- Email: [email protected]
Identification of heritage chicken breeds with diminished susceptibility to intestinal colonization by multiple antibiotic-resistant Salmonella spp.
Livestock Science 182 (2015) 34–37
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Identification of heritage chicken breeds with diminished susceptibility to intestinal colonization by multiple antibiotic-resistant Salmonella spp. Kristi L. Anderson a, Matthew T. Brewer b, Mark A. Rasmussen c, Steve A. Carlson a,n a
Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA c The Leopold Center for Sustainable Agriculture, Iowa State University, Ames, IA 50011, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2015 Received in revised form 20 October 2015 Accepted 22 October 2015
Salmonella are commensal bacteria frequently present in the intestinal tracts of commercial poultry. Unfortunately, these microbes will contaminate the flesh of the bird during slaughter, thus creating one of the most important food safety hazards. It is clear that commercial broilers are susceptible to intestinal colonization by Salmonella, but it is unclear if this susceptibility is related to selective breeding for rapid growth and increased feed efficiency. Since non-commercial heritage breeds have genetic profiles diverging from the commercial broiler, we hypothesized that some heritage breeds may exhibit diminished susceptibility to Salmonella colonization of the intestine. To test this hypothesis, we experimentally infected 38 heritage breeds and one commercial breed (Cobb 500) with Salmonella enterica. One week after infection, we enumerated the Salmonella present in the intestinal tracts of the birds.Four breeds (Dark Cornish, New Hampshire Red, Red Ranger, and Sicilian Buttercup) exhibited diminished susceptibility while four other breeds (Anacona, Black Australorpe, Blue Andalusian, and Cuckoo Maran) demonstrated elevated susceptibility. Thirty-one breeds (including the commercial chicken breed) demonstrated a moderate susceptibility to Salmonella colonization. It thus appears that a few heritage breeds are hyposusceptible to colonization by Salmonella while most of the tested heritage breeds exhibited susceptibility that is similar to or greater than that observed in a commercial breed of chicken. & 2015 Elsevier B.V. All rights reserved.
Keywords: Salmonella Food safety Heritage breed Chicken
1. Introduction Most Salmonella, except for serovars Pullorum and Gallinarum (Wilson et al., 2000), act as commensal microbes in the intestinal tracts of poultry. These microbes colonize the distal part of the intestinal tract (Barrow et al., 1994) and are shed in the feces (Traub-Dargatz et al., 2006). Unfortunately, these microbes will contaminate the flesh of the bird during slaughter, thus creating one of the most important food safety hazards. In the U.S. there are just a few breeds of commercial broilers that represent birds with the genetic capacity for rapid growth and enhanced feed efficiency. Selecting for these traits does not prevent the intestinal colonization of Salmonella which is unfortunate since Salmonella can have a deleterious impact upon the production efficiency of the bird (Wang et al., 2014). Therefore, identification of breeds that are resistant to colonization is of interest
because incorporation of these genetics into commercial broilers would result in both economic and food safety benefits. Heritage breeds of chickens are those in which genetic selection has not necessarily focused on maximizing production traits (Schmidt et al., 2009). These birds have been selected for unique phenotypes such as feather color, egg laying, thermotolerance, etc. Studies with Salmonella colonization in the intestines of heritage breeds are lacking. In light of this gap in knowledge and the ubiquity of Salmonella in the commercial broiler, this study focused on determining the scope of Salmonella susceptibility in a significant number of heritage breeds. The susceptibilities were compared to that observed in a single representative commercial breed.
2. Methods 2.1. Animal care
n
Corresponding author. E-mail address: [email protected] (S.A. Carlson).
http://dx.doi.org/10.1016/j.livsci.2015.10.018 1871-1413/& 2015 Elsevier B.V. All rights reserved.
