Campylobacter jejuni is a pathogenic bacterium that causes human foodborne gastroenteritis [1]. In the United States, Campylobacter is responsible for an estimated 2.1-2.4 million cases of foodborne illnesses each year [2-4] resulting in 13,000 hospitalizations, 100 deaths and an estimated cost of over $1 billion annually [3,5]. FoodNet [6] reported that the number of infections and incidence of Campylobacter per 100,000 persons were 6,621 and 13.82 respectively in 2013. Of these infected persons, 1,010 (15%) were hospitalized and 12 (0.2%) died from contaminated food [6].

Approximately 70% of human illnesses due to Campylobacter are caused by the consumption or handling of raw or undercooked poultry [2,7]. Additionally, Campylobacter can be transmitted via contact with infected animals or their feces. Many animals carry Campylobacter asymptomatically and shed the bacterium in their feces. Poultry, particularly broiler chickens, also frequently harbor the bacterium. Because of the threat to public health, serious efforts are being made to prevent the colonization and spread of C. jejuni in poultry production [8-10]. A reduction in numbers of Campylobacter in poultry, production can lead to a corresponding reduction in human infections. Quantitative risk assessment models have indicated that a reduction of 2 log units on a broiler carcass could result in 30 times less prevalence of Campylobacteriosis [11]. Therefore, reduction or elimination of C. jejuni in the poultry reservoir is an essential consideration in the control of this food safety problem.

Although there are multiple levels at which Campylobacter contamination can be targeted, on-farm control of Campylobacter has the greatest impact because the living poultry intestine is the primary amplification point for Campylobacter throughout the food chain [12,13]. Therefore, the use of various antimicrobial therapies to control Campylobacter infection in poultry production is worthy of exploration.

Antimicrobial therapy is a potentially important tool in reducing the prevalence and enumeration of C. jejuni in poultry. Studies have addressed the use and efficacy of antibiotics on an array of poultry infections including C. jejuni producing varied results. An in vivo study [14] reported that a three-phage lytic cocktail administered to chickens resulted in a 2 log CFU/g reduction in C. jejuni. In another in vivo study using turkeys [15], the administration of enrofloxacin, neomycin and vancomycin resulted in a respective decrease of 1, 2 and 4 log CFU/g in C. jejuni. In a recent study [16] the live bacterium Enterococcus faecalis was administered to inhibit C. jejuni in chickens but was proved ineffective in preventing growth. Thus, more studies are needed to explore possible alternative antibiotics that can reduce the population of C. jejuni in poultry production.

Sulfonamides were first used to treat upper respiratory [17] and coccidial infections caused by Eimeria tenella and Eimeria necatrix in poultry [18,19]. The commonly used sulfonamide in poultry production is sulfadimethoxine and therefore is appropriate for in vivo testing [20]. Sulfadimethoxine has been used alone or in combination with other antibiotics and coccidiostats to improve weight gain and final body weight [21].

Sulfadimethoxine is also commonly used as an antimicrobial for the treatment and/or prevention of coccidiosis, fowl cholera, and coryza in poultry [22,23]. The previous study [24] supported that the use of sulfadimethoxine can reduce the load of C. jejuni in turkey.

To date, there have been no in vivo studies on the efficacy of sulfadimethoxine in the control of C. jejuni in broilers in small-scale poultry operations. Although largely unknown, the positive potential effects of sulfadimethoxine antibiotic treatment to control C. jejuni infection in chickens should be explored.

The purpose of this study is to determine the effects of sulfadimethoxine antibiotic on the enumeration of Campylobacter spp. and the prevalence on both Campylobacter spp. And C. jejuni in growing broilers.

Broiler production

The McNeese State University Animal Care and Use Committee approved the methods related to animal care that were used in this experiment.

Two treatments with two replications each using 300 broiler chickens (Ross × Ross) obtained from a commercial hatchery were used. These experiments were conducted from January 2014 to May 2014. Replication I was initiated on January, 2014 and replication II on March, 2014. Birds were housed in a controlled environment and maintained in Petersime®Battery Cages (32°C) with raised wire flooring (Petersime Incubator Co., Gettysburg, OH). Each cage was divided into 12 pens of equal size (74.7 cm × 99.1 cm × 24.13 cm). Each pen housed twenty-five birds. Individual open water and feed troughs were provided for each pen. Individual water and feed troughs were provided for each pen and feed was supplied ad libitum. Feed was procured from the Texas Farm Products Company. This feed contains 18% protein chick grower crumbles and no antibiotics.

