Introduction: After more than 20 years of exploration and practice, Phytobiotics GmbH of Germany, as the global patent owner of Boluohui extract , has become the leader in the application of Boluohui extract in animal production. Added to more than 2.5% of the world's feed, endorsed by world-renowned manufacturers.
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Valeria Artuso-Ponte,1* Steven Moeller,2 Paivi Rajala-Schultz,1 Julius J. Medardus,1 Janet Munyalo,1 Kelvin Lim,1 and Wondwossen A. Gebreyes1
1, Department of Veterinary Preventive Medicine, The Ohio State University College of Veterinary Medicine, Ohio Department of Animal Sciences, College of Food, Agricultural and Environmental Sciences, The Ohio State University, Columbus, Ohio, United States.
2, Ohio State University Department of Animal Sciences, College of Food, Agricultural and Environmental Sciences, Columbus, OH, United States.
*任仹平台, Bavaria , Wallover Street 10A 65343 Wortweiler, Germany.
Abstract
This study aimed to evaluate the effect of herbal extract additives, specifically benzophenanthridine alkaloids (QBA), which have previously been shown to have anti-inflammatory, antibacterial and immunomodulatory effects. We verified the effect of QBA on stress secretion and Salmonella infection during the preparation of pigs for transport to the slaughterhouse. A total of 82 pigs were gavaged with Salmonella mixed media containing Salmonella Meleagridis, Hartford, Bo-vismorbificans and Newport serovars ( Day 0), and were randomly assigned to three treatment groups 2 weeks later (Day [D] 14): T1, feed supplemented with QBA; T2, feed and water-soluble QBA; CON, control, no challenge). Challenge Pigs were transported to the slaughterhouse after poisoning (D 28) and slaughtered nearly 19 hours later (D 29). Saliva, fecal samples and carcass swabs were collected from all pigs. Only the CON control group showed post-transport salivary cortisol increased (5.48 ng/mL; p 0.0001), compared to higher concentrations than T1 (2.73 ng/mL; p = 0.0002) and T2 (1.88ng/mL; p 0.0001). In QBA-supplemented pigs, post-transport Salmonella The prevalence and infection rate decreased (p 0.05), while the control group showed a significant increase in Salmonella infection rate after transport (p = 0.04). On D 28, pigs in T2 excreted lower numbers of Salmonella than T1 (1.3E + 02 CFU/mL vs. 8E + 03 CFU/mL; p = 0.002). Furthermore, carcass contamination with Salmonella was higher in the CON control group than in the treatment group (p = 0.01). Research results show that the addition of QBA can effectively reduce transportation stress in pigs, thereby reducing salmonella infections and having a positive impact on animal welfare and pork safety.
01
Introduction
Salmonellosis is the foodborne bacterial disease with the highest rates of hospitalization and mortality in the United States, with annual treatment-related costs of up to $2.5 billion (Hoelzer, 2011). In addition, the number of people infected with Salmonella after eating contaminated pork accounts for 1% of the total Salmonella infection cases (Guo, 2011). Pork can be contaminated with Salmonella at any point in the food chain; however, contamination before and after entry into the slaughterhouse is considered a major factor in pig carcass contamination (Davies, 2011). In pigs, salmonellosis is usually subclinical, with infected pigs shedding Salmonella in their feces intermittently over long periods of time (Kranker, 2003). Transport-induced stress has been shown to increase the chance of Salmonella infection, even in people with subclinical infection of at the farm stage, thus posing a food safety risk (Larsen, 2003; Verbrugghe, 2011). Antimicrobial strategies using antibiotics have been widely adopted and antibiotics reduce the incidence of foodborne pathogens at the farm level (Doyle and Erickson, 2012; Looft et al., 2012). However, their widespread use has been directly linked to the emergence and spread of -resistant bacteria (Oliver, 2009). The use of herbal extracts, such as sanguinarine and cleidouracil, has been proposed as a good alternative to the use of in-feed antibiotics to enhance growth without causing the development of antibiotic resistance (Yakhkeshi, 2011; Robbins , 2013).These compounds are benzo(c)phenanthridine alkaloids (QBA) and the alkaloid active constituents of several plants, including Macleaya cordata extract, which have been shown to have anti-inflammatory, antibacterial and immunomodulatory effects (Lenfeld, 1981; Colombo and Bosisio, 1996). Furthermore, QBA can inhibit aromatic l-amino acid decarboxylase, which catalyzes the decarboxylation of the aromatic amino acid into its biogenic amines; therefore, the availability of aromatic amino acids such as tryptophan is increased (Drsata, 1996). Tryptophan is an essential amino acid . Less than 5% of total tryptophan is metabolized via the methoxyindole pathway to synthesize the neutrophil transmitter serotonin, which is known to enhance the ability of animals to adapt to stress conditions. . Therefore, countries that have approved the use of QBA include QBA in pig and poultry diets to improve the utilization of amino acid and promote growth (Vieira, 2008; Yakhkeshi, 2011). Furthermore, our research group previously reported that the addition of QBA reduced Salmonella infection and improved intestinal permeability during the brooding stage of pigs (Robbins, 2013). However, the effect of QBA additives on the stress response of fattening pigs during transportation and its correlation with pork safety have not been extensively studied.