One-day old chicks were obtained from five different
K.L. Anderson et al. / Livestock Science 182 (2015) 34–37
hatcheries (Murray McMurray, eFowl, Backyard Chickens, My Pet Chicken, and Welp), and shipped to the research facility via the U. S. Postal Service. Birds were housed indoors in plastic totes in humidity- ( 40%) and temperature-controlled (35 °C for three days then 31 °C for the remainder of the study) rooms in a BL-2 facility at Iowa State University (approved by the Iowa State University Animal Care and Use Committee, protocol number 11-147901-G). Breeds included: Ancona, Araucana, Barred Rock, Black Australorp, Black Cochin, Black Giant, Black Jersey, Black Langshans, Black Minorca, Black Star, Blue Andalusian, Buckeye, Buff Brahma, Buff Orpington, Cobb 500, Columbian Wyandotte, Cornish Game Hen, Cuckoo Maran, Cubalaya, Dark Cornish, Delaware, Easter Egg, Egyptian, Faroulle, Giant White Cochin, Gold Polish, Iowa Blue, Jumbo Cornish Cross, Light Brahma, New Hampshire Red, Red Ranger, Red Silver Laced Wyandotte, Salmon Faverolle, Sicilian Buttercup, Silver Lakenvelder, Silver Spangled, Speckled Sussex, Welsummer, and White Sultan. 2.2. Initial experimental challenge with Salmonella enterica serotype Typhimurium At day two of age, all chicks were verified to be Salmonella-free by fecal culture in which approximately 5 gm of feces were collected into 10 mL of Lennox broth (Invitrogen) of which 100 μL was plared on XLD agar (Becton Dickinson) and incubated overnight at 37 °C. At three days of age, chicks (n ¼15/breed, three from each commercial source whenever possible) were orally inoculated with 2 108 colony-forming units (CFUs) of Salmonella enterica serotype Typhimurium strain LNWI (Wu et al., 2002). 2.3. . Experimental challenge with multiple doses of S. enterica serotype Typhimurium At three days of age, chicks (n ¼3–5/breed/dose) were orally inoculated with 107 to 1012 CFUs of Salmonella enterica serotype Typhimurium strain LNWI. For the eight individual breeds demonstrating low or high susceptibility to colonization to the single dose (2 108 CFUs) of S. enterica serotype Typhimurium (Dark Cornish, New Hampshire Red, Red Ranger, Sicilian Buttercup, Anacona, Black Australorp, Blue Andalusian, and Cuckoo Maran), five birds were used for each dose. For the 31 breeds demonstrating moderate susceptibility to colonization, three birds were used for each dose.
35
this Lennox broth (i.e., dilution factor equal to 200) was plated on XLD agar plates containing two antibiotics (ampicillin and florfenicol) selective for strain LNWI. Bacteria plated were incubated 16 h at 37 °C for colony enumeration the next day. Colonization was calculated by (200)*(number of colonies recovered)/(weight of the intestinal section). 2.6. Statistical analyses Statistical comparisons were made using an analysis of variance with Tukey's post hoc test for multiple comparisons. p o0.05 was deemed to indicate statistical significance.
3. Results 3.1. Diminished and elevated susceptibilities to intestinal colonization by Salmonella enterica serotype Typhimurium in eight heritage breeds of chickens In order to determine the differential susceptibilities to colonization by Salmonella, we experimentally infected 39 different breeds of chickens. For initial screening purposes we used Salmonella enterica serovar Typhimurium which is a common serovar linked to food-associated salmonellosis in humans (Jackson et al., 2013). At one week post-infection, birds were euthanized and Salmonella intestinal colonization was evaluated. As shown in Fig. 1, four breeds (Dark Cornish, New Hampshire Red, Red Ranger, and Sicilian Buttercup) exhibited diminished susceptibility while four other breeds (Anacona, Black Australorpe, Blue Andalusian, and Cuckoo Maran) demonstrated elevated susceptibility. The other 31 breeds, including the commercial chicken breed, demonstrated moderate susceptibility. 3.2. Dose-dependent diminished and elevated susceptibility to
2.4. Experimental challenge with multiple serovars of S. enterica At three days of age, chicks (n ¼3–5/breed/dose) were orally inoculated with 2 108 CFUs of 10 different poultry-associated serovars of Salmonella enterica - Anatum, Cerro, Enteriditis, Give, Heidelberg, Infantis, Kentucky, Mbandaka, Meleagridis, and Muenster. The isolates representing each serovar were resistant to ampicillin and florfenicol. For the eight individual breeds demonstrating low or high susceptibility to colonization, five birds were used for each dose. For the 31 breeds demonstrating moderate susceptibility to colonization, three birds were used for each dose. 2.5. Enumeration of Salmonella in the chick intestinal tract At 7 days post-inoculation (a time frame that insures colonization based on empirical studies), chicks were euthanized (130 mg pentobarbital, intraperitoneal) and a 5–10 cm section of large intestine (between the ceca and the cloaca) intestine was removed from each chick and placed in 20 mL of Lennox broth. The intestinal sections were cut longitudinally, weighed, and vortexed while present in the Lennox broth. After 1 h, 100 μL of
Fig. 1. Salmonella enterica serotype Typhimurium intestinal colonization of 38 heritage-type breeds of chickens. One day-old chicks were obtained from five commercial sources (Murray McMurray, eFowl, Backyard Chickens, My Pet Chicken, and Welp). At three days of age, chicks (n ¼15/breed, three from each commercial source whenever possible) were orally inoculated with 2 108 colony-forming units (CFUs) of Salmonella enterica serotype Typhimurium strain LNWI. At 7 days post-inoculation, chicks were euthanized and a 5–10 cm section of intestine was removed from each chick and subjected to Salmonella enumeration. The eight breeds highlighted all exhibited a susceptibility to colonization that was significantly different (p o 0.05) than the 31 moderately-colonized breeds.
