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Efficacy of Iron Chelators on Campylobacter Concentrations in Turkey Semen¹

K. Cole^*, A. M. Donoghue, I. Reyes-Herrera^*, N. Rath and D. J. Donoghue^*,2

^* Department of Poultry Science, University of Arkansas, Fayetteville 72701; and Poultry Production and Product Safety Research Unit, ARS, USDA, Fayetteville, AR 72701

² Corresponding author: ddonogh{at}uark.edu

	ABSTRACT

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Campylobacter is a leading bacterial cause of human foodborne infections in the United States. Recent studies suggest that the organism is highly prevalent in poultry semen and may contribute to vertical transmission between the breeder hen and offspring. Because Campylobacter requires iron for its growth and survival, the objective of this study was to determine if the addition of natural and synthetic chelators such as ovotransferrin, desferrioxaime, EDTA, or 2,2'-dipyridyl could reduce or eliminate Campylobacter in turkey semen. In a preliminary study without semen, a commercial poultry semen extender was supplemented with various concentrations of ovotransferrin, desferrioxaime, EDTA, or 2,2'-dipyridyl and inoculated with an average of 10⁸ cfu/mL of a wild-type Campylobacter coli turkey semen isolate. At 6 and 24 h of storage at 4°C, a sample was taken from each treatment group and enumerated for Campylobacter. In all 3 trials, Campylobacter was undetectable (<10²) in the commercial poultry semen extender supplemented with 20 mg/mL of 2,2'-dipyridyl. There were no differences observed in Campylobacter concentrations in the commercial poultry semen extender supplemented with ovotransferrin, desferrioxaime, or EDTA compared with unsupplemented controls. In a follow-up study, pooled semen samples were randomly collected from toms, diluted with a commercial poultry semen extender supplemented with 5, 10, or 20 mg/mL of 2,2'-dipyridyl and inoculated with an average of 10⁸ cfu/mL of a wild-type C. coli turkey semen isolate. At 6 and 24 h of storage at 4°C, samples were taken from each treatment group, enumerated for Campylobacter, and evaluated for sperm viability. In all 3 trials, supplementing the commercial poultry semen extender with 20 mg/mL of 2,2'-dipyryidyl significantly reduced (3 to 4 logs) Campylobacter concentrations when compared with the positive controls. Sperm viability was also reduced with this treatment, and, therefore, the use of 2,2'-dipyridyl may not be a practical treatment for reducing Campylobacter in poultry semen.

Key Words: Campylobacter • iron • turkey • semen

	INTRODUCTION

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Campylobacter is one of the leading bacterial causes of human foodborne infections in the United States (Friedman et al., 2000; Centers for Disease Control and Prevention, 2005). Epidemiological evidence has emphasized the importance of poultry products as a significant source of human Campylobacter infection (Jacobs-Reitsma, 2000; Corry and Attabay, 2001). Recent studies suggest that the organism is highly prevalent in turkey semen and may contribute to vertical transmission between the breeder hen and offspring (Cox et al., 2002; Cole et al., 2004a,b). Semen on commercial turkey farms is routinely pooled and used to inseminate multiple hens, and, therefore, may be a potential source of Campylobacter contamination in the female reproductive tract and subsequent eggs. Unfortunately, strategies to reduce or eliminate Campylobacter in poultry semen, such as aeration, reduced storage temperatures, and dilution with extenders containing antibiotics have not been completely effective (Cole et al., 2004b; Donoghue et al., 2004).

Campylobacter, like most organisms, requires iron for its growth and survival (van Vliet et al., 2002; Palyada et al., 2004). Numerous in vitro studies have demonstrated that limiting the availability of iron in the environment can inhibit certain strains of Escherichia coli (Chart and Rowe, 1993), Salmonella (Chart and Rowe, 1993; Lisiecki et al., 2000; Ho et al., 2004), and Campylobacter (Field et al., 1986; Holmes et al., 2005). Limiting iron can be accomplished by the addition of natural or synthetic chelators such as ovotransferrin, desferrioxaime, EDTA, and 2,2'-dipyridyl to the growth media (Chart and Rowe, 1993; Bergan et al., 2001; Ho et al., 2004). Theoretically, supplementing semen diluents with these iron-chelating agents should reduce or eliminate Campylobacter concentrations in poultry semen. In addition, desferrioxaime has been used to improve sperm viability during in vitro storage in some species (Vishwanath et al., 1994). Therefore, the objective of the present study was to determine whether supplementation with natural and synthetic chelators could reduce Campylobacter concentrations in pooled turkey semen.