The purpose of this study was to (1) evaluate the effect of QBA additive on salivary cortisol monitoring; (2) determine the correlation between cortisol levels and Salmonella infection; (3) evaluate the inhibitory effect of QBA additive on Salmonella infection and carcass contamination.
02
Methods
2.1 Animals and Facilities
A total of 82 pigs (initial weight: 47.9 – 7.2 kg) were selected for a randomized controlled antibacterial study. The animals were grouped according to litter, breed and sex and assigned to nine pens. Each treatment group was allocated three adjacent pens, separated from other treatment groups by two empty pens and a solid physical barrier to prevent direct contact between pigs in different treatment groups. The pigs are housed in a partially slatted barn at Ohio State University's Ohio Agricultural Research and Development Center's Western Agricultural Research Station. Provide feed and water ad libitum. Implement a biosecurity control plan to minimize human exposure to Salmonella and prevent its spread to other animals and facilities on farms and in slaughterhouses. All procedures described below were approved by the Ohio State University Institutional Animal Care and Use Committee.
2.2 Salmonella challenge
On day 0 (D 0), all pigs received 15 ml of bacterial culture medium, each injection containing a mixture of multiple Salmonella broths from 2 weeks before the start of the study. Isolated from fecal samples collected from research pigs on the same farm (Bager and Peterson, 1991). According to the Clinical Laboratory Standas Institute (CLSI), resident Salmonella culture media include Bovis-morbificans, Newport, Hartford, and Meleagridis. All isolates were resistant to 12 antibiotics: ampicillin (10 lg), amoxicillin-clavulanic acid (30 lg), amikacin (30 lg), ceftriaxone (30 lg), cephalothin (30 lg), chloramphenicol (30 lg), ciprofloxacin (5 lg), gentamicin (10 lg), kanamycin (30 lg), chain Mycomycin (10 lg), sulfisoxazole (250 lg) and tet-racycline (30 lg), 2012). uses resident Salmonella to challenge viruses in order to better simulate real epidemiological scenarios in commercial farms. Cultured strains of Salmonella have been identified as being of low pathogenicity and causing mild and transient symptoms of illness (Wood, 1991; Vigo, 2009; Ngoc et al., 2013; Jackson, 2013); therefore, All pigs received a booster dose (15 ml) 9 days after (D 9) to ensure colonization of colonies with . Both initial and challenge booster doses were prepared in Luria-Bertani broth and contained approximately 108 colony units (CFU)/mL (McFarland scale of 0.5).