36
K.L. Anderson et al. / Livestock Science 182 (2015) 34–37
Fig. 2. Dose-dependent diminished suceptibility to Salmonella enterica serotype Typhimurium intestinal colonization of four heritage-type breeds of chicks. At three days of age, chicks (n ¼5/breed) were orally inoculated with 2 108 CFUs of Salmonella enterica serotype Typhimurium strain LNWI. Colonization was determined as indicated in the caption for Fig. 1. For the moderately- and highlycolonized breed groups, each point represents three chicks from each breeds. The four breeds highlighted all exhibited a susceptibility to colonization that was significantly different (p o 0.05) than the 31 moderately-colonized breeds and the four highly-colonized breeds.
Fig. 3. Diminished Salmonella enterica intestinal colonization of four heritage-type breeds of chicks across 10 different serovars. At three days of age, chicks (n¼ 5/ breed) were orally inoculated with 2 108 CFUs of Salmonella enterica. Colonization was determined as indicated in the caption for Fig. 1. For the moderately- and highly-colonized breed groups, each point represents three chicks from each breed. The four breeds low colonization breeds all exhibited a susceptibility to colonization that was significantly different (po 0.05) than the 31 moderately colonized breeds and the four highly colonized breeds.
intestinal colonization by Salmonella enterica serotype Typhimurium in eight heritage breeds of chickens
Buttercup. Dark Cornish are a British/Indian breed with excellent meat and egg production characteristics. The New Hampshire Red is a U.S. breed that also has excellent meat and egg production characteristics. The Red Ranger is a heat-tolerant meat bird with moderate egg production. The Sicilian Buttercup is a Mediterranean breed with moderate egg production and poor meat production (brief descriptions of each breed can be found on the Murray McMurray website- mcmurrayhatchery.com). That is, there is no known common background for the four breeds with diminished susceptibility. For the four breeds with elevated susceptibility to Salmonella colonization (Anacona, Black Australorp, Blue Andalusian, and Cuckoo Maran), there also is no common thread. The Anacona is a Mediterranean breed with excellent egg and meat characteristics. The Black Australorp (aka, the Black Australian Orpington) is an Australian breed with superior egg production and excellent meat production. The Blue Andalusian is a Spanish breed with moderate egg and meat production capacities. The Cuckoo Maran is a French breed with excellent egg production and moderate meat production (brief descriptions of each breed can be found on the Murray McMurray website- mcmurrayhatchery.com). Therefore, meat and egg production characteristics do not predict susceptibility to colonization by Salmonella in these heritage breeds. The results of this study identify susceptible or resistant breeds of chickens, however, the molecular mechanisms resulting in these phenotypes has not yet been elucidated. It is possible that the host proteins exploited by Salmonella during the colonization process (Schleker et al., 2012) are minimally expressed in the breeds with diminished susceptibility. Conversely, it is possible that the proteins exploited by Salmonella during the colonization process are hyper-expressed in the breeds with elevated susceptibility. Ongoing studies will address these hypotheses. Another possibility is supported by a study in which a commercial breed was found to have a longer intestinal tract than a heritage breed (Schmidt et al., 2009), raising the possibility of greater surface area for Salmonella colonization.
Since the studies presented in Fig. 1 revealed differing susceptibilities to a single dose of Salmonella enterica serovar Typhimurium, these studies were repeated using six doses of the same microbe. As shown in Fig. 2, the susceptibility pattern was observed across all six doses of Salmonella enterica serovar Typhimurium. 3.3. diminished and elevated susceptibilities to intestinal colonization by ten different serotypes of Salmonella enterica in eight heritage breeds of chickens Although Salmonella enterica serovar Typhimurium is a major poultry-associated serovar, other serovars are frequently isolated from chickens. To examine the extension of the susceptibility pattern across an array of serovars, studies presented in Fig. 1 were repeated with ten different serovars of S. enterica (Anatum, Cerro, Enteriditis, Give, Heidelberg, Infantis, Kentucky, Mbandaka, Meleagridis, and Muenster). As shown in Fig. 3, the breed susceptibility pattern extended across all 10 serovars.
