal calves (≤3 weeks of age and ~68 kg) were obtained from a local dairy producer and were transported by truck to the Kansas State University Meat Laboratory abattoir holding unit, under a protocol approved by the Kansas State University Institutional Animal Care and Use Committee. Each calf was slaughtered within 6 h using USDA-approved practices, and carcasses were immediately used for research purposes. This study was repeated on three different days (i.e., replications) using four animals per day, for a total of 12 animals. For each of the three replications, a fresh STEC surrogate inoculum cocktail and fresh antimicrobial solutions were prepared.
Five strains of nonpathogenic, surrogate E. coli approved for in-plant use by the USDA (38) were obtained from the American Type Culture Collection (ATCC accession numbers BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431, ATCC, Manassas, VA) and were transformed to demonstrate rifampin resistance (18). All strains were grown separately in 10 ml of tryptic soy broth (Difco, BD, Sparks, MD) with 0.1 mg/ml rifampin added (Fisher Scientific, Fair Lawn, NJ) for 24 h at 37°C. Following incubation, the entire contents of each tube were combined to form a mixed inoculum containing approximately equal numbers of each of the five strains, from which 1 ml was placed into 1,000 ml of sterile 0.1% peptone (Bacto, BD, Sparks, MD) water immediately prior to use as the mixed inoculum solution (containing 6.15 ± 0.11 CFU/ml).
The five-strain surrogate cocktail was applied evenly to the exterior of the entire carcass using a 7.62-cm foam paintbrush (Jen Manufacturing, Inc., Millbury, MA), achieving a target concentration of 3.0 ± 0.2 log CFU/cm2. Foam brush inoculation has been shown to adequately deliver a desired and consistent inoculum level to meat products (11, 20, 22) and presumably would work equally well for inoculating veal carcass surfaces. The veal carcass inoculation procedure was performed immediately after USDA zero-tolerance trimming, and inoculated carcasses were left undisturbed on the slaughter line for 30 min at ~25°C to allow for inoculum attachment before proceeding with the different experimental treatments.
Solutions of 4.5% L-lactic acid (pH 2.0; Birko Corporation, Henderson, CO), 2.25% Beefxide (pH 2.3; Birko Corporation), and Citrilow (pH 1.2; Safe Foods Corporation, North Little Rock, AR) were prepared according to manufacturers' recommendations. Lactic acid 88% is a concentrated mixture of lactic acid in water. Beefxide is a proprietary mixture of lactic and citric acids in water. Citrilow is a proprietary mixture of citric acid and hydrochloric acid in water. Each chemical was mixed with ambient temperature tap water to achieve targeted concentrations that comply with USDA-FSIS Directive 7120 (37). Lactic acid and Beefxide concentrations were confirmed by titrating 10 ml of the antimicrobial solution with 0.1 N NaOH (Fisher Scientific) using 1% phenolphthalein (Fisher Scientific) as an indicator; and Citrilow potency was confirmed by pH (Oakton Instruments, Vernon Hills, IL) before veal carcass treatment. Fresh antimicrobial solutions were prepared for each experimental replication, and a fresh solution was prepared within each replication for the treatment after 24 h of chilling.
Veal carcass treatment.
Each replication consisted of four bob veal calves that received one of four treatments: control (no antimicrobial treatment), lactic acid, Beefxide, or Citrilow. Following the 30-min inoculum attachment period, each veal carcass was subjected to a standard water spray wash (~50°C for 1 min) to remove bone dust and residual blood spots using a handheld hose with spray nozzle (70 lb/in2, 26.5 liter/min, narrow spray setting; M-70 nozzle, Strahman Valves, Inc., Bethlehem, PA). After water was allowed to drip from the carcasses for 5 min, the control carcass was moved to the carcass cooler, and the other three carcasses were treated with an assigned antimicrobial spray. Antimicrobial sprays were applied (~326 ml per carcass; ~20°C) using an 11.4-liter poly pump sprayer (i.e., hand-pump garden sprayer, Chapin International, Inc., Batavia, NY), and carcasses were allowed to drip for 5 min at abattoir room temperature (~25°C) before being placed in the −2°C cooler for 24 h. Following the 24-h chill, an additional spray of the same assigned chemical, under the abovementioned conditions, was applied to each chilled veal carcass.
