2024 年 61 巻 論文ID: 2024016
Lactobacillus spp. inhibit the growth of Campylobacter spp. in vitro. However, in chicken crops, in which Lactobacillus spp. predominate, such inhibition of Campylobacter has not been confirmed. In our previous study, feeding paddy rice to broiler chicks increased the residence time of the food, which might enhance the bactericidal activity of the crop. Here, the bactericidal activity against the remaining Campylobacter spp. in broiler crops was evaluated. A suspension prepared by mixing Campylobacter jejuni and titanium dioxide (TiO2) was inoculated into the pharynx of 26-day-old broiler chicks fed a paddy rice-based diet. The crop contents were sampled at 20-min intervals. The TiO2 residual ratio in the crop gradually decreased with time after inoculation, with 57% of the inoculated TiO2 remaining in the crop 60 min after inoculation. The survival fraction of C. jejuni in the crops was 11% at 40 min, only 1% at 60 min, and was undetectable at 80 min. Most of the inoculated C. jejuni died in the crop before entering the next segment. These data indicated that bacterial death occurred between 30 min and 40 min after inoculation. The average survival time of C. jejuni in the crop was calculated to be 37.1 min. Thus, C. jejuni remaining in a chicken crop for more than 40 min died.
Campylobacter is a leading cause of enteric zoonotic infections[1]. Campylobacter infection is a major public health challenge with a complex epidemiology involving widespread animal and environmental reservoirs and multiple risk factors[2,3]. Campylobacter has been detected in soil, groundwater, wild animals, and insects[4,5,6,7], indicating its transmission through footwear and clothing of farm workers, flies, and small animals.
Domestic poultry (e.g., broilers, layers, turkeys, and ducks) have been identified as natural reservoirs of Campylobacter[8,9]. Broiler chicks are colonized by Campylobacter primarily in their ceca between 2 and 4 weeks of age[10]. Once some chickens are infected with Campylobacter, most broiler flock birds become colonized within a few days and remain infectious until age of slaughter[11,12,13].
Although broiler chicks are natural reservoirs of Campylobacter, accidental ingestion of Campylobacter does not always lead to colonization of the lower gastrointestinal tract. Chickens have several natural defense barriers in the upper digestive tract that eliminate pathogens invading the oral cavity[14]. Gastric juice secreted from the proventriculus kills microorganisms by lowering the pH of the gizzard[15]. This natural defense barrier is enhanced by the addition of insoluble dietary fiber to the feed[14]. In our previous report[16], the diluted nutrients and hardness of insoluble fiber equivalent to 20% of paddy rice accelerated the grinding activity of the gizzard, which homogenized the pH value in the gizzard and eliminated areas of higher pH where Campylobacter survives. This pH homogenization in the gizzard could prevent Campylobacter colonization of the cecum[17].
The second defense mechanism involves Lactobacillus spp., which predominantly inhabit chicken crops. Lactobacillus spp. are known to produce organic acids, such as lactic acid, acetic acid, hydrogen peroxide, and an antibacterial peptide, that inhibits Campylobacter growth under in vitro conditions[18,19,20,21]. However, Campylobacter inhibition has not been confirmed in vivo because animal experiments cannot maintain a sufficient residence time to kill Campylobacter in chicken crops. In our previous report[16], paddy rice fed to broiler chicks increased the residence time in the crop, resulting in an average retention time of 115 min, which allowed observation of the bactericidal activity of the inoculum in the crop. In this study, the survival of Campylobacter remaining in broilers was evaluated by taking advantage of the increased residence time in broiler crops fed paddy rice.
This study was approved by the Animal Use and Care Committee of Kyoto Prefectural Agriculture, Forestry, Fisheries, and Livestock Technology Center (approval number: 6-116).
Microorganisms, culture conditions, and preparation of the inoculumCampylobacter jejuni GTC 03263, a strain used previously, was used in this study[17]. For the preparation of inoculum, a bacterial culture was revived from the stock bacterial suspension stored at −80 °C in the Micro Bank (Iwaki Co., Tokyo, Japan). Two micro beads were inoculated in 5 mL brain heart infusion (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and incubated at 37 °C for 48 h under microaerobic conditions (5% oxygen, 10% carbon dioxide, and 85% nitrogen) and then 100 µL of the suspension was spread onto nutrient agar plates (Nissui). Plates were then incubated at 37 °C for 48 h under microaerobic conditions, and two platinum loops of the cultured bacteria were harvested after incubation and resuspended in 5 mL sterile saline solution. This suspension contained approximately 1 × 108 colony-forming units [CFU]/mL) and was used for Campylobacter oral inoculation.