2.3 Benzophenanthridine alkaloid (QBA) treatment
QBA treatment consists of feed or water additives containing botanical ingredients and natural extracts of Macleaya cordata (Sangrovit®S and Sangrovit® WS; Phytobiotics Germany GmbH, Ertewil ,Germany). Fourteen days after the initial gavage challenge (D 14), treatment and control groups were randomly assigned to group pens. Pigs in treatment T1 (T1; n = 27) received QBA (equivalent to commercial QBA® S) mixed with 450 g/ton of feed for 2 weeks (Shen, 2012a). Pigs in treatment T2 (T2; n = 27) received QBA (equivalent to commercial QBA® S) at 450 g/ton feed for 2 weeks together with water-soluble QBA (equivalent to QBA® WS) 300g/1000L added to drinking water to enhance the effect of QBA in feed. Pigs in the control group were not supplemented with any QBA product (CON control group; n = 28).
2.4 Transportation to Slaughterhouse
On day 28 (D 28), all pigs were transported to the Ohio State University Meat Laboratory slaughterhouse. In the trailers, physical barriers were set up to prevent direct contact between pigs from different treatment groups. After a transport period of approximately 45-50 minutes, all pigs are unloaded into slaughter pens and slaughtered approximately 19 hours later (D 29). Pigs from different treatment groups are not allowed to have direct (nose-to-nose) contact while awaiting slaughter, and treatment groups are alternated during loading and slaughtering to avoid potential cross-infection of and due to differences in loading and onset times, which may Affect the stress state of pigs.
2.5 Sample collection and processing
Saliva collection from all pigs was performed using cotton swabs (Salivette® Cortisol, Sarstedt AG & Co., Germany) at D 0, D 14, D 27 (before transport) and D28 (after transport) collect. To avoid confounding due to the circadian rhythmicity of cortisol , all samples were collected between 1 PM and 3 PM, alternating the order of treatment groups. Two weeks before the start of the study, all pigs were introduced and trained with cotton swabs to minimize stress due to the collection procedure, which may affect the pig's stress profile and thus salivary cortisol. Let the pig chew the cotton for 60 seconds or until the cotton is completely saturated with saliva. The swab is then placed inside the collection tube and transported on ice to the laboratory for further processing. Upon sample arrival, all samples were incubated at 1500 g for 10 min at 4°C to separate saliva from the swabs (Shen, 2012b). Finally, the saliva was stored at -80°C until further processing. A total of 310 saliva samples (T1, n = 107; T2, n = 104; CON control group, n = 99) were analyzed in duplicate using commercially available enzyme immunoassay kits (Salimetrics LLC, State College, PA) Determination of sali-changing cortisol concentrations. The intraclass coefficient of variation (CV) for each sample was calculated as: standard deviation /mean salivary cortisol (ng/mL)*100. Then, calculate the average CV% for the 310 samples. Inter-assay CV was calculated using Standard deviation/mean salivary cortisol (ng/mL)*100 from known low and high salivary cortisol controls included in each plate as well as standards and unknown samples. Based on this method, the intra-assay and inter-assay coefficients of variation of and were 6% and 2.9%, respectively. Fresh fecal material was aseptically collected from the rectum of all pigs on D 0, D 3, D 14, D 21, D 27, and D 28 and placed individually into sterile bags (Nasco Whirl-Pak® Easy-Close; Wisconsin Fort Atkinson) were kept refrigerated until processing in the laboratory. A total of 384 stool samples (T1, n = 129; T2, n = 130; CON control group, n = 125) and genomic DNA were collected using the QIAamp® Fast DNA Stool Mini Kit (, Qiagen, Valencia, CA) extraction, and the purified DNA was stored at 4°C until further analysis.
Carcass swabs were collected from all carcasses (internal and external surfaces) using a sterile sponge (Nasco Whirl-Pak® Speci-Sponge®) after evisceration and before final washing and cooling at slaughter (D 29) and washed with 10 mL Buffered peptone water pre-moistened. Sponges were kept on ice until processed in the laboratory.To extract genomic DNA from carcass swabs, a total of 72 sponges (T1, n = 25; T2, n = 23; CON control group, n = 24) were placed in an incubator with 50 mL of 0.02% Tween 20 (Sigma, St. Louis, MO) solution in a single sterile container and stir in a shaker at 37°C for 30 min. Subsequently, 15 mL of liquid was poured into sterile 15-mL Falcon sample tubes (BD Falcon®; Franklin Lakes, NJ) and centrifuged at 1500 g for 10 min to obtain pellets (Guy, 2006). Finally, DNA was extracted using the DNeasy® Blood and Tissue Kit (Qiagen) following the instructions of the manufacturing method, and the DNA was stored at 4°C until further analysis.