4. Discussion Salmonella colonization of the chicken intestinal tract is a critical food safety hazard. In the U.S., the commercial breeds of chicken have been selected for optimal growth characteristics to maximize production efficiencies. Unfortunately this selection process does not prevent Salmonella colonization since this microbe is mostly a commensal in birds (Foley et al., 2013). However, discovery of genetic lines associated with resistance to intestinal colonization is highly desirable so that commercial flocks could overcome this problem. The studies presented herein reveal a diminished susceptibility to Salmonella colonization in four non-commercial heritage breeds of chickens. This diminished susceptibility was manifested by a quantitative (Figs. 1–3) and a qualitative (Table 1) decrease in Salmonella recovered from these breeds. These breeds include the Dark Cornish, New Hampshire Red, Red Ranger, and Sicilian
5. Conclusions In summary, this study reveals differing susceptibilities to Salmonella colonization in heritage breeds of chickens. While we did
K.L. Anderson et al. / Livestock Science 182 (2015) 34–37
37
Table 1 Prevalences of birds experimentally colonized by Salmonella across the different breeds, doses of S. enterica serotype Typhimurium, and the 10 other serovars. Values are the mean 7 sem representing the percentage of birds that yielded at least one CFU of Salmonella from the intestinal tract. Breed(s)
Dark Cornish New Hampshire Red Red Ranger Sicilian Buttercup 31 moderately susceptible breeds Anacona Black Australorp Blue Andalusian Cuckoo Maran
Dose of S. enterica serotype Typhimurium (CFUs/bird)
10 other serovars
107
108
2 108
109
1010
1011
1012
0* 0* 0* 0* 127 4 107 4 87 2 107 5 117 3
67 2* 127 3* 127 2* 67 2* 187 1 267 3* 327 4* 187 4 267 3*
127 4* 127 4* 127 4* 67 2* 237 3 357 5* 427 7* 367 5* 297 5
187 4* 127 4* 187 4* 127 2* 287 1 447 6* 487 9* 517 14* 437 9*
247 6* 12 74* 187 4* 247 6* 47 712 100* 100* 100* 100*
247 6* 337 5* 337 5* 337 6* 677 13 100* 100* 100* 100*
24 76* 33 75* 33 75* 33 76* 100 100* 100* 100* 100*
67 2* 127 4* 247 8 67 2* 257 5 377 12 357 1* 447 6* 397 3*
*p o0.05 versus the moderately susceptible breeds.
not examine all heritage breeds, we found four breeds with diminished susceptibility when compared to 35 other breeds including a commercial chicken breed. This work will provide the basis for identifying traits that could possibly be incorporated into the commercial breeds in order to minimize the colonization of Salmonella in the intestinal tracts of a major protein source.
Conflict of interest The authors declare no competing interests.
Author's contributions KLA performed the bacteriology work. MTB assisted in the in vivo studies. MAR participated in the design of the study and provided funding. SAC conceived the study, assisted in both the in vitro and in vivo phases, and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgments The study was funded through a grant provided by the Leopold Center for Sustainable Agriculture (DSP2013-01) at Iowa State University.
