J APPL POULT RES 2007. 16:456-463
© 2007 Poultry Science Association
Evaluation of a Competitive Exclusion Culture and Megan Vac 1 on Salmonella Typhimurium Colonization in Neonatal Broiler Chickens
J. L. McReynolds*,1,
R. W. Moore
,
A. P. McElroy
,
B. M. Hargis
and
D. J. Caldwell||
* USDA, Agricultural Research Service, Southern Plains Agricultural Research Center, College Station, TX 77845; USDA, Agricultural Research Service, Russell Research Center, Athens, GA 30605; Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg 24061; Department of Poultry Science, University of Arkansas, Fayetteville 72701; and || Departments of Poultry Science and Veterinary Pathobiology, Texas A&M University, College Station 77843
Correspondence: 1 Corresponding author: mcreynolds{at}ffsru.tamu.edu
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SUMMARY
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The present study was designed to evaluate the effect of individual or simultaneous application of 2 products, a competitive exclusion culture (CEC) or Megan Vac 1 (MV), for bioefficacy in reducing Salmonella Typhimurium (ST) cecal colonization in broiler chicks following experimental challenge. In experiment 1, CEC and MV were applied to day-of-hatch broiler chicks, and chicks were experimentally challenged with ST approximately 48 h later. In control chicks, ST was recovered at a level of 3.84 log10 cfu/g following direct plating of cecal contents, and 12 of 19 (63.15%) cultured individual ceca were positive following selective enrichment. In chicks receiving CEC by spray application on day of hatch, a numerical reduction in ST recovered from cecal contents (2.63 log10 cfu/g) and a significant reduction (P < 0.05) in recovery of ST from cultured ceca following selective enrichment (4 of 18, or 22.2%) was observed when compared with controls. Significant reductions in ST cecal colonization in chicks treated with MV alone were not observed in experiment 1. In experiment 2, chicks received day-of-hatch spray application of CEC alone, MV alone, or CEC and MV as a combined application immediately prior to or within 24 h of chick placement. When chicks were experimentally challenged with ST 48 h posthatch, significant reductions (P < 0.05) in cecal colonization by ST were observed with each experimental group when compared with nontreated controls. These data suggest that both commercially available products, alone or in combination, are efficacious in reducing cecal colonization in broiler chicks challenged with ST.
Key Words: Salmonella • chicken • competitive exclusion • vaccine
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DESCRIPTION OF PROBLEM
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Based on the concept of competitive exclusion culture (CEC) as described by Nurmi and Rantala in 1973 [1], several laboratories have developed and used CEC to reduce enteric pathogen colonization or infection in commercial poultry production systems [2, 3, 4]. Our laboratories have previously reported the efficacy of a CEC for reducing intestinal colonization of paratyphoid salmonellae in commercial meat-type or egg-laying chickens following application under laboratory and field conditions. The culture is produced by using continuous flow fermentation methodologies and contains 29 distinct obligate and facultative anaerobic bacteria. Among the various proposed mechanisms of action, the CEC works by competing for nutrients or intestinal attachment sites, or by producing bacterial substances such as volatile fatty acids. The CEC consistently produces distinguishable levels of propionic acid, which has been proposed as a marker for the establishment of this CEC and efficacy, because propionic acid as a marker applies only to this CEC [5, 6]. None of the proposed methods for the actions of CEC has been assigned to a definitive list. However, under laboratory and field conditions, the administration of CEC to neonatal chickens on day of hatch has been associated with reductions in cecal colonization or organ invasion from experimental or natural Salmonella challenge [7, 8, 9, 10].
Although CEC presumably prevent salmonellae from colonizing in the lower ileum or cecum of poultry, stimulation of the immune system in neonatal poultry through vaccination represents another potentially viable method of control that has recently received widespread industry interest. Vaccination against salmonellae in the commercial poultry industry dates back to the early 1950s, when a vaccine strain of Salmonella gallinarum, strain 9R, was developed to prevent the horizontal spread of fowl typhoid [11]. More recently, other vaccine preparations, such as oil emulsion bacterins, have been used with some degree of success for vaccinating against paratyphoid salmonellae. Through the use of these bacterins, several laboratories have reported measurable levels of protective immunity against paratyphoid salmonellae when vaccinating egg-laying poultry prior to a Salmonella Enteritidis experimental challenge [12, 13, 14, 15]. Currently, the use of more recently developed gene-deletion bacterial mutants as modified live vaccines appears to be a promising mechanism for vaccinating poultry against salmonellae. Deletion mutants of salmonellae are bacterial mutants that have been rendered less virulent or less persistent by a genetic point mutation that inhibits the bacterium’s ability to synthesize a certain nutritional enzyme or virulence factor. Based partly on detailed reports on the efficacy of such bacterial strains as vaccine candidates [16, 17], the deletion mutant vaccine Megan Vac 1 (MV) is currently available for commercial use in the United States to vaccinate poultry against paratyphoid salmonellae challenge [18].