All fecal samples and carcass swabs were analyzed in triplicate sections to quantify Salmonella by performing a quantitative real-time polymerase chain reaction assay (qPCR) on an Mx3005P machine (Agilent Technologies, Santa Clara, CA). The primer set was chosen to amplify a 119 base pair fragment of the invA gene as previously described (forward: 5'-TCGTCATTCCATTACCTACC-3' and reverse: 5'-AAACGTTGAAAACTGAGGA-3'; Hoorfar, 2000) . Thermal cycling conditions were as follows: 1 cycle at 95°C for 15 min (hot start), followed by 55 cycles at 95°C for 15 s (denaturation), 55°C for 15 s (annealing), and 72°C for 30 s. (extension) and one cycle at 75°C for 15 s (additional data acquisition step), and 1 cycle at 72°C for 5 min (final extension). Standard curves were constructed by plotting known CFU/mL of Salmonella Enteritidis ATCC 13076 against threshold values (Ct) as previously described, and linear regression analysis was performed using MxPro® QPCR software (Agilent Technologies) to quantify Salmonella The amount (CFU/g or CFU/mL).
03
Statistical analysis
From the 82 pigs initially enrolled in the study, 6 pigs (T1, n = 1; T2, n = 2; CON control group, n = 3) were removed because they were removed before the end of the study period. died or received antibiotic treatment. For all continuous data, SAS' univariate procedures were used (SAS 9.4; Cary, NC) prior to statistical analysis. The mean procedure of SAS (SAS 9.4) is used to calculate the mean and the standard error of the mean. Salivary cortisol was analyzed using the Mixed procedure of SAS (SAS 9.4). Concentrations of salivary cortisol over time were analyzed as repeated measurements. Pigs and pig pens were treated as random effects. The final model included treatment effects, sample collection day, their interaction, and sex. For Salmonella detection, non-parametric testing was performed using SAS software (SAS 9.4). The Kruskall-Wallis test was performed to analyze Salmonella detection at D0, D 3, D 14, D 21, D 27, D 28 and carcass contamination between treatment groups. In addition, the Wilcoxon rank sum (Mann-Whitney) test was performed to perform pairwise comparisons between treatment groups. Pairwise comparisons were also performed within treatment groups and used the Wilcoxon signed rank test. Logistic regression analysis was performed using the GLIMMIX procedure of SAS (SAS 9.4) to test for differences in Salmonella prevalence between and within treatments. Pigs and pig pens were treated as random effects. The final model included treatment effects, sample collection day, and their interaction. Due to convergence, Fisher's exact test was performed to test for significant differences in Salmonella prevalence between D 27 and D 28 treatment groups and between D 3 and D 28 treatment groups. The prevalence of Salmonella in carcass swabs was analyzed by performing Fisher's exact test. For all analyses, a p value of 0.05 was considered significant, and Tukey-Kramer tests were performed to adjust for multiple comparators. Spearman rank correlation coefficients (rs) were calculated to determine the association between salivary cortisol and Salmonella detection at D28, and the magnitude of the correlation coefficient was interpreted as previously described (Mukaka, 2012).
04
Results
Spearman Rank correlation coefficient showed a highly significant positive correlation between salivary cortisol and post-transport detection of Salmonella in all groups, with the highest rs in the CON control group (Fig. 1; rs = 0.93, p 0.001), followed by T1 (rs = 0.85, p = 0.0002) and T2 (rs = 0.82, p = 0.0006).