References Barrow, P.A., Huggins, M.B., Lovell, M.A., 1994. Host specificity of Salmonella infection in chickens and mice is expressed in vivo primarily at the level of the reticuloendothelial system. Infect. Immun. 62, 4602–4610. Foley, S., Johnson, T., Ricke, S., Nayak, R., Danzeisen, J., 2013. Salmonella pathogenicity and host adaptation in chicken-associated serovars. Microbiol. Mol. Biol. Rev. 77, 582–607. Jackson, B., Griffin, P., Cole, D., Walsh, K., Chai, S., 2013. Outbreak-associated Salmonella enterica serotypes and food Commodities, United States, 1998–2008. Emerg. Infect. Dis. 19, 1239–1344. Schleker, S., Sun, J., Raghavan, B., Srnec, M., Müller, N., Koepfinger, M., Murthy, L., Zhao, Z., Klein-Seetharaman, J., 2012. The current Salmonella-host interactome. Proteom. Clin. Appl. 6, 117–133. Schmidt, C., Persia, M., Feierstein, E., Kingham, B., Saylor, W., 2009. Comparison of a modern broiler line and a heritage line unselected since the 1950s. Poult. Sci. 88, 2610–2619. Traub-Dargatz, J., Ladel, S., Dargatz, D., Fedorka-Cray, P., 2006. Impact of heat stress on the fecal shedding patterns of Salmonella enterica Typhimurium DT104 and Salmonella enterica infantis by 5-week-old male broilers. Foodborne Pathog. Dis. 3, 178–183. Wang, C., Fan, Y., Chun-Hsien, T., Chiu, C., Tsai, H., Chou, C., 2014. Salmonella Enteritidis infection slows steroidogenesis and impedes cell growth in hen granulosa cells. Avian Dis. 58, 511–517. Wilson, R., Elthon, J., Clegg, S., Jones, B., 2000. Salmonella enterica serovars gallinarum and pullorum expressing Salmonella enterica serovar typhimurium type 1 fimbriae exhibit increased invasiveness for mammalian cells. Infect. Immun. 63, 4782–4785. Wu, M., Carlson, S.A., Meyerholz, D.K., 2002. Cytopathic effects observed upon expression of a repressed collagenase gene present in Salmonella and related pathogens: mimicry of a cytotoxin from multiple antibiotic-resistant Salmonella enterica serotype Typhimurium phagetype DT104. Microb. Pathog. 32, 1–9.
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Identification of heritage chicken breeds with diminished susceptibility to intestinal colonization by multiple antibiotic-resistant Salmonella spp. Kristi L. Anderson a, Matthew T. Brewer b, Mark A. Rasmussen c, Steve A. Carlson a,n a
Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA c The Leopold Center for Sustainable Agriculture, Iowa State University, Ames, IA 50011, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2015 Received in revised form 20 October 2015 Accepted 22 October 2015
Salmonella are commensal bacteria frequently present in the intestinal tracts of commercial poultry. Unfortunately, these microbes will contaminate the flesh of the bird during slaughter, thus creating one of the most important food safety hazards. It is clear that commercial broilers are susceptible to intestinal colonization by Salmonella, but it is unclear if this susceptibility is related to selective breeding for rapid growth and increased feed efficiency. Since non-commercial heritage breeds have genetic profiles diverging from the commercial broiler, we hypothesized that some heritage breeds may exhibit diminished susceptibility to Salmonella colonization of the intestine. To test this hypothesis, we experimentally infected 38 heritage breeds and one commercial breed (Cobb 500) with Salmonella enterica. One week after infection, we enumerated the Salmonella present in the intestinal tracts of the birds.Four breeds (Dark Cornish, New Hampshire Red, Red Ranger, and Sicilian Buttercup) exhibited diminished susceptibility while four other breeds (Anacona, Black Australorpe, Blue Andalusian, and Cuckoo Maran) demonstrated elevated susceptibility. Thirty-one breeds (including the commercial chicken breed) demonstrated a moderate susceptibility to Salmonella colonization. It thus appears that a few heritage breeds are hyposusceptible to colonization by Salmonella while most of the tested heritage breeds exhibited susceptibility that is similar to or greater than that observed in a commercial breed of chicken. & 2015 Elsevier B.V. All rights reserved.
Keywords: Salmonella Food safety Heritage breed Chicken
1. Introduction Most Salmonella, except for serovars Pullorum and Gallinarum (Wilson et al., 2000), act as commensal microbes in the intestinal tracts of poultry. These microbes colonize the distal part of the intestinal tract (Barrow et al., 1994) and are shed in the feces (Traub-Dargatz et al., 2006). Unfortunately, these microbes will contaminate the flesh of the bird during slaughter, thus creating one of the most important food safety hazards. In the U.S. there are just a few breeds of commercial broilers that represent birds with the genetic capacity for rapid growth and enhanced feed efficiency. Selecting for these traits does not prevent the intestinal colonization of Salmonella which is unfortunate since Salmonella can have a deleterious impact upon the production efficiency of the bird (Wang et al., 2014). Therefore, identification of breeds that are resistant to colonization is of interest
because incorporation of these genetics into commercial broilers would result in both economic and food safety benefits. Heritage breeds of chickens are those in which genetic selection has not necessarily focused on maximizing production traits (Schmidt et al., 2009). These birds have been selected for unique phenotypes such as feather color, egg laying, thermotolerance, etc. Studies with Salmonella colonization in the intestines of heritage breeds are lacking. In light of this gap in knowledge and the ubiquity of Salmonella in the commercial broiler, this study focused on determining the scope of Salmonella susceptibility in a significant number of heritage breeds. The susceptibilities were compared to that observed in a single representative commercial breed.