Surrogate E. coli bacterial populations on each carcass were determined by sampling at five points during processing: after inoculation with a 30-min attachment period, after the standard water wash, after the prechill carcass antimicrobial spray application, after 24 h of chilling, and after the postchill carcass antimicrobial spray application. Five anatomical locations were delineated and randomly assigned to treatments for sampling both sides of each veal carcass. Two Whirl-Pak sponges (Nasco, Fort Atkinson, WI), one per carcass side, were used to swab 100 cm2 of the matched anatomical locations at each of the different sampling points. Matched anatomical locations for each sampling point on the paired bob veal carcass sides can be seen in Figure 1. Both sponges were combined into a single stomacher bag (Fisher Scientific) containing 40 ml of Dey-Engley neutralizing broth (BD, Franklin Lakes, NJ) with 0.1 mg/ml rifampin added and then were mixed by hand massaging for 1 min. Samples were serially diluted using 0.1% peptone water containing 0.1 mg/ml rifampin, and dilutions were plated in duplicate onto APC Petrifilm (3M, Saint Paul, MN). Petrifilm plates were incubated at 37°C for 24 h and were counted per manufacturer's instructions.
Carcass color was measured using a HunterLab MiniScan XE Plus spectrophotometer (15-mm-diameter aperture, 10° standard observer, illuminant A; model 45/0 LAV, Hunter Associated Laboratories Inc., Reston, VA). The Hunter MiniScan was placed below the hipbone on a smooth surface of the veal carcass that was not sampled for microbial populations (Fig. 1). Two readings were taken on both sides of the carcass and averaged (n = 4) for L*, a*, and b* values. Veal carcass color was measured at five different processing points: pretreatment (immediately prior to application of inoculum), after prechill antimicrobial spray treatment, after a 1-h chill, after a 24-h chill, and after the antimicrobial spray application done after the 24-h chill.
The experiment consisted of three replications, each using four bob veal calves. The microbial counts were transformed into log CFU per square centimeter format prior to statistical analyses. Microbial counts and color values were analyzed using SAS version 9.4 (SAS Institute Inc., Cary, NC); this was a repeated measurement analysis with the repeated measurements over treatments at a significance of P ≤ 0.05.
RESULTS AND DISCUSSION
Antimicrobial effectiveness of chemical sprays.
Surrogate E. coli levels at each sampling point are provided in Table 1 for each of the four treatments. The standard water wash (~50°C) provided an effective primary reduction step for controlling the surrogate STEC populations, reducing levels by an average of 0.9 log CFU/cm2. Little research has been reported regarding the antimicrobial impact of ambient or warm water washes on meat animal carcasses. Castillo et al. (4, 5) found that a 35°C water wash could reduce E. coli O157:H7 counts by ~2.2 log CFU/cm2 on beef carcass tissues. The higher reduction observed by Castillo et al. (4, 5) may be due to differences in how the meat was washed. The standard water wash in the current study sprayed the entire veal carcass for 1 min using a water hose and hand nozzle at 70 lb/in2, whereas Castillo et al. (4, 5) applied a 1.5-liter hand spray wash (90 s at 10 lb/in2) followed by a 5-liter automated cabinet wash (9 s at 250 to 400 lb/in2) on individual sections of the carcass (inside round, outside round, brisket, flank, and clod). The water wash in this study was applied over a shorter time and was not focused on individual pieces of meat. Differences in experimental design and/or experimental methods that are not easily comparable (e.g., Castillo et al. (4, 5) utilized a 2-log CFU/cm2 higher meat inoculation level) could also have led to the greater observed reductions in E. coli O157:H7 populations. Phebus et al. (24) also evaluated a 35°C water wash applied at 38 to 40 lb/in2 for 23 s onto individual beef carcass surfaces that were hanging in a cabinet. This water wash was more comparable to the one evaluated in the current study and resulted in a similar E. coli O157:H7 reduction (0.8- and 0.9-log CFU/cm2 reductions, respectively).