A chicken crop is a diverticulum of the esophagus that temporarily stores the unsorted flow of ingested food[22]. This property enables the use of titanium dioxide (TiO2), which is used as an indicator substance in digestion tests, to simultaneously monitor the dynamics of retention and excretion of dosed substances in the crop. A TiO2-water suspension (0.5 g/mL) was prepared by mixing TiO2 (Wako Pure Chemical Industries, Osaka, Japan) in sterile saline.
The oral inoculation used in this study was prepared by mixing the Campylobacter and TiO2-water suspensions at a ratio of 1:9, and was stored in a plastic tube for several minutes before inoculation.
Experimental animals and the challenge modelTwenty 1-day-old female broiler chicks (Chunky) were raised in a flock-rearing area, measuring approximately 2 m2 and covered with sawdust, in a windowless broiler house. The rearing area temperature was set at 34 °C on the first day and was gradually lowered to 25 °C until the chicks reached 14 days of age. The room temperature was maintained between 20–25 °C after 15 days of age. The chicks were fed a starter feed (crushed corn at a weight ratio of 65%) until 14 days of age (Table 1). The chickens were fed a diet containing 60% whole-grain paddy rice after 15 days of age (Table 1). The feedstuff did not contain any antimicrobials or coccidiostats. All chicks had free access to food and water throughout the study. The duration of light exposure was continuously controlled until 7 days of age, and then 20 h of light exposure per day was provided. All chicks were housed in an experimental cage at 26 d of age. The birds were confirmed to be culture-negative for Campylobacter prior to inoculation.
| Ingredients (%) | 1 to 14 days | 15 to 26 days |
| Ground corn | 65 | 10 |
| Paddy rice | 60 | |
| Soybean meal | 20 | 20 |
| Fish meal (65% crude protein) | 7 | 5 |
| Corn gluten meal | 2 | |
| Soybean oil | 3 | 2 |
| Calcium carbonate | 0.5 | 0.5 |
| Tricalcium phosphate | 0.6 | 0.6 |
| Dicalcium phosphate | 0.5 | 0.5 |
| Manganese sulfate | 0.015 | 0.015 |
| Sodium chloride | 0.25 | 0.19 |
| DL-methionine | 0.3 | 0.25 |
| L-lysine HCl | 0.5 | 0.4 |
| Riboflavin | 0.0004 | 0.00025 |
| Copper sulfate | 0.0005 | 0.001 |
| Zinc sulfate | 0.0005 | 0.004 |
| Folacin | 0.00004 | 0.00003 |
| L-threonine | 0.25 | 0.25 |
| Choline chloride | 0.05 | |
| Calcium pantothenate | 0.0004 | |
| Nicotinamide | 0.003 | |
| Vitamin/mineral premixa | 0.3 | 0.3 |
| Calculated | ||
| Crude protein (%) | 21.1 | 17.5 |
| Metabolizable energy (kcal/kg) | 3,056 | 2,748 |
a) Vitamin and mineral premix including the following(per kg of the diet): retinol (retinyl acetate), 3,500,000 IU; cholecalciferol, 700,000 IU; vitamin E (DL-α-tocopheryl acetate), 600 mg; menadione, 250 mg; thiamine, 500 mg; riboflavin, 450 mg; pyridoxine, 350 mg; cyanocobalamin, 0.8 mg; nicotinamide, 1,700 mg; D-pantothenic acid, 750 mg; choline chloride, 35,000 mg; ZnCO3, 5,700 mg; MnSO4, 8,250 mg; FeSO4, 3,890 mg; CuSO4, 1,160 mg; CoSO4, 17 mg.