4.1 Salivary cortisol
Overall, the mean wrinkle-reducing concentration of salivary cortisol was significant in all groups from D 0 to D 27 (Fig. 2; p0.0001). After transport to the slaughterhouse (D 28), only the CON control group had a significant increase in salivary cortisol with D 27 (1.87 ng/mL to 5.48 ng/mL, p 0.0001). Furthermore, mean salivary cortisol in CON control group D 28 was significantly higher than T1 (5.48 ng/mL vs 2.73 ng/mL, p = 0.0002) and T2 (5.48 ng/mL vs 1.88 ng/mL, p 0.0001).
Figure 1. Comparison of average cortisol in each treatment group. Correlation between Salmonella detection (colony forming units [CFU]/g feces) and salivary cortisol centers (ng cortisol/mL saliva) between treatment and control groups. CON control group (control with basal diet); T1 (diet benzophenanthrene alkaloid [QBA]) and T2 (diet + water-soluble QBA).
4.2 Salmonella stool detection
The prevalence of Salmonella reached the range of 71.4% to 100%, and there was no difference between the treatment groups of D0, D 3, D 14, D 21 and D 27 (p 0.05), indicating that the challenge was effective. and Salmonella was consistently detected throughout the study period (data not shown). On D 28, Salmonella prophase was significantly higher in CON control (100%) pigs compared to T1 (73.7%, p = 0.02) and T2 (71.4%, p = 0.02). Furthermore, the proportion of Salmonella-positive pigs after transport decreased significantly in T2 compared to D 27 (95.5% to 71.4%, p = 0.05).
Significant differences in Salmonella detection were detected in the groups D 27 (Fig. 3; p = 0.0002) and D 28 (p = 0.01). On D 27 (before transport stress), Salmonella detections were lower in the CON control group compared to pigs receiving QBA in feed (p 0.0001) or feed and water-soluble QBA (p = 0.001). On D 28 (after transport stress treatment), pigs in T2 shed significantly lower numbers of Salmonella compared to T1 (1.3E+02 CFU/g vs. 8E+03 CFU/g, p=0.002). Meaning that when QBA is added to drinking water, it may be due to higher bioavailability (del Castillo, 1998). However, the difference between the T2 group and CON control group was not statistically significant (1.3E+02 CFU/g vs. 5.9E+02 CFU/g, p=0.08). Additionally, CON control pigs showed a significant increase in Salmonella detection after transport (6E + 01 CFU/g to 5.9E + 02 CFU/g, p = 0.04). In contrast, pigs in T2 showed a significant reduction in Salmonella detection after transfer compared to pre-transfer levels (3.8E + 02 CFU/g to 1.3E + 02 CFU/g, p = 0.03) .
4.3 Salmonella carcass detection
Salmonella was detected in 37.5% (27/72) of all carcasses. There were no significant differences in Salmonella prevalence between any of the treatment groups: QBA (T1 and T2 combined) vs. CON (p = 0.32), T1 vs. CON (p = 0.07), T2 vs. CON (p = 1 ) or T1 versus T2 (p = 0.07). However, the number of Salmonella in contaminated carcasses varied significantly between treatment groups (Fig. 4; p = 0.03). The amount of Salmonella contaminating carcasses was significantly higher in the CON control group compared to T1 and T2 (3.7E + 01 CFU/mL compared to 9E + 00 CFU/mL and 1E + 01 CFU/mL respectively, p = 0.01).
Figure 2. Comparison of salivary cortisol concentrations (ng cortisol/mL saliva) between the treatment and control groups. CON control (control with basal diet); T1 (one quarter of the benzophenanthridine alkaloids [QBA] in the feed) and T2 (in the feed + water-soluble QBA). D 27 (day 27, before transport) and D 28 (day 28, after transport to the slaughterhouse). a,bDifferent letters indicate significance between treatment groups at p0.05. *Indicates significance at P 0.05 among treatments between D 27 and D 28.
Figure 3. Comparison of Salmonella detection levels (colony units [CFU]/g feces) in the feces of the treatment group and the control group. CON control (control with basal diet); T1 (benzo(c)phenanthridine alkaloids [QBA]) and T2 (in-feed + water-soluble QBA). D 27 (day 27, before transport) and D 28 (day 28, after transport to the slaughterhouse). a,bDifferent letters indicate significance between treatment groups at p0.05. *Denotes significance at P 0.05 in treatments between D 27 and D 28.