Because CEC and deletion mutant vaccines use different mechanisms of action and both represent viable methods for controlling paratyphoid salmonellae in commercial poultry flocks, an investigation of their efficacy and compatibility is warranted. In the current investigation we evaluated the efficacy of an individual or combined administration of CEC and MV against a subsequent Salmonella Typhimurium (ST) challenge. The objective of this research was to determine whether these products should be considered for dual use in live production operations.
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MATERIALS AND METHODS
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Experimental Animals
For experiment 1, straight-run Ross x Ross broiler chicks (n = 80, 20/treatment) were obtained from a local commercial hatchery. After the completion of experiment 1, we were informed by the hatchery that the chicks had received gentamicin sulfate by in ovo injection on d 18 of embryonic life. Because in ovo antibiotic administration has been shown to negatively affect CEC establishment in the gut of neonatal poultry, a different hatchery was chosen as the source of experimental animals for the second experiment. For experiment 2, straight-run Hubbard x Hubbard broiler chicks (n = 200, 20/treatment) that had not received in ovo antibiotic administration were obtained on day of hatch. In each experiment, chicks were placed in individual rearing pens on day of hatch at an age-appropriate rearing temperature on clean pine shaving litter material. All birds were reared in 2.4 x 1.2 m pens, with 0.12 m2 of pen space per bird in an environmentally controlled house. All chicks were provided a nonmedicated corn-soybean diet that met or exceeded NRC guidelines [19] and water ad libitum.
Experimental Design
In experiment 1, CEC and MV were administered individually or in combination on day of hatch and evaluated for efficacy in protecting chicks from a naladixic acid- and sodium novobiocin-resistant ST challenge. Route of administration for both products was spray application with a commercial spray cabinet common to commercial hatcheries. Approximately 48 h posthatch, prior to experimental challenge with ST, cecal contents were collected from a subset of 10 chicks from each experimental group to determine cecal propionate levels (described below). All chicks received 0.5 mL of 2 x 104 ST via crop gavage approximately 48 h postadministration of CEC or MV. Five days postexperimental challenge, chicks (n = 20) in each experimental group were euthanized by cervical dislocation and necropsy was performed.
In experiment 2, chicks received day-of-hatch application of CEC alone, MV alone, or CEC and MV as a combined application (CEC + MV) separated by 30 min, 16 h, or 24 h between product administrations. The CEC was given first in the sequence, except for the 24-h time point in which it was administered 24 h post MV spray. Individually, CEC was administered via one of several routes, which included oral gavage (CEC-G), drinking water for 24 h (CEC-DW, 24 h), or by spray (CEC-S) application. Megan Vac 1 was administered only through spray application. On day of hatch, chicks were randomly divided into 10 experimental groups (Table 2
) corresponding to treatment, with n = 20 chicks per treatment group. The ST experimental challenge, cecal propionate levels, and bacteriologic recovery were performed as described below to determine ST incidence and log10 colony-forming units per gram of cecal contents.
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Table 2. The effect of administration of competitive exclusion culture, Megan Vac 1 (MV), or both to neonatal broiler chickens in experiment 2 on the recovery of Salmonella Typhimurium (ST) from the ceca of challenged animals
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CEC
Broiler chicks received CEC administration by spray application, crop gavage, or drinking water administration [20]. How each route of application relates to specific experimental groups in each experiment is described in the experimental design section above. Regardless of the route of administration, CEC was prepared in sterile, deionized water by the manufacturer’s supplied instructions to achieve a single manufacturer’s recommended dose of CEC per chick within the respective experimental groups. The presence of a selected profile of volatile fatty acids in the chick cecum after CEC administration has been associated with protection against experimental and natural Salmonella challenge [10]. In these investigations, CEC establishment was associated with elevated levels of propionic acid in chick cecal contents. In the current study, to measure propionic acid in chick cecal contents as a potential indicator of CEC establishment, 10 chicks from each experimental group were randomly selected and euthanized by cervical dislocation 48 h post placement (d 3 of age). Approximately 0.2 g of cecal contents from each chick was added to a sterile polypropylene tube containing 1.8 mL of double-distilled H2O. Propionic acid concentration of the cecal contents present in each tube was determined by gas-liquid chromatography as previously described [2].