2. Methods 2.1. Animal care
n
Corresponding author. E-mail address: [email protected] (S.A. Carlson).
http://dx.doi.org/10.1016/j.livsci.2015.10.018 1871-1413/& 2015 Elsevier B.V. All rights reserved.
One-day old chicks were obtained from five different
K.L. Anderson et al. / Livestock Science 182 (2015) 34–37
hatcheries (Murray McMurray, eFowl, Backyard Chickens, My Pet Chicken, and Welp), and shipped to the research facility via the U. S. Postal Service. Birds were housed indoors in plastic totes in humidity- ( 40%) and temperature-controlled (35 °C for three days then 31 °C for the remainder of the study) rooms in a BL-2 facility at Iowa State University (approved by the Iowa State University Animal Care and Use Committee, protocol number 11-147901-G). Breeds included: Ancona, Araucana, Barred Rock, Black Australorp, Black Cochin, Black Giant, Black Jersey, Black Langshans, Black Minorca, Black Star, Blue Andalusian, Buckeye, Buff Brahma, Buff Orpington, Cobb 500, Columbian Wyandotte, Cornish Game Hen, Cuckoo Maran, Cubalaya, Dark Cornish, Delaware, Easter Egg, Egyptian, Faroulle, Giant White Cochin, Gold Polish, Iowa Blue, Jumbo Cornish Cross, Light Brahma, New Hampshire Red, Red Ranger, Red Silver Laced Wyandotte, Salmon Faverolle, Sicilian Buttercup, Silver Lakenvelder, Silver Spangled, Speckled Sussex, Welsummer, and White Sultan. 2.2. Initial experimental challenge with Salmonella enterica serotype Typhimurium At day two of age, all chicks were verified to be Salmonella-free by fecal culture in which approximately 5 gm of feces were collected into 10 mL of Lennox broth (Invitrogen) of which 100 μL was plared on XLD agar (Becton Dickinson) and incubated overnight at 37 °C. At three days of age, chicks (n ¼15/breed, three from each commercial source whenever possible) were orally inoculated with 2 108 colony-forming units (CFUs) of Salmonella enterica serotype Typhimurium strain LNWI (Wu et al., 2002). 2.3. . Experimental challenge with multiple doses of S. enterica serotype Typhimurium At three days of age, chicks (n ¼3–5/breed/dose) were orally inoculated with 107 to 1012 CFUs of Salmonella enterica serotype Typhimurium strain LNWI. For the eight individual breeds demonstrating low or high susceptibility to colonization to the single dose (2 108 CFUs) of S. enterica serotype Typhimurium (Dark Cornish, New Hampshire Red, Red Ranger, Sicilian Buttercup, Anacona, Black Australorp, Blue Andalusian, and Cuckoo Maran), five birds were used for each dose. For the 31 breeds demonstrating moderate susceptibility to colonization, three birds were used for each dose.
35
this Lennox broth (i.e., dilution factor equal to 200) was plated on XLD agar plates containing two antibiotics (ampicillin and florfenicol) selective for strain LNWI. Bacteria plated were incubated 16 h at 37 °C for colony enumeration the next day. Colonization was calculated by (200)*(number of colonies recovered)/(weight of the intestinal section). 2.6. Statistical analyses Statistical comparisons were made using an analysis of variance with Tukey's post hoc test for multiple comparisons. p o0.05 was deemed to indicate statistical significance.