The housing system was emptied of birds, feed, and litter and cleaned with hot water wash and disinfect. Animal care givers monitored feed and water and removed litter trays daily. Normal pest and rodent control was maintained throughout the experiment. The temperature and % RH during time period was 32°C and 58%, respectively.

Birds were allotted to one of two treatments: 1) control (drinking water) and 2) drinking water + 0.05% (wt/vol) sulfadimethoxine (Durvet Inc., Blue Springs, MO). Drinking water was refreshed every day in both treatment groups. Each week, 150 individual broilers were randomly tested via cloacal sterile rayon tipped swabs and assayed for the presence of Campylobacter spp. [25] and C. jejuni [26]. The swabs were placed in a tube containing 3 ml of sterile tryptone soy broth (TSB) for further analysis.

Bacterial isolation and identification

Immediately upon arrival in the laboratory, the swab samples were whirl-mixed in a shaker incubator (Excella E24/E24R Temperature- Controlled Benchtop Shaker, New Brunswick Scientific, Edison, NJ) for approximately 1 h at 37°C and then mixed with a vortexer for 2 min to release the bacteria. Each 0.1 ml of swab sample was aseptically transferred and directly spread onto modified charcoal cefoperazone deoxycholate agar (mCDDA). The inoculated plates were then incubated at 42°C for 48 h in a microaerophilic environment (5% O2, 10% CO2, 85%N2) [27]. Campylobacter spp. was verified via latex agglutination [28,29] with a Microgen M46 Campylobacter Assay Kit (Microgen Bioproducts Ltd., Camberley, Surrey, UK) by manufactures instructions and C. jejuni was confirmed via hippurate hydrolysis [26].

Statistical analysis

Statistical procedures were performed using SAS Windows [30]. Day old broilers were randomly allotted to two treatments (with and without the antibiotic sulfadimethoxine administration in chicken production) with two replications. All calculations were performed with Proc GLM procedures (SAS 2003) using P=0.05 for significance of Least Squares Means with a model of the antibiotic sulfadimethoxine administration in chicken production and week of testing. When treatment difference is detected, specific comparisons between treatment means at that time point were made with the PDIFF option of LSMEANS.

Enumeration of Campylobacter spp.

Our results showed that the counts of Campylobacter spp. steadily increased from week 1 through week 6 in both the control and antibiotic treatments (Figure 1). The counts of Campylobacter spp. in the control treatment increased from an initial value of 3.58 log CFU/broiler in week one to a maximum value of 6.05 log CFU/broiler in week six. This represents a total increase of 2.47 log CFU/broiler during the course of the experiment. In the antibiotic treatment the initial value of 3.44 log CFU/broiler in week one increased to a maximum value of 5.12 log CFU/broiler in week six (Figure 1). This represents a total increase of 1.68 log CFU/broiler.

Figure 1: Campylobacter species bacterial counts in live broilers from the control and antibiotic treatments from weeks 1 through 6. Data are means from two replications. SEM=4.9738. Superscript letters a and b show treatment means with different superscripts for the same week are significantly different (P<0.05).

There was no significant difference in the enumeration of Campylobacter spp. in the antibiotic treatment (P>0.05) and the control treatment in weeks 1 through 5. However, the Campylobacter spp. counts were significantly higher in the control treatment than the antibiotic treatment (P<0.05) at week 6 (Figure 1).

For the overall experiment, the Campylobacter spp. counts in the antibiotic treatment were lower than in the control treatment (Figure 1). These results suggest that the antibiotic sulfadimethoxine, as applied in this experiment can reduce the counts of Campylobacter spp. in the broilers.

Prevalence of Campylobacter spp. and C. jejuni

For each of the test weeks, the prevalence of Campylobacter spp. in individual broilers from the control treatment ranged from 37.3% (112 of 300) to 66.7% (200 of 300). From the antibiotic treatment, the prevalence ranged from 13.3% (40 of 300) to 65.3% (196 of 300) (Table 1). At week 1, the prevalence of Campylobacter spp. was significantly lower in the antibiotic treatment (P<0.05) than in the control treatment (Table 1). In weeks 2 and 3, the prevalence of Campylobacter spp. declined by 63.0% in the antibiotic treatment but increased by 43.0% in the control treatment.

Table 1: The prevalence of Campylobacter spp. in live broilers from the control and antibiotic treatments from weeks 1 through 6.

In week 3, the prevalence of Campylobacter spp. was significantly higher in the control treatment than the antibiotic treatment (P<0.05) (Table 1). Specifically, there was a 13.3% (40 of 300) incidence in the antibiotic treatment and a 53.3% (160 of 300) incidence in the control treatment. These finding showed that the antibiotic sulfadimethoxine can reduce the prevalence of Campylobacter spp. in broilers especially in week 3 (Table 1).

