A human–food web–animal interface on the prevalence of food-borne pathogens (Clostridia and Enterococcus) in mixed veterinary farms


In the present work, we addressed the impact of a human–food web–animal interface on the prevalence of food-borne pathogens in mixed farms of Tamil Nadu, India. We have isolated and identified six strains of Clostridium sp. and five strains of Enterococcus sp. from food and animal sources disposed near to the veterinary and poultry farms. Phylogenetic relationships of these strains were inferred from their homologies in 16S rDNA sequences and rRNA secondary structures. The strain PCP07 was taxonomically equivalent to C. botulinum confirmed by neurotoxin-specific PCR primers, followed by mouse bioassay. Other Clostridial and Enterococcal isolates have shown a phylogenetic similarity to the C. bifermentans and E. durans isolated from veterinary farms, respectively. Results of our study revealed that a human–food web–animal interface has influenced the disease incidence and prevalence of these isolates in the poultry to veterinary farms, where human food acted as a likely transmittance vehicle for their infections.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Allen HA. Characterizing zoonotic disease detection in the United States: who detects zoonotic disease outbreaks & how fast are they detected? J. Infect. Public Health 8:194–201 (2015)

    Article  Google Scholar 

  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402 (1997)

    CAS  Article  Google Scholar 

  3. Bagge E, Persson M, Johansson KE. Diversity of spore-forming bacteria in cattle manure, slaughterhouse waste and samples from biogas plants. J. Appl. Microbiol. 109: 1549–1565 (2010)

    CAS  PubMed  Google Scholar 

  4. Bano L, Drigo I, Tonon E, Agnoletti F, Giovanardi D, Morandini E. Rice hulls as a possible source of Clostridium botulinum type C spores for poultry. Vet. Rec. 173: 427–428 (2013)

    CAS  Article  Google Scholar 

  5. Bekele B, Ashenafi M. Distribution of drug resistance among enterococci and Salmonella from poultry and cattle in Ethiopia. Trop. Anim. Health Prod. 42: 857–864 (2010)

    Article  Google Scholar 

  6. Brodskiĭ LI, Ivanov VV, Kalaĭdzidis IL, Leontovich AM, Nikolaev VK, Feranchuk SI, Drachev VE. GeneBee-NET: internet-based server for analyzing biopolymers structure. Biokhimiia 60: 1221–1230 (1995)

    PubMed  Google Scholar 

  7. Chellapandi P, Prisilla A. PCR-based molecular diagnosis of botulism outbreaks in aquatic birds. Ann. Microbiol. 68: 835–849 (2018)

    CAS  Article  Google Scholar 

  8. Cho JI, Joo IS, Choi JH, JungEun KH, Kyung JC, Jeong HJ, Ho RS, Hwang LG. Prevalence and antimicrobial resistance of Enterococcus spp. isolated from meat and fishery production in Korea. Food Sci. Biotechnol. 22: 161–165 (2013)

  9. Damborg P, Top J, Hendrickx AP, Dawson S, Willems RJ Guardabassi L. Dogs are a reservoir of ampicillin-resistant Enterococcus faecium lineages associated with human infections. Appl. Environ. Microbiol. 75: 2360–2365 (2009)

  10. Daniel A, Rapose A. The evaluation of Clostridium difficile infection (CDI) in a community hospital. J. Infect. Public Health. 8: 155–160 (2015)

    Article  Google Scholar 

  11. Daniel DS, Lee SM, Gan HM, Dykes GA, Rahman S. Genetic diversity of Enterococcus faecalis isolated from environmental, animal and clinical sources in Malaysia. J. Infect. Public Health 10: 617–623 (2017)

    Article  Google Scholar 

  12. Dhama K, Rajagunalan S, Chakraborty S, Verma AK, Kumar A, Tiwari R, Kapoor S. Food-borne pathogens of animal origin-diagnosis, prevention, control and their zoonotic significance: a review. Pak. J. Biol. Sci. 16: 1076–1085 (2013)