3. Results 3.1. Diminished and elevated susceptibilities to intestinal colonization by Salmonella enterica serotype Typhimurium in eight heritage breeds of chickens In order to determine the differential susceptibilities to colonization by Salmonella, we experimentally infected 39 different breeds of chickens. For initial screening purposes we used Salmonella enterica serovar Typhimurium which is a common serovar linked to food-associated salmonellosis in humans (Jackson et al., 2013). At one week post-infection, birds were euthanized and Salmonella intestinal colonization was evaluated. As shown in Fig. 1, four breeds (Dark Cornish, New Hampshire Red, Red Ranger, and Sicilian Buttercup) exhibited diminished susceptibility while four other breeds (Anacona, Black Australorpe, Blue Andalusian, and Cuckoo Maran) demonstrated elevated susceptibility. The other 31 breeds, including the commercial chicken breed, demonstrated moderate susceptibility. 3.2. Dose-dependent diminished and elevated susceptibility to
2.4. Experimental challenge with multiple serovars of S. enterica At three days of age, chicks (n ¼3–5/breed/dose) were orally inoculated with 2 108 CFUs of 10 different poultry-associated serovars of Salmonella enterica - Anatum, Cerro, Enteriditis, Give, Heidelberg, Infantis, Kentucky, Mbandaka, Meleagridis, and Muenster. The isolates representing each serovar were resistant to ampicillin and florfenicol. For the eight individual breeds demonstrating low or high susceptibility to colonization, five birds were used for each dose. For the 31 breeds demonstrating moderate susceptibility to colonization, three birds were used for each dose. 2.5. Enumeration of Salmonella in the chick intestinal tract At 7 days post-inoculation (a time frame that insures colonization based on empirical studies), chicks were euthanized (130 mg pentobarbital, intraperitoneal) and a 5–10 cm section of large intestine (between the ceca and the cloaca) intestine was removed from each chick and placed in 20 mL of Lennox broth. The intestinal sections were cut longitudinally, weighed, and vortexed while present in the Lennox broth. After 1 h, 100 μL of
Fig. 1. Salmonella enterica serotype Typhimurium intestinal colonization of 38 heritage-type breeds of chickens. One day-old chicks were obtained from five commercial sources (Murray McMurray, eFowl, Backyard Chickens, My Pet Chicken, and Welp). At three days of age, chicks (n ¼15/breed, three from each commercial source whenever possible) were orally inoculated with 2 108 colony-forming units (CFUs) of Salmonella enterica serotype Typhimurium strain LNWI. At 7 days post-inoculation, chicks were euthanized and a 5–10 cm section of intestine was removed from each chick and subjected to Salmonella enumeration. The eight breeds highlighted all exhibited a susceptibility to colonization that was significantly different (p o 0.05) than the 31 moderately-colonized breeds.
36
K.L. Anderson et al. / Livestock Science 182 (2015) 34–37
Fig. 2. Dose-dependent diminished suceptibility to Salmonella enterica serotype Typhimurium intestinal colonization of four heritage-type breeds of chicks. At three days of age, chicks (n ¼5/breed) were orally inoculated with 2 108 CFUs of Salmonella enterica serotype Typhimurium strain LNWI. Colonization was determined as indicated in the caption for Fig. 1. For the moderately- and highlycolonized breed groups, each point represents three chicks from each breeds. The four breeds highlighted all exhibited a susceptibility to colonization that was significantly different (p o 0.05) than the 31 moderately-colonized breeds and the four highly-colonized breeds.
Fig. 3. Diminished Salmonella enterica intestinal colonization of four heritage-type breeds of chicks across 10 different serovars. At three days of age, chicks (n¼ 5/ breed) were orally inoculated with 2 108 CFUs of Salmonella enterica. Colonization was determined as indicated in the caption for Fig. 1. For the moderately- and highly-colonized breed groups, each point represents three chicks from each breed. The four breeds low colonization breeds all exhibited a susceptibility to colonization that was significantly different (po 0.05) than the 31 moderately colonized breeds and the four highly colonized breeds.
intestinal colonization by Salmonella enterica serotype Typhimurium in eight heritage breeds of chickens
Buttercup. Dark Cornish are a British/Indian breed with excellent meat and egg production characteristics. The New Hampshire Red is a U.S. breed that also has excellent meat and egg production characteristics. The Red Ranger is a heat-tolerant meat bird with moderate egg production. The Sicilian Buttercup is a Mediterranean breed with moderate egg production and poor meat production (brief descriptions of each breed can be found on the Murray McMurray website- mcmurrayhatchery.com). That is, there is no known common background for the four breeds with diminished susceptibility. For the four breeds with elevated susceptibility to Salmonella colonization (Anacona, Black Australorp, Blue Andalusian, and Cuckoo Maran), there also is no common thread. The Anacona is a Mediterranean breed with excellent egg and meat characteristics. The Black Australorp (aka, the Black Australian Orpington) is an Australian breed with superior egg production and excellent meat production. The Blue Andalusian is a Spanish breed with moderate egg and meat production capacities. The Cuckoo Maran is a French breed with excellent egg production and moderate meat production (brief descriptions of each breed can be found on the Murray McMurray website- mcmurrayhatchery.com). Therefore, meat and egg production characteristics do not predict susceptibility to colonization by Salmonella in these heritage breeds. The results of this study identify susceptible or resistant breeds of chickens, however, the molecular mechanisms resulting in these phenotypes has not yet been elucidated. It is possible that the host proteins exploited by Salmonella during the colonization process (Schleker et al., 2012) are minimally expressed in the breeds with diminished susceptibility. Conversely, it is possible that the proteins exploited by Salmonella during the colonization process are hyper-expressed in the breeds with elevated susceptibility. Ongoing studies will address these hypotheses. Another possibility is supported by a study in which a commercial breed was found to have a longer intestinal tract than a heritage breed (Schmidt et al., 2009), raising the possibility of greater surface area for Salmonella colonization.