ST
Experimental challenge and subsequent bacteriologic recovery of naladixic acid- and sodium novobiocin-resistant ST from experimental animals was performed according to previously published methods [8], with slight modification. Recovery of the ST challenge isolate was completed by aseptically removing 1 intact cecum from each chick. Tissues were minced with scissors and incubated in 20 mL of tetrathionate broth base at 42°C for 24 h. After incubation, 10 µL of each sample was streaked onto a brilliant green agar plate containing 20 µg/mL of naladixic acid and 25 µg/mL of sodium novobiocin, and plates were incubated for 24 h at 37°C.
To enumerate cecal ST, 0.2 g of contents from the remaining intact cecum was serially diluted at 1:10, 1:100, 1:1,000, and 1:10,000. From each dilution, 100 µL was directly plated onto brilliant green agar plates and incubated at 37°C for 24 h. The number of colony-forming units of ST recovered from each experimental animal was determined by manually counting ST colonies identified on the respective plates at each dilution. Representative ST colonies were selected and confirmed by biochemical and serological methods. Cecal contents that cultured negative at the 1:10 dilution but were positive following selective enrichment in tetrathionate were arbitrarily assigned a value of 1.50 log10 ST/g of cecal contents [8, 9]. Cecal contents that were culture negative at the 1:10 dilution and remained negative following selective enrichment in tetrathionate were arbitrarily assigned a value of 0 log10 ST/g of cecal contents.
MV
In both experiments, MV was prepared in sterile, deionized water and administered by spray application to chicks according to the manufacturer’s supplied instructions. To assess MV establishment in vaccinated animals, at necropsy the cecal tonsils and a combined sample of liver and spleen tissue from a subset of 20 chicks from each experimental group were cultured to recover the vaccine isolate of ST present in MV. After selective enrichment, 10 µL from each cecal tonsil or liver-spleen sample was plated on MacConkey agar supplemented with 1% maltose according to previously published methods [21].
Statistical Analysis
Incidence (+/–) of recovery of ST from enriched cecal cultures was compared by using the chi-squared test of independence [22]. Numeric data, including log10 colony-forming units of ST per gram of cecal contents and propionate values, were analyzed by using the GLM procedure for ANOVA with SAS software; statistically different means (P < 0.05) were further separated by using Duncan’s multiple range test [23].
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RESULTS AND DISCUSSION
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Recovery and incidence of ST for experiment 1 are presented in Table 1. For the control group, ST counts averaged 3.98 log10 cfu/g, with 12 of the 19 (63.15%) cecal tissues testing positive for ST following enrichment. Spray application of CEC and CEC + MV to day-of-hatch broiler chicks resulted in a lower incidence (P < 0.05) of cecal ST compared with controls. Average ST counts of 2.63 log10 cfu/g of cecal contents were estimated for those treated with a single application of CEC. Following tissue enrichment, a significant reduction (P < 0.05) was seen, with 4 of 18 (22%) chicks testing positive for ST compared with the 63.2% of control chicks that tested positive. Also differing from control chicks, those treated with a single application of CEC + MV had a lower (P < 0.05) incidence of ST, with 5 of 20 (25%) chicks testing positive for the ST challenge and an estimated 2.09 log10 cfu/g of cecal contents. The incidence of ST cecal colonization in MV-treated chicks was similar to that of control chicks. Importantly, MV was recovered from experimental animals only in groups that received MV by spray application in the present experiment (Table 1).
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Table 1. The effect of administration of competitive exclusion culture, Megan Vac 1 (MV), or both to neonatal broiler chickens in experiment 1 on the recovery of Salmonella Typhimurium (ST) from the ceca of challenged animals
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Cecal propionate levels were similar between the experimental groups, which was unexpected. Typically, we have seen increased levels of propionate in birds that have been given the CEC. Short-chain fatty acids, which are created by commensal bacteria as an end product of microbial fermentation, have been used as an indicator for the establishment of this CEC in the gastrointestinal tract. However, in this study and in the following study, several experimental groups had suppressed levels of propionate. Volatile fatty acids such as acetic, propionic, and butyric acids have been shown to be biological indicators of a healthy microbial ecosystem, as well as having inhibitory effects on Salmonella colonization in chickens [5, 6, 7, 8, 9]. Although we saw suppressed levels of propionate in our study, in several groups we still saw protection against ST in all groups that received the CEC. There are myriad reasons why the CEC product is efficacious in reducing pathogens; therefore, we should be careful when using stereotypical characteristics.