In week 4, the prevalence of Campylobacter spp. increased to 52.0%, (156 of 300) in the antibiotic treatment but it was unchanged at 53.3% (160 of 300) in the control treatment. In week 5, the prevalence of Campylobacter spp. declined somewhat in both treatments. Specifically, the prevalence was measured at 41.3% (124 of 300) in the antibiotic treatment and at 42.7% (128 of 300) in the control treatment (Table 1). In week 6, the prevalence of Campylobacter spp. in the control treatment was 66.7% (200 of 300) and in the antibiotic treatment was 65.3% (196 of 300) (Table 1). Overall, for the six-week period of testing, the prevalence of Campylobacter spp. in the antibiotic treatment was lower (P<0.05) than in the control treatment (Table 1).

Additionally, the overall prevalence of C. jejuni in the control treatment ranged from 33.3% (100 of 300) to 56.0% (168 of 300) and from 13.3% (40 of 300) to 50.0% (150 of 300) in the antibiotic treatment (Table 2). In week 1, the prevalence of C. jejuni was significantly higher (P<0.05) in the control treatment at 41.3% (124 of 300) than in the antibiotic treatment at 25.3% (76 of 300) (Table 1). In week 2, the prevalence of C. jejuni was the same in both control and antibiotic treatments at 33.3% (100 of 300) (Table 2). In week 3, the prevalence of C. jejuni declined in the antibiotic treatment to 13.3% (40 of 300) whereas, it increased to 45.3% (136 of 300) in the control treatment. These values represent a significant difference (P<0.05) (Table 2).

Table 2: The prevalence of C. jejuni in live broilers from the control and antibiotic treatments from weeks 1 through 6.

In week 4, the prevalence of C. jejuni was 45.3% (136 of 300) in the antibiotic treatment and 48.0% (144 of 300) in the control treatment (Table 2). These values are not significantly different (P>0.05) (Table 2). In week 5, the prevalence of C. jejuni was at 41.3% (124 of 300) in the antibiotic treatment and at 42.7% (128 of 300) in the control treatment (Table 2). These values are not significantly different (P>0.05) (Table 2). In week 6, the prevalence of C. jejuni was significantly higher (P<0.05) in the control treatment at 56.0% (168 of 300) than in the antibiotic treatment at 50.0% (150 of 300) (Table 2). For the overall experiment, the prevalence of C. jejuni in the antibiotic treatment was significantly lower (P<0.05) than in the control treatment (Table 2). These results suggest that the antibiotic sulfadimethoxine, as applied in this experiment can reduce the prevalence of C. jejuni in the broilers.

This study suggests that the antibiotic sulfadimethoxine can reduce the bacterial counts of Campylobacter spp. and the prevalence of Campylobacter spp. and C. jejuni in small-scale poultry farming. This is the first study in which sulfadimethoxine has been used in an in vivo setting to control Campylobacter.

Results from previous studies whose aim was to control C. jejuni in poultry are mixed. For example, the previous research [16] attempted to inhibit C. jejuni in chickens through the administration of the live bacterium Enterococcus faecalis. As they reported, this bacterium failed to inhibit the growth of C. jejuni. In a second study using a bacteriophage lytic cocktail administered to chickens resulted in a 2 log CFU/ml reduction in the counts of C. jejuni [14]. In a third study using turkeys, the administration of the antibiotics enrofloxacin, neomycin and vancomycin resulted in a respective decrease of 1, 2 and 4 log CFU/ml of C. jejuni [15]. For purposes of comparison, the present study found that the use of sulfadimethoxine resulted in a decrease of 0.93 log CFU/broiler in the experimental group as compared to the control group. Therefore, the positive results from this study compare favorably with the previously mentioned second and third studies.

The quantitative risk assessment model [31] suggests that reducing Campylobacter spp. levels by 1, 2 and 3 log CFU/ml could result in a reduction in the prevalence of Campylobacter spp. by 55%, 81% and 94% respectively. However, this model is not supported by the present study. Specifically, the enumeration of Campylobacter spp. in the experimental group decreased by 0.93 log CFU/ml as compared to the control group but the prevalence of Campylobacter spp. was only reduced by 1.34%. Subsequent studies may investigate the addition of sulfadimethoxine at varied levels or in combination with other antimicrobials to control or prevent C. jejuni during the growing phase of broilers.

This work was partly funded by the McNeese State University Juliet Hardtner Women in Science and Technology Endowed Professorship. We thank Dr. Ray Neyland and Mrs. Jan Prudhomme who provided assistance for this study.