	MATERIALS AND METHODS

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In preliminary dose titration studies conducted without semen, 0.25 mL of Campylobacter enrichment broth (CEB) containing an average of 10⁸ cfu/mL of a wild-type Campylobacter coli semen isolate was added to 1.75 mL of Field Ready Green Extender (no antibiotics; IMV International Corp., Maple Grove, MN) supplemented with either 0.5, 5, or 50 mg/mL of ovotransferrin (Sigma Chemical Co., St. Louis, MO); 0.1, 1, or 10 mg/mL of desferrioxaime (Desferal, Ciba-Geigy Corp. Ltd., Basel, Switzerland); 0.1, 1, or 10 mg/mL of EDTA (Sigma Chemical Co.); or 0.2, 2, or 20 mg/mL of 2,2'-dipyridyl (Sigma Chemical Co.) in 3 separate trials. Campylobacter concentrations ranging from 10² to 10⁶ cfu/mL in turkey semen have been previously reported (Cole et al., 2004a); however, we have found levels as high as 10⁸ cfu/mL in turkey semen samples since that report. We chose this higher concentration for challenge because we believed that if we could demonstrate efficacy at the highest levels of Campylobacter in semen this would also be effective at lower concentrations. In the present studies, 1.75 mL of Field Ready Green Extender (IMV International Corp.) inoculated with 0.25 mL of CEB containing an average of 10⁸ cfu/mL of a wild-type C. coli semen isolate served as the positive controls, whereas 1.75 mL of Field Ready Green Extender (IMV International Corp.) inoculated with 0.25 mL of CEB alone served as the negative controls. Each treatment was incubated at 4°C for 24 h with agitation (150 rpm; Thurston et al., 1998). At 6 and 24 h, a 0.1-mL sample was taken from each treatment group and diluted with 0.9 mL of CEB, 10-fold serial dilutions in CEB were performed, and 0.1 mL of each dilution was plated on Campy-Line agar (CLA; Line, 2001) and incubated at 42°C for 48 h in a microaerophilic environment (5% O₂, 10% CO₂, 85% N₂). After incubation, characteristic colonies were confirmed as Campylobacter by observation of typical cellular morphology using a phase contrast microscope and with a commercial latex agglutination test kit (Pan Bio Inc., Columbia, MD) specific for Campylobacter jejuni, C. coli, and Campylobacter laridis. The colonies on each CLA plate were counted on a Leico Darkfield plate colony counter (Leica Inc., Buffalo, NY), and the direct counts were converted to log₁₀ colony-forming units per milliliter of extender.

In a follow-up study, pooled semen samples from commercial toms were randomly collected by abdominal massage (Burrows and Quinn, 1937) and aspirated into sterile test tubes. In 3 separate trials, the pooled semen samples were diluted 1:4 (vol:vol) with Field Ready Green Extender (no antibiotics; IMV International Corp.) and divided into 5 treatment groups. The positive and negative control groups received no 2,2'-dipyridyl, whereas the remaining 3 treatment groups were supplemented with either 5, 10, or 20 mg/mL of 2,2'-dipyridyl. Each treatment group, except for the negative control, was then inoculated with 0.25 mL of CEB containing an average of 10⁸ cells/mL of a wild-type C. coli semen isolate. Each treatment was incubated at 4°C for 24 h with agitation (150 rpm; Thurston et al., 1998). At 6 and 24 h of storage, a 0.1-mL sample was taken from each treatment group and serially diluted with CEB. The dilutions were plated on CLA and evaluated for Campylobacter as previously described. Sperm motility was assessed for each treatment group at 6 and 24 h of storage according to the hanging drop method of Wishart and Wilson (1997).

Data were analyzed by ANOVA using the Statistical Analysis System (SAS Institute Inc., 1998) GLM program. The numbers of Campylobacter colonies were logarithmically transformed (log₁₀ cfu/mL) prior to analysis to achieve homogeneity of variance (Byrd et al., 2003; Cole et al., 2004b; Farnell et al., 2005). Treatment means were partitioned by LSMEANS analysis (SAS Institute Inc., 1998). Sperm motility data expressed as percentages (Table 1) were arc sine transformed before analysis. A probability of P < 0.05 was required for statistical significance. The data in Tables 2 and 3 are shown as arithmetic means for clarity of presentation.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

In a follow-up study, pooled turkey semen was diluted with a commercial poultry semen extender supplemented with 5, 10, or 20 mg/mL of 2,2'-dipyridyl. Similar to the results in the previous study, the only reduction in Campylobacter concentrations was observed at 6 or 24 h of storage in commercial poultry semen and extender supplemented with 20 mg/mL of 2, 2'-dipyridyl (Table 3). However, the presence of 2,2'-dipyridyl adversely affected sperm motility (Table 1).

The mechanism by which 2,2'-dipyridyl reduced Campylobacter concentrations in these studies is unclear. It has been reported that 2,2'-dipyridyl can cause lysis in bacterial cells (Neilands, 1982; Chart et al., 1986). In addition, 2,2'-dipyridyl is able to bind cellular iron effectively, producing reactive oxygen species that lead to apoptosis in cancer cells (Yuan et al., 2004). Although reactive oxygen species production was not measured in the semen in this study, this may be a possible explanation for the bactericidal effects observed in this study. The production of free radicals may also explain the spermicidal effects observed in this study, as they can cause irreversible damage in sperm membranes (Wishart, 1984; Ravie and Lake, 1985).

In conclusion, Campylobacter concentrations were significantly reduced after 6 or 24 h of storage at 4°C in a commercial poultry semen extender supplemented with 20 mg/mL of 2,2'-dipyridyl. This approach, however, is not a practical solution to reduce Campylobacter concentrations in semen, because this treatment also reduced sperm motility. Further studies are needed to find a practical means of reducing or eliminating pathogens in poultry semen without adversely affecting sperm viability and subsequent function.

	FOOTNOTES

 
¹ This research has been supported in part by the U.S. Egg and Poultry Association (project #394) and the Food Safety Consortium. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that are suitable.

Received for publication January 11, 2006. Accepted for publication March 20, 2006.

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