Since the studies presented in Fig. 1 revealed differing susceptibilities to a single dose of Salmonella enterica serovar Typhimurium, these studies were repeated using six doses of the same microbe. As shown in Fig. 2, the susceptibility pattern was observed across all six doses of Salmonella enterica serovar Typhimurium. 3.3. diminished and elevated susceptibilities to intestinal colonization by ten different serotypes of Salmonella enterica in eight heritage breeds of chickens Although Salmonella enterica serovar Typhimurium is a major poultry-associated serovar, other serovars are frequently isolated from chickens. To examine the extension of the susceptibility pattern across an array of serovars, studies presented in Fig. 1 were repeated with ten different serovars of S. enterica (Anatum, Cerro, Enteriditis, Give, Heidelberg, Infantis, Kentucky, Mbandaka, Meleagridis, and Muenster). As shown in Fig. 3, the breed susceptibility pattern extended across all 10 serovars.
4. Discussion Salmonella colonization of the chicken intestinal tract is a critical food safety hazard. In the U.S., the commercial breeds of chicken have been selected for optimal growth characteristics to maximize production efficiencies. Unfortunately this selection process does not prevent Salmonella colonization since this microbe is mostly a commensal in birds (Foley et al., 2013). However, discovery of genetic lines associated with resistance to intestinal colonization is highly desirable so that commercial flocks could overcome this problem. The studies presented herein reveal a diminished susceptibility to Salmonella colonization in four non-commercial heritage breeds of chickens. This diminished susceptibility was manifested by a quantitative (Figs. 1–3) and a qualitative (Table 1) decrease in Salmonella recovered from these breeds. These breeds include the Dark Cornish, New Hampshire Red, Red Ranger, and Sicilian
5. Conclusions In summary, this study reveals differing susceptibilities to Salmonella colonization in heritage breeds of chickens. While we did
K.L. Anderson et al. / Livestock Science 182 (2015) 34–37
37
Table 1 Prevalences of birds experimentally colonized by Salmonella across the different breeds, doses of S. enterica serotype Typhimurium, and the 10 other serovars. Values are the mean 7 sem representing the percentage of birds that yielded at least one CFU of Salmonella from the intestinal tract. Breed(s)
Dark Cornish New Hampshire Red Red Ranger Sicilian Buttercup 31 moderately susceptible breeds Anacona Black Australorp Blue Andalusian Cuckoo Maran
Dose of S. enterica serotype Typhimurium (CFUs/bird)
10 other serovars
107
108
2 108
109
1010
1011
1012
0* 0* 0* 0* 127 4 107 4 87 2 107 5 117 3
67 2* 127 3* 127 2* 67 2* 187 1 267 3* 327 4* 187 4 267 3*
127 4* 127 4* 127 4* 67 2* 237 3 357 5* 427 7* 367 5* 297 5
187 4* 127 4* 187 4* 127 2* 287 1 447 6* 487 9* 517 14* 437 9*
247 6* 12 74* 187 4* 247 6* 47 712 100* 100* 100* 100*
247 6* 337 5* 337 5* 337 6* 677 13 100* 100* 100* 100*
24 76* 33 75* 33 75* 33 76* 100 100* 100* 100* 100*
67 2* 127 4* 247 8 67 2* 257 5 377 12 357 1* 447 6* 397 3*
*p o0.05 versus the moderately susceptible breeds.
not examine all heritage breeds, we found four breeds with diminished susceptibility when compared to 35 other breeds including a commercial chicken breed. This work will provide the basis for identifying traits that could possibly be incorporated into the commercial breeds in order to minimize the colonization of Salmonella in the intestinal tracts of a major protein source.
Conflict of interest The authors declare no competing interests.
Author's contributions KLA performed the bacteriology work. MTB assisted in the in vivo studies. MAR participated in the design of the study and provided funding. SAC conceived the study, assisted in both the in vitro and in vivo phases, and wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgments The study was funded through a grant provided by the Leopold Center for Sustainable Agriculture (DSP2013-01) at Iowa State University.
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