    CAS  Article  Google Scholar 

  13. Dmochewitz L, Fortsch C, Zwerger C, Vaeth M, Felder M, Lang MH, Barth H. Recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages. PlosOne. 8: e54517 (2013)

    CAS  Article  Google Scholar 

  14. Espelund M, Klaveness D. Botulism outbreaks in natural environments—an update. Front Microbiol. 5: 287 (2014)

    Article  Google Scholar 

  15. Facklam RR, Carvalho MGS, Teixeira LM. Enterococcus 9th ed, pp. 430–442. In: Manual of clinical microbiology. American Society for Microbiology (2007)

  16. Haines MD, Parker HM, McDaniel CD, Kiess AS. Impact of 6 different intestinal bacteria on broiler breeder sperm motility in vitro. Poult. Sci. 92: 2174–2181 (2013)

    CAS  Article  Google Scholar 

  17. Hayes J, English L, Carter P, Proescholdt T, Lee K, Wagner D, White D. Prevalence and antimicrobial resistance of Enterococcus species isolated from retail meats. Appl. Environ. Microbiol. 69: 7153–7160 (2003)

    CAS  Article  Google Scholar 

  18. Hogg RA, White VJ, Smith GR. Suspected botulism in cattle associated with poultry litter. Vet. Record. 126: 476–479 (1990)

    CAS  Google Scholar 

  19. Iweriebor BC, Obi LC, Okoh AI. Virulence and antimicrobial resistance factors of Enterococcus spp. Isolated from fecal samples from piggery farms in Eastern Cape, South Africa. BMC Microbiol. 15: 2–11 (2015)

  20. Keller A, Förster T, Mülle TJ, Dandekar M, Schultz, W. Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees. Biol. Direct. 15: 1–12 (2015)

    Google Scholar 

  21. Kimura K, Kubotaa T, Ohishib I, Isogaic E, Isogaid H, Nobuhiro F. The gene for component-II of botulinum C2 toxin. Vet. Microbiol. 62: 27–34 (1998)

    CAS  Article  Google Scholar 

  22. Kjeldsen KU, Kjellerup BV, Egli K, Frølund B, Nielsen PH, Ingvorsen K. Phylogenetic and functional diversity of bacteria in biofilms from metal surfaces of an alkaline district heating system. FEMS Microbiol. Ecol. 61: 384–397 (2007)

    CAS  Article  Google Scholar 

  23. Lawson PA, Rainey FA. Proposal to restrict the genus Clostridium (Prazmowski) to Clostridium butyricum and related species. Int. J. Syst. Evol. Microbiol. 66: 1009–1016 (2016)

    CAS  Article  Google Scholar 

  24. Lindstrom M, Korkeala H. Laboratory diagnostics of botulism. Clin. Microbiol. Rev. 19: 298–314 (2006)

    CAS  Article  Google Scholar 

  25. Liu M, Zhao K, Tang Y, Ren D, Yao W, Tian X, Zhang X, Bin Y, Deng B. Analysis of Clostridium cluster I community diversity in pit mud used in manufacture of Chinese Luzhou-flavor liquor. Food Sci. Biotechnol. 24: 995–1000 (2015)

    CAS  Article  Google Scholar 

  26. Nobuhiro F, Kubota T, Shirakawa S, Kimura K, Ohishi I, Moriishi K, Isogai E, Isogai, H. Characterization of component-I gene of botulinum C2 toxin and PCR detection of its gene in Clostridial species. Biochem. Biophys. Res. Commun. 220: 353–359 (1996)

    Article  Google Scholar 

  27. Oguma K, Yamaguchi T, Sudou K, Yukosawa N, Fujikawa Y. Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivates. Appl. Environ. Microbiol. 51: 256–260 (1986)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Olsen I, John L, Johnson VH, Moore WE, Moore C. Rejection of Clostridium putrificum and conservation of Clostridium botulinum and Clostridium sporogenes. Int. J. Syst. Evol. Microbiol. 49: 339 (1999)