Figure 4. Overall comparison of Salmonella (colony forming units [CFU]/mL) carcass contamination in treatment and control groups. CON Control Control (control with basal diet); T1 (alkaloids added to feed [QBA]) and T2 (feed + water-soluble QBA). a,b Different values indicate the significance between treatment groups at p0.05.
05
Discussion
The main findings of this study are as follows: (1) Transportation is a stress condition for pigs, which may lead to an increase in salivary cortisol and salmonella detection rates; (2) Adding QBA to feed and drinking water can be effective Modulates the transport stress response and reduces the rate of Salmonella infection and the amount of Salmonella excreted in the feces.
Slaughterhouse transport is one of the greatest stress conditions for pigs, which may negatively affect the normal functioning of the gastrointestinal tract (Webster Marketon and Glaser, 2008; Rostagno, 2009). Furthermore, stress is associated with increased detection of Salmonella and increased food safety risks (Rostagno, 2009). Our results are consistent with previous reports showing that only pigs in CON controls showed an increase in salivary cortisol after transport, suggesting that transport is indeed a stressor for grooming pigs. Furthermore, only unsupplemented pigs showed a significant increase in Salmo-nella detection after transport. Furthermore, our results, in line with other studies (Hurd, 2002; Ver-brugghe, 2011), show a high positive correlation between salivary cortisol and post-transport Salmonella detection, suggesting that modulating stress is a way to reduce Salmonella Choose your strategy wisely.
In addition to other physiological functions, QBA has been shown to reduce the detection rate of Salmonella in growing pigs (Robbins, 2013). In agreement with Robbins (2013), the current results show that when QBA is included in feed and drinking water, the proportion of Salmonella-positive pigs and the number of Salmonella shed through feces after transfer to the slaughterhouse significantly decreases. In the present study, approximately 38% of the carcasses were contaminated with Salmonella and confirmed similar results previously reported (Botteldoorn, 2003; Arguello et al., 2013). Number of carcasses contaminated with Salmonella in the CON control group compared with T1 and T2 significantly increased. In addition, compared with the CON control group, T2 pigs shed less Salmonella in their feces after transportation, This indicates that the infection level of T2 pigs entering the slaughter line is less than that of the CON control group, thereby reducing the risk of carcasses being contaminated by feces. In this study, analysis of salivary cortisol concentrations revealed that T1 pigs had significantly reduced post-transduction stress responses compared with CON controls. However, the lower stress response in T1 did not lead to a reduction in Salmonella detection after transport to the slaughterhouse. The significant reduction in carcass contamination in T1 indicates a better adaptation to stress during preparation, which may reduce the number of Salmonella colonies in the excreta of pigs arriving at the slaughterhouse, thereby reducing the risk of carcass contamination. More research is needed to elucidate the mechanism by which QBA additives improve the regulation of stress during slaughter and the detection of Salmonella.
In summary, the results of this study indicate that adding QBA to the feed and drinking water of fattening pigs can effectively reduce the proportion of Salmonella-positive pigs and the amount of Salmonella excreted into the slaughterhouse after transfer to the slaughterhouse, thereby reducing carcass contamination. probability. Furthermore, the results show that the program using sanguinarine and chelerythrine additives to reduce salmonella contamination of carcasses is successful. In addition, QBA additives have a positive impact on the stress regulatory response caused by transportation, which may reduce the negative impact of stress on the gastrointestinal tract and reduce the detection of Salmonella. Further research is needed to determine the underlying mechanisms of reduced Salmonella detection and reduced salivary cortisol. Additionally, more research is needed to evaluate the impact of QBA additives on Salmonella antibiotic resistance.
Special thanks to
The authors would like to express their gratitude to the staff of the Laboratory of Molecular Epidemiology of Infectious Diseases for their technical assistance.This work was supported by intramural funding from Phytobiotics Fut-terzusatzstoffe GmbH, Germany, and the U.S. Department of Agriculture Animal Health, and received support from the Ohio State University College of Veterinary Medicine.