In experiment 2, chicks received CEC alone by day-of-hatch spray application, drinking water application, or crop gavage. Additionally, other experimental groups consisted of MV spray application alone or in association with CEC as a simultaneous or delayed application by one of the above-mentioned routes of administration. Results are presented in Table 2
. Recovery of ST for all treated groups following cecal enrichment differed from that of the controls (P < 0.05). Salmonella Typhimurium detected in the cecal contents of control chicks averaged 2.33 log10 cfu/g, with 19 of 19 (100%) chicks testing positive for ST following tissue enrichment. As expected, mean propionate levels were 0.0 µmol/g of cecal contents for these animals.
With the direct plating method, ST was not recovered from cecal contents of chicks treated by CEC-S. However, when ceca were enriched, 3 of 20 (15%) tested positive, which resulted in a significantly lower recovery (P < 0.05) of ST when compared with controls. Mean cecal propionate levels for the CEC-S experimental group were 3.91 µmol/g of cecal contents. The CEC-G treatment resulted in a lower (P < 0.05) incidence of recovered ST (2/19) as well as a decrease (P < 0.05) in colonies of ST when directly plated. When CEC was administered at hatch in the drinking water for the first 24 h post placement (CEC-DW, 24 h) we found 0.30 cfu/g of contents, with 3 of 20 (15%) tissues testing positive for ST following enrichment.
When the different routes of administration were evaluated, all 3 methods presented here showed that the CEC did significantly reduce ST. Many researchers have shown that this product does significantly reduce the ST challenge; however, they have all shown that increased volatile fatty acid levels were correlated with ST protection [8, 9]. In the CEC-S treatment group, the propionate levels where significantly reduced compared with those of the CEC-G or CEC-DW, 24 h groups, with mean levels of 3.91, 13.2, and 11.17 µM/g, respectively. However, this group did have the lowest colony-forming unit level and one of the lowest levels of ST, indicating that this CEC was still efficacious in reducing ST. The precise mechanisms used by CE cultures to exclude Salmonella are not fully understood. The first line of defense against enteric pathogens is the normal gut microflora. These bacteria have many different mechanisms, which they can use to defend the host’s gastrointestinal tract. It has been postulated that CE cultures provide protection against pathogens in several ways. One mechanism involves competition for intestinal attachment sites on the mucosa of the intestine [24]. If the microorganisms of the CE culture can fill intestinal attachment sites prior to challenge with a pathogen, it is believed that the pathogen will not have a site to bind, and thus will pass through the animal. Other products such as hydrogen peroxide are produced by commensal bacteria. Production of hydrogen peroxide results in the peroxidation of lipid membranes and increased bacterial membrane permeability. Any of these mechanisms could possibly explain why the CEC product was still effective, even with low propionate levels.
Spray application of MV alone at hatch resulted in a lower (P < 0.05) incidence of ST when compared with controls. Salmonella Typhimurium counts averaged 0.6 log10 cfu/g following direct plating of cecal contents, with 4 of 19 (21%) cecal tissues testing positive following selective enrichment. Population counts of the recovered challenge dose were similar regardless of the products used (i.e., CEC-S vs. MV). Mean cecal propionate levels for this experimental group were 0.0 µmol/g of cecal contents. Salmonella Typhimurium recovered in chicks receiving MV in a single spray application 16 h posthatch, averaged 1.20 log10 cfu/g, with 74% of cecal tissues testing positive for ST following enrichment, which differed (P < 0.05) from control chicks but was similar to MV-treated chicks.