The prepared inoculum suspension (1 mL) was inoculated into the pharynx of all broilers using a 1 mL plastic syringe attached to a flexible tube. After inoculation, groups of four broiler chicks were randomly selected at 20-min intervals until 80 min and euthanized by anesthesia overdose (intravenous injection of sodium pentobarbital, Somnopentyl; Kyoritsu Pharmacy, Tokyo, Japan). The broiler crops were immediately excised with sterilized surgical scissors and the contents were transferred into 100 mL sterilized beakers. The weight of the crop contents was measured, and approximately 1 g of the contents was collected aseptically into sterile plastic tubes. The samples were stored in an icebox for approximately 2 h prior to bacterial detection. The remaining portion of the crop content was added to two volumes of deionized water in a 50 mL centrifuge tube and mixed using a vortex mixer for 1 min. The pH of the diluted mixture was repeatedly measured using a glass electrode pH meter (9615-10D; Horiba, Kyoto, Japan). The contents in the beaker were dried at 105 °C overnight using a ventilation dryer. The dried content was crushed using a food mill and subjected to quantitative determination of TiO2 using the method described by Short et al.[23].
Enumeration of bacteriaFor Campylobacter jejuni enumeration, the culture methods described below were used based on the fact that Campylobacter enters a viable but non-culturable state that has not been confirmed in vivo.
The preserved crop contents (1 g) were diluted 1:10 (w/v) using dilution anaerobic buffer solution (composition: KH2PO4, 4.5 g; Na2HPO4, 6.0 g; L-cysteine hydrochloride, 0.5 g; Tween 80, 0.5 g; and agar, 1 g in 1000 mL of purified water) in a plastic tube. The first suspension was serially diluted 10-fold with a diluted anaerobic buffer solution. The suspensions were spread onto modified charcoal cefoperazone deoxycholate agar (CCDA) plates (Oxoid, Basingstoke, United Kingdom), whereas modified Lactobacillus Selective agar (mLBS) was used for Lactobacillus sp. The CCDA medium consisted of a Campylobacter blood-free selective agar base (CM739; Oxoid) with a Campylobacter selective supplement (SR155; Oxoid) and a Campylobacter growth supplement (SR084; Oxoid). The plates were incubated at 42 °C under microaerophilic conditions (85% N2, 10% CO2, and 5% O2) for 48 h. The isolated colonies were confirmed to be C. jejuni using standard microbiological methods (International Standards Organization, 2006), including Gram staining, catalase and oxidase tests, specific spiral morphology, and corkscrew motility observed using phase-contrast microscopy. The number of bacteria in the samples of 60 min and 80 min after the inoculation was measured using an enrichment culture. Briefly, 1 g of crop content was added to 10 mL Preston broth (Nutrient broth No. 2 [CM67; Oxoid] supplemented with SR117, SR084, and 5% [vol/vol] lysed horse blood, Oxoid) and cultured at 42 °C under microaerobic conditions for 24 h. One platinum loop of Preston broth was plated onto a CCDA plate. The plates were incubated at 42 °C under microaerophilic conditions (85% N2, 10% CO2, and 5% O2) for 48 h. Colonies suspected to be C. jejuni were isolated and incubated on blood agar base No. 2 (Oxoid) containing 5% lysed horse blood. The isolated colonies were confirmed to be C. jejuni using standard microbiological methods (International Standards Organization, 2006), including Gram staining, catalase and oxidase tests, specific spiral morphology, and corkscrew motility observed using phase-contrast microscopy. Lactobacillus spp. were quantified using mLBS, i.e., prepared by supplementing LBS agar (BD, Becton Dickinson and Company, Sparks, MD, USA) with 0.8% Lab-Lemco Powder (Oxoid), 0.1% sodium acetate trihydrate, and 0.37% acetic acid. The plates were incubated at 38 °C for 48 h under anaerobic conditions, as described above, and the colonies were counted.
Modeling the Campylobacter survival curveA crop nonselectively passes food components to the next segment[22]; therefore, the mixed suspension of C. jejuni and TiO2 remains in the same ratio in the crop at any elapsed time. Therefore, the fraction of the amount of remaining TiO2 in the crop to the dosed TiO2 (TiO2 in the crop/dosed TiO2) may be substituted for the fraction of the remaining C. jejuni in the crop to the dosed C. jejuni (C. jejuni in the crop/dosed C. jejuni) in each sample of crop contents at the same elapsed time.
That is:
TiO2 in the crop/dosed TiO2 = C. jejuni in the crop/dosed C. jejuni (1)In the above formula, the units TiO2 and C. jejuni present the weight and number of bacteria, respectively.