The three prerigor chemical sprays applied after the water wash resulted in additional surrogate E. coli population reductions of 0.6, 0.5, and 0.4 log CFU/cm2 for lactic acid, Beefxide, and Citrilow, respectively. No differences (P > 0.05) were observed among the three antimicrobial sprays, but all were different (P < 0.05) from the nontreated control. Studies have shown similar efficacy of lactic acid when applied to beef carcasses and beef subprimals (6, 7, 25, 43). Castillo et al. (6) determined the efficacy of a 4% lactic acid spray on beef carcasses. Their in-plant study resulted in a 3-log reduction of aerobic bacteria and undetectable levels (1.4-log CFU/cm2 detection limit) of E. coli and coliforms. In a related study, Castillo et al. (7) achieved a 2.4-log reduction in E. coli O157:H7 numbers using a 4% lactic acid spray on chilled outside rounds that had previously been treated with a prerigor water wash. This reduction was larger than the one observed in the present study and could be attributed to the method in which the lactic acid was delivered and to differences in experimental design. Particularly relevant in the study by Castillo et al. (7) is that the 4.0% lactic acid spray was applied at 55°C compared to ~20°C, as in the current study. In the present study, ~326 ml of 4.5% lactic acid was sprayed across the entire veal carcass, whereas Castillo et al. (7) sprayed 500 ml on a smaller portion of the carcass. Pittman et al. (25) showed the efficacy of a 5% lactic acid spray on chilled beef subprimals, resulting in a 2.4-log reduction of surrogate E. coli. The greater reduction could be attributed to differences in the volume and application parameters of lactic acid used to treat each subprimal and to their use of a custom-built spray cabinet. However, Wolf et al. (43) reported similar results to the ones found in the present study, achieving a 0.5-log reduction of E. coli O157:H7 on chilled beef trim treated with a 4.4% lactic acid solution.
Studies have evaluated the efficacy of Beefxide on beef cheek meat (26) and beef bottom sirloin butts (19). Schmidt et al. (26) reported a 1.0-log reduction of E. coli O157:H7 and a 1.2-log reduction of non-O157 populations when prerigor beef cheek meat was submerged (1.5 liters of antimicrobial poured over cheek meat in a sterile beaker) in a 2.25% Beefxide solution for 1 min. Laury et al. (19) reported similar results, with a 1.4-log reduction of E. coli O157:H7 counts, when prerigor beef bottom sirloin butts were treated with a 2.25% Beefxide solution in a spray cabinet. Both studies started with similar initial bacterial loads, and both studies observed greater targeted microbial reductions than the present study. This could be attributed to the method of treating beef surfaces with Beefxide. Schmidt et al. (26) completely submerged the cheek meat for 1 min, whereas Laury et al. (19) sprayed the beef bottom sirloin butts in a cabinet at a rate of 1 ft/2.5 s (0.3048 m/2.5 s) at 40 lb/in2. Both studies treated a small portion of the carcass with high volumes of chemical antimicrobial at longer exposure times.
The efficacy of Citrilow was also evaluated for microbial control on chilled beef subprimals (2, 9). Beers et al. (2) showed a 1.8-log reduction of aerobic bacteria on sections of chilled beef brisket subprimals treated with a Citrilow solution at pH 1.5. Cook et al. (9) reported a 1.1-log reduction of E. coli O157:H7 on chilled beef subprimals treated with Citrilow solution at pH 1.3, a reduction substantially higher than that observed in the present study. This could be due to the way the meat was treated with Citrilow, along with other notable experimental design differences. Cook et al. (9) delivered approximately 2.6 ml (5 s spray at a rate of 1.9 liter/h) onto a piece of meat (12.7 by 12.7 cm). This application would likely deliver a greater volume of Citrilow per cm2 onto the subprimal surface compared to the present study, in which only ~326 ml was sprayed onto the surface of an entire veal carcass.