Salmonella Typhimurium was not recovered from chicks treated with CEC and MV by using spray application (CEC-S + MV) with a 30-min delay between applications. In addition, none of the enriched cecal tissues from treated chicks tested positive for ST, which was significantly lower (P < 0.05) than the incidence of ST measured in the control chicks but was similar to that found in the CEC-S-treated group. The mean cecal propionate level for the CEC-S + MV experimental group was 3.86 µmol/g of cecal contents. Chicks treated with MV via spray application followed by CEC-G 24 h postplacement (MV + CEC-G, 24 h), had lower (P < 0.05) recoveries of ST (0.30 cfu/g) compared with controls (2.33 cfu/g). However, recovery of ST did not differ from the CEC-G-treated group. Following enrichment, the number of cecal tissues testing positive was also lower for the MV + CEC-G, 24 h group (26%) compared with controls (100%). Proprionate levels (Table 2) for animals receiving treatments (CEC and MV alone, or in combination) via drinking water or oral gavage were similar between treatments but differed from control animals. Similar to experiment 1, MV was recovered only from experimental animals that received MV by spray application in the present experiment (Table 2). The combination of CEC-S and MV, with a 16-h lag period between applications (CEC-S + MV, 16 h), resulted in fewer (P < 0.05) ST colonies compared with control chicks, with an average 1.21 log10 cfu/g. However, bacterial counts of ST were greater (P < 0.05) than those observed in the CEC-S + MV experimental group and were similar to those detected for the MV, 16 h chicks. Following tissue enrichment, 12 of the 20 (60%) chicks tested positive for cecal ST. Mean cecal propionate levels for the CEC-S + MV, 16 h experimental group were 3.07 µmol/g of cecal contents. However, the decreased levels of propionate, once considered to be indicative of reduced CEC establishment in the cecum of treated chicks, did not accurately predict the effectiveness of CEC to reduce or eliminate ST colonization in treated chicks. The present investigation raises questions about using cecal propionate levels as an indicator of CEC establishment and efficacy. The combination of CEC-DW, 24 h and MV (CEC-DW, 24 h + MV) produced results similar to CEC-DW, 24 h but differed from control animals. After direct plating, recovery of ST from the cecal contents of chicks averaged 0.15 log10 cfu/g of cecal contents, with 5 of 19 (26%) enriched cecal samples testing positive for ST.
Hassan and Curtiss [16] investigated the efficacy of an ST x3985 deletion mutant vaccine against an experimental challenge with the ST progenitor field strain x3761. Data from this investigation demonstrated that the x3985-treated chicks were protected from the experimental challenge of x3761. This technology is now commercially available in the United States to poultry producers as the deletion mutant vaccine known as MV [16, 17]. The results in our experiment are consistent in showing protection from an experimental ST challenge. Other scientists have used oil immersion vaccines for controlling SE in chickens. Gast and coworkers showed an acetone-killed oil immersion bacterin that was prepared from a different strain of SE, SE PT13a, to be efficacious against SE infection in vaccinated animals [12, 13]. The initial vaccination occurred at 23 wk and a booster vaccination was given at 29 wk of age. A significant reduction in SE infection in the vaccinated hens was reported when the liver, spleen, ovaries, and oviduct were cultured for SE and compared with the non-treated controls. The results from the experiment also showed a significant reduction in the incidence of fecal shedding and intestinal colonization of SE.
The present study evaluated 2 products, CEC or MV, for efficacy in reducing paratyphoid salmonellae colonization in neonatal broiler chickens. Importantly, the efficacy in reducing ST colonization in 2 experiments was observed when each product was applied both individually and simultaneously. Such observations are important because both products use presumably different modes of action to exclude intestinal salmonellae. As such, they may be considered for dual use in commercial poultry production because of the apparent lack of complication associated with the efficacy of both products when administered concurrently. These data suggest that both products can significantly reduce ST and should be compatible for dual implementation into an integrated Salmonella control program.
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CONCLUSIONS AND APPLICATIONS
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- Competitive exclusion culture and MV were efficacious in reducing ST colonization in an experimental setting when each product was applied individually or simultaneously.
- These products may be considered for dual use in a commercial poultry setting because of the apparent lack of complications associated with the efficacy of both products administered concurrently.
- These data show that there are alternatives to antibiotics on the commercial market that can reduce the establishment of pathogens in the gastrointestinal tract. As antibiotic-free animals continue to be promoted through consumer pressure, these alternatives may become a valuable resource for our industry.
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REFERENCES AND NOTES
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- Hassan, J. O., and R. Curtiss, III. 1997. Efficacy of a live avirulent Salmonella Typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella Typhimurium and Salmonella enteritidis. Avian Dis. 41:783–791.[ISI][Medline]
- Megan Vac 1, No. LV:5000-J, K, L, Lohmann Animal Health International, Cuxhaven, Germany.
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SUMMARY
DESCRIPTION OF PROBLEM