    Google Scholar 

  29. Princewill TJ, Agba M, Jemitola SO. Animal feeds as likely vehicles of clostridial infections in livestock. J. Micobiol. 42: 155–162 (1985)

    CAS  Google Scholar 

  30. Prisilla A, Chellapandi P. Cloning and expression of immunogenic Clostridium botulinum C2I mutant proteins designed from its evolutionary imprints. Comp. Immunol. Microbiol. Infect. Dis. https://doi.org/10.1016/j.cimid.2019.01.012 (2019)

  31. Razia M, Padmanaban K, Raja KL, Chellapandi P, Sivaramakrishnan S. 16S rDNA-based phylogeny of non-symbiotic bacteria of entemopathogenic nematodes from infected insect cadavers. Genom. Proteom. Bioinform. 9: 104–112 (2011)

    CAS  Article  Google Scholar 

  32. Repizo GD, Espariz M, Blancato VS, Suárez CA, Esteban L, MagniC. Genomic comparative analysis of the environmental Enterococcus mundtii against enterococcal representative species. BMC Genom. 15: 489 (2014)

  33. Sasaki Y, Yamamoto K, Kojima A, Norimats M, Tamura Y. Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region. Res. Vet. Sci. 69: 289–294 (2000)

    CAS  Article  Google Scholar 

  34. Souillard R, Le Maréchal C, Hollebecque F, Rouxel S, Barbé A, Houard E, Léon D, Poëzévara T, Fach P, Woudstra C, Mahé F, Chemaly M, Le Bouquin S. Occurrence of C. botulinum in healthy cattle and their environment following poultry botulism outbreaks in mixed farms. Vet. Microbiol. 180: 142–155 (2015)

    CAS  Article  Google Scholar 

  35. Stalker MJ, Brash ML, Weisz A, Ouckama RM, Slavic D. Arthritis and osteomyelitis associated with Enterococcus cecorum infection in broiler and broiler breeder chickens in Ontario, Canada. J. Vet. Diagn. Invest. 22: 643–645 (2010)

    Article  Google Scholar 

  36. Tamura K, Stecher G, Peterson D, Filipskimm A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30: 2725–2729 (2013)

  37. Tavakoli HR, Meshgi MA, Jafari NJ, Izadi M, Ranjbar R, Fooladi AA. A survey of traditional Iranian food products for contamination with toxigenic Clostridium botulinum. J. Infect. Public Health. 2: 91–95 (2009)

    CAS  Article  Google Scholar 

  38. Thompson DJ, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res. 24: 4876–4882 (1997)

    Article  Google Scholar 

  39. Triplett MD, Parker HM, McDaniel D, Kiess AS. Influence of 6 different intestinal bacteria on Beltsville Small White turkey semen. Poult. Sci. 95: 1918–1926 (2016)

    CAS  Article  Google Scholar 

  40. Williamson JL, Rocke TE, Iken JMA. In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay. Appl. Environ. Microbiol. 65: 3240–3242 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


This work was financially supported by Life Science Research Board-Defense Research and Development Organization, New Delhi, India (No. DLS/81/48222/LSRB-249/BTB/2012).

Author information



Corresponding author

Correspondence to P. Chellapandi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Appendix B. Mouse bioassay (MP4 101571 kb)

Appendix A.

Supplementary data (DOCX 4169 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Prisilla, A., Deena Remin, M., Roja, B. et al. A human–food web–animal interface on the prevalence of food-borne pathogens (Clostridia and Enterococcus) in mixed veterinary farms. Food Sci Biotechnol 28, 1583–1591 (2019). https://doi.org/10.1007/s10068-019-00595-8

Download citation


  • Avian botulism
  • Poultry disease
  • Molecular diagnosis
  • Phylogeny
  • Clostridium
  • Enterococcus