Treating the veal carcasses after 24 h of chilling with a second application of the same designated chemical spray provided no additional STEC surrogate population reductions. Similar results were found by Acuff et al. (1) and Dixon et al. (10), showing little efficacy of lactic acid on chilled beef subprimals; however, these studies evaluated the effect of the antimicrobial on total bacterial numbers, not on STEC or USDA-approved STEC surrogates. Pittman et al. (25) and Castillo et al. (7) showed notable reductions (~2.4 log CFU/cm2) of E. coli O157:H7 on chilled beef subprimals treated with 5 and 4% lactic acid solutions, respectively. The lack of surrogate microbial reductions observed in the current study, when spraying antimicrobials on chilled beef surfaces, could be due to the population level of E. coli available to be treated and/or due to differences in experimental design, methods of inoculation, and application of treatments. In the current study, there was only ~1.5 log CFU/cm2 of surrogate E. coli on the chilled veal carcasses before the second antimicrobial treatment, which is much lower than the ~4.0-log CFU/cm2 inoculation level that Pittman et al. (25) and Castillo et al. (7) treated with lactic acid. Differences in initial E. coli population levels may not be the only contributing factor leading to the higher reductions observed by Pittman et al. (25) and Castillo et al. (7) on chilled beef tissue; however, the ~1.5 log CFU/cm2 initial population level at the time of the second antimicrobial spray application in the current study was considerably lower, which possibly limited demonstration of a larger reduction capability of the antimicrobial spray at the chilled carcass level. Further research should be conducted to evaluate the efficacy of chemical antimicrobials against STEC on chilled beef and veal carcasses with higher initial E. coli populations.
Carcass color impact of antimicrobial treatments.
The Hunter MiniScan produces color readings based on L*, a*, and b* values. L* measures lightness to darkness on a scale of 0 to 100; 100 is perfect white and 0 is black. The a* value measures redness when positive, gray when zero, and greenness when negative. The b* measures yellowness when positive, gray when zero, and blueness when negative. Carcass L*, a*, and b*values were not different (P > 0.05) among the four treatments (no-treatment control, 4.5% L-lactic acid, 2.25% Beefxide, and pH 1.2 Citrilow). There were significant differences (P ≤ 0.05) across the sampling times throughout the veal slaughter process. Mean L*, a*, and b* results were combined from each treatment and can be seen in Table 2. Each of the treatments caused similar color fluctuations through the five sampling points, becoming lighter immediately after prechill antimicrobial spray treatment and then darker throughout the chilling process. Following the 24-h chilling period, all carcass L* values were similar to the pretreatment meat and were not different (P > 0.05) from each other. The a* values were not different among any of the treatments; the carcass became less red immediately after prechill antimicrobial treatment but then increased in redness following the 24-h chill period. The b* values were not different among any of the treatments; the carcasses became less yellow after prechill antimicrobial treatments, but then yellowness increased following the 24-h chill period. L*, a*, and b* values did not change significantly between the 24-h chill sampling and the second antimicrobial spray applied to chilled carcasses. It was determined that none of the antimicrobial sprays had a negative impact on the veal carcass color compared to the no-treatment control and that the only factors significantly (P ≤ 0.05) affecting carcass color in this experiment were the different stages throughout the slaughter process.
In summary, the results from the present carcass-level study can be difficult to compare to the supporting literature, which predominately addressed chilled beef subprimal applications. There are also few published data validating intervention strategies specifically for the veal industry. Two studies evaluated interventions for veal but do not directly apply to this research. Wang et al. (41) evaluated in-plant interventions on veal calf hides (because hide-on carcass chilling has been occasionally practiced in veal slaughter), whereas Penney et al. (23) evaluated peroxyacetic acid in reducing E. coli on hot-boned beef and veal. With the rising concern related to STEC prevalence associated with raw and processed veal products, more research must be done to validate different antimicrobial intervention strategies. The present study showed that a standard water wash (~50°C) provides an effective primary reduction step for controlling E. coli surrogates (and STEC by inference) in veal processing. The application of an organic acid antimicrobial spray (4.5% lactic acid, 2.25% Beefxide, and/or pH 1.2 Citrilow) to dehided, prerigor carcasses following a water wash can serve as an effective step to control STEC contamination, with a 1.4-log CFU/cm2 population reduction being achieved by the combination of both processes. This study also indicated that a second acid-based antimicrobial spray onto chilled veal surfaces did not provide any additional benefit in controlling STEC contamination. Further research should be done to identify and validate the effectiveness of various antimicrobial sprays or other forms of intervention on chilled veal carcasses and fabricated products.
This is contribution no. 16-094-J from the Kansas Agricultural Experiment Station (Manhattan, KS). This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award no. 2012-68003-30155. Special thanks to Mr. John Wolf (Kansas State University Meat Abattoir Manager), the entire Kansas State University meat laboratory group, and Drs. Ron Pope and Luis Mendonca for their help in the execution of this project. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA or other participating academic institutions.
† Present address: Department of Poultry Science, University of Georgia, Athens, GA 30602, USA.