In addition, if the fraction of live C. jejuni to the remaining fraction (live or dead) in the crop at some time t after the challenge is defined as the survival fraction Scrop(t), the number of live bacteria in every sample at some time t is expressed by the following formula:
Live C. jejuni in the crop = C. jejuni in the crop × Scrop(t)Substituting the above formula into formula (1) and arranging it:
Scrop(t) = (live C. jejuni in the crop/dosed C. jejuni)/ (TiO2 in the crop/dosed TiO2) (2)The survival curve of Scrop(t) is obtained by fitting the cumulative form of the Weibull distribution[24].
Scrop(t) = exp(−btn) (3)where b and n are constants. b and n were derived using nonlinear least-squares analysis in Microsoft EXCEL (2010). The values of b and n are used to generate the frequency distribution using the following equations:
| (4) |
where
| (5) |
| (6) |
where Γ is the gamma function. The mean t
corresponds to the inactivation time on average with its variance,
The Wilcoxon rank-sum test was used to test for significant differences in the survival fraction, Scrop(t), of C. jejuni at the time of inoculation and at each time point.
Bacterial counts (C. jejuni and Lactobacillus. spp.), along with the pH of the crop contents sampled at 20-min intervals after inoculation, are presented in Table 2. C. jejuni was not detected in two out of the four samples at 60 min in the crop contents, but was not detected in any of the four samples at 80 min after inoculation, even using the enrichment culture method. When no C. jejuni was detected in a sample, the colony count of C. jejuni was deemed to be zero.
| Time after inoculation |
C. jejuni | Lactobacillus spp. | pH |
| (min) | (log10 cfu/g) | (log10 cfu/g) | |
| 0 | 5.93 | - | - |
| 5.87 | - | - | |
| 6.00 | - | - | |
| 5.85 | - | - | |
| 20 | 5.88 | 7.69 | 5.49 |
| 5.77 | 7.45 | 5.80 | |
| 5.93 | 6.48 | 4.81 | |
| 5.92 | - | - | |
| 40 | 4.03 | 7.45 | 4.75 |
| 5.12 | - | - | |
| 4.79 | - | - | |
| 4.66 | - | - | |
| 60 | 3.74 | 8.98 | 4.73 |
| 2.89 | 8.36 | 4.87 | |
| ND | 7.97 | 4.70 | |
| ND | 8.49 | 4.64 | |
| 80 | ND | 8.67 | 4.80 |
| ND | 8.72 | 4.70 | |
| ND | 8.54 | 4.66 | |
| ND | 8.92 | 4.58 |
ND: no detected colonies; -: not examined.
The TiO2 residual ratio (TiO2 in the crop/TiO2 dose) and Scrop(t) in the content of the crop sampled at each elapsed time are listed in Table 3. The TiO2 residual ratio gradually decreased over time after inoculation, with 57% of the inoculated TiO2 remaining in the crop after 60 min. In contrast, the survival fraction of the dosed C. jejuni in the crop (Scrop(t)) decreased 20 min–40 min after inoculation. A statistically significant difference was observed between Scrop(0) and the survival fraction Scrop(t) at 40, 60, and 80 min after inoculation using the nonparametric test (Table 3).
| Time after inoculation (min) | |||||
| 0 | 20 | 40 | 60 | 80 | |
| TiO2 residual ratio | 1.00 ± 0.00 | 0.89 ± 0.09 | 0.71 ± 0.12 | 0.57 ± 0.17 | 0.66 ± 0.05 |
| Scrop(t)c | 1.00 ± 0.14a | 1.13 ± 0.11a | 0.11 ± 0.07b | 0.01 ± 0.01b | 0.00 ± 0.00b |
Data are presented as mean ± SD (n=4)
a-b) Means within the same row with different superscripts are significantly different (p<0.01)
c) Scrop(t) = (lived C. jejuni in the crop/dosed C. jejuni)/ (TiO2 in the crop/dosed TiO2)
Equation (1) was used to calculate the value
of Scrop(t) for each sample, and the parameters
b and n were determined to be 0.0261317.6826 and 17.6826, respectively, using the
least-squares method. The survival fraction
Scrop(t) for each elapsed time and the
survival curve obtained by substituting parameters b and n into Equation (3) are depicted in Fig. 1. The frequency distribution of the survival fraction
Scrop(t) is obtained by substituting these
parameters into Equation (4), as illustrated
in Fig. 2. Furthermore, the mean survival time
(
The survival fraction Scrop(t) and the survival curve of Scrop(t) in the crop for each elapsed time after C. jejuni inoculation (white dots, n = 4).
The survival curve of Scrop(t) is calculated from equation (2); Scrop(t) = exp(−btn), n = 17.6826, b = 0.02613n.
The frequency distribution of lethal events corresponding to time after C. jejuni inoculation.
The frequency
distribution (
The number of Lactobacillus spp. in the crop contents of chickens inoculated with the mixed suspension varied in the range of 7.45–8.71 log CFU/mL at each elapsed time. The bacterial load is similar to that of previous reports[26,27,28]. The pH of the crops measured at each elapsed time point ranged from 4.69–5.37.
The levels of remaining TiO2 in the crop at 20, 40, 60, and 80 min after inoculation were 89%, 71%, 57%, and 66% of the inoculated amount, respectively (Table 3). Most of the TiO2 persisted in the crop for 80 min, indicating that the inoculatedC. jejuni also remained in a live or dead state at approximately the same rate in the crop. However, the survival fraction ofC. jejuni remaining in the crops was 11% at 40 min, only 1% at 60 min, and was undetectable at 80 min using the culture method (Table 3). Most of the inoculated C. jejuni died in the crop before entering the next segment. These data indicated that a lethal event occurred intensively over a certain period, between 30 min and 40 min, as revealed in Figs. 1 and 2. The mean survival time of C. jejuni in the crop was calculated to be 37.1 min. These data suggest that C. jejuni remaining for more than 40 min in a chicken crop died.
No lethal events occurred up to 20 min after inoculation of the crop (Figs. 1, 2). Therefore, these results indicate that a crop residence time of less than 20 min cannot prevent C. jejuni infection. Broilers raised with free access to nutritious feeds rarely store ingested feeds in their crops[29,30]. For example, 78% of ad libitum-fed birds store less than 5 g dry matter of feed in the crop[29]. Additionally, a small amount of storage in the crop corresponds to a short retention time. The feed retention time in the crop was only 5–7 min in broilers with free access to feed, as reported in previous studies[31,32]. Therefore, if broilers are provided with conventional free feeding, the short residence time of crop contents will promote early passage to the next segment and allow C. jejuni to survive. Thus, the residence time of the crop content must be treated as an important factor affecting the bactericidal role of the crop.
Lactobacillus inhibits the growth of co-cultivated Campylobacter in vitro[18,19,20,21]. In this experiment, Lactobacillus formed the dominant bacterial flora (106–108 CFU/g) at any time after oral inoculation with Campylobacter, demonstrating the qualitative bactericidal activity of Lactobacillus in broiler crops. The crop showed bactericidal activity (89% of the inoculated Campylobacter was eliminated in 40 min and 99% in 60 min), but no such bactericidal activity was observed in in vitro experiments in which Campylobacter and Lactobacillus were co-cultivated[33] or in an exposed substrate of organic acids and bacteriocins produced by Lactobacillus[18,19,20,21]. However, the planktonic phase formed using broth media in these reports seems to be quite different from the co-cultivated phase chicken crops in the present experiment, because it has been observed that Lactobacillus attached to epithelial cells on the inner wall of the crop produces exopolysaccharide and forms biofilms[34]. Stationary biofilms of lactic acid bacteria likely inhibit pathogenic bacterial invasion through spatial or nutritional competitive exclusion[35]. Therefore, this experiments using chicken crops suggest that antimicrobial metabolites (organic acids or antibacterial peptides) produced by Lactobacillus and spatial or nutritional competitive exclusion by Lactobacillus-formed biofilms might enhance the bactericidal effect against challenged C. jejuni.
These data revealed that the frequency of C. jejuni death occurred intensively between 30 min and 40 min after broiler crop inoculation. Further investigations simulating the spatial and microbial characteristics of chicken crops are required to elucidate the factors contributing to the intense elimination of C. jejuni from the crop.
We thank former Professor Dr. Takayuki Ezaki, Graduate School of Medicine, Gifu University, for providing the Campylobacter strain GTC 03263 (GTC: Gifu Type Culture Collection) used in this study.
Mari Nishii conducted the experiments and drafted the original manuscript. Masaharu Yasutomi analyzed the data. Both authors critically discussed and reviewed the manuscript.
The authors declare no conflict of interest.
