Isolation and identification of Brachyspira pilosicoli from laying hens flocks, using conventional culture and molecular methods in Mashhad, Iran

Document Type : Research Articles


Ferdowsi University of Mashhad


Avian intestinal spirochaetosis (AIS) is an intestinal infection caused by anaerobic spirochaetes of the genus Brachyspira, including B. pilosocoli. The purpose of this study was
isolation and identification of B. pilosocoli from laying hen flocks, located in Mashhad suburb, Khorasan Razavi province, Iran, and investigating the frequency of the infection. One hundred and eighty cloacal swab samples from 18 randomly selected flocks (10 samples /flock) were cultured anaerobically on selective agar and confirmed as intestinal spirochaete by its spirichaetal form using phase contrast microscopy. Then, the samples were subjected to
PCR amplification followed by DNA sequencing. A total of 24 samples from 8 flocks were selected as suspected cases by culture and phase contrast microscopy. Upon PCR amplification by specific primers, only 9 cultures belonged to 3 flocks appeared to be B.pilosicoli. Sequence analysis of the amplicons confirmed the identity of all isolated ones.
Based on the results obtained, it was concluded that B. pilosicoli might be strongly involved in AIS among laying hen flocks of this geographical region. The results could also be considered as an indicator for large scale investigation into the true prevalence of the infection. This study is the first report of infection in laying hens flocks of Iran.


Main Subjects

Atyeo, R. F., Oxberry, S. L., Combs, B. G. and Hampson, D. J. (1998). Development and evaluation of polymerase chain
reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis. Letters in Applied Microbiology 26, 126–130.
Barrett S.P. (1990). Intestinal spirochaetes in a Gulf Arab population. Epidemiology and Infection 104, 261-266.
Calderaro, A., Bommezzadri, S., Piccolo, G.,Zuelli, C., Dettori, G., Chezzi, C (2005).Rapid isolation of Brachyspira hyodysenteriae and Brachyspira pilosicoli from pigs. Veterinary Microbiology 105, 229-234.
Choi C., Han D.U., Kim J., Cho W.S., Chung H.K., Jung T., Yoon B.S. ,Chae C.(2002). Prevalence of Brachyspira pilosicoli in Korean pigs, determined using a nested PCR, Veterinary Record
150, 217–218.
Davelaar, F.G., Smit, H.F., Hovind-Hougen,K., Dwars, R.M. and van der Valk, P.C. (1986). Infectious typhlitis in chickens caused by spirochaetes. Avian Pathology 15, 247–258.
Dwars, R.M., Smit, H.F. and Davelaar, F.G.(1990). Observations on avian intestinal spirochaetosis. Veterinary Quarterly 12, 51–55.
Dwars, R.M., Davelaar, F.G. and Smit, H.F.(1992a). Influence of infection with avian intestinal spirochaetes on the faeces of laying hens. Avian Pathology 21, 427– 429.
Dwars, R.M., Davelaar, F.G. and Smit, H.F.(1992b). Spirochaetosis in broilers.Avian Pathology 21, 261–273.
Dwars, R.M., Davelaar, F.G. and Smit, H.F.(1993). Infection of broiler parent hens (Gallus domesticus) with avian intestinal spirochaetes: effects on egg production and chick quality. Avian
Pathology 22, 693–701.
Dwars, R.M., Smit, H.F., Davelaar, F.G. and Van‘t Veer, W. (1989). Incidence of spirochaetal infections in cases of
intestinal disorder in chickens. Avian Pathology 18, 591 -595.
Griffiths, I.B., Hunt, B.W., Lister, S.A. and Lamont, M.H. (1987). Retarded growth rate and delayed onset of egg production associated with egg production in pullets. Veterinary
Record 121, 35–37.
Hampson, D.J., Duhamel, G.E (2006). Porcine colonic spirochetosis/intestinal spirochetosis. Chapter 46. In: Diseases
of Swine 9th Edition. Blackwell Publishing, Oxford, UK.PP:755-767.
Hampson, D.J. and McLaren, A.J. (1999).Experimental infection of laying hens with Serpulinaintermediacauses reduced egg production and increased faecal water content. Avian Pathology
28, 113–117.
Hopwood, D.E., Pethick, D.W and Hampson, D.J (2002). Increasing the viscosity of the intestinal contents stimulates
proliferation of enterotoxigenic Escherichia coli and
Brachyspirapilosicoli in weaner pigs.British Journal of Nutrition 88, 523–532.
Jamshidi, A. and Hampson, D. J. (2002). Zinc bacitracin enhances colonization by the intestinal spirochaete
Brachyspirapilosicoli in experimentally infected layer hens. Avian Pathology 31, 293–298.
Jamshidi A., Hampson D.J. (2003).Experimental infection of layer hens with a human isolate of Brachispira Pilosicoli: Journal of Clinical Microbiology. 52, 361-364.
La T., Collins M.A., Phillips N.D., Oksa A,Hampson DJ (2006). Development of a multiplex-PCR for rapid detection of
the enteric pathogens Lawsonia intracellularis, Brachyspira
hyodysenteriae, and Brachyspira pilosicoli in porcine faeces, Letters in Applied Microbiology 42, 284–288.
La T., Phillips N.D. ,Hampson D.J. (2003). Development of a duplex PCR assay for the detection of Brachyspira
hyodysenteriae and Brachyspira pilosicoli in pig feces, Journal of
Clinical Microbiology 41, 3372–3375.
McLaren, A.J., Trott, D.J., Swayne, D.E.,Oxberry, S.L. andHampson, D.J. (1997). Genetic and phenotypic
characterisation of intestinal spirochetes colonizing chickens, and
allocation of known pathogenic isolates to three distinct genetic groups. Journal of Clinical Microbiology 35, 412– 417.
Mikosza, A.S.J., ArifMunshi, M., Hampson,D.J. (2004). Analysis of genetic variation in Brachyspiraaalborgi and related spirochaetes determined by partial sequencing of the 16S rRNA
and NADH oxidase genes. Journal of Medical Microbiology 53, 333–339.
Mikosza A.S.J., Hampson D.J. (2001). Human intestinal spirochaetosis:Brachyspiraaalborgiand/or Brachyspirapilosicoli. Animal Health Research Reviews 2,101-110.
Mikosza, A.S.J., La,T., Margawani, K.R., Brooke, C. J., Hampson, D.J(2001).PCR detection of Brachyspira alborgi and Brachyspira pilosicoli in human faeces. FEMS Microbiology Letters
197, 167–170.
Nyree D. Phillips, Tom La and David J.Hampson (2006). Development of a two-step nested duplex PCR assay for
the rapid detection of Brachyspira pilosicoli and Brachyspira. Veterinary Microbiology 116, 239-245.
Simon, M.C., Gray, D.I and Cook, N. (1996) DNA Extraction and PCR Methods for the Detection of Listeria monocytogenes in Cold-Smoked Salmon. Applied and Environmental
Microbiology 62(3), 822-824.
Smit, H.F., Dwars, R.M., Davelaar, F.G. and Wijtten, A.W. (1998). Observations on the influence of intestinal spirochaetosis in broiler breeders on the performance of their progeny and
on egg production. Avian Pathology 27, 133–141.
Stanton, T.B., Fourni´e-Amazouz, E., Postic,D., Trott, D.J., Grimont, P.A.D.,Baranton, G., Hampson, D.J. and Saint
Girons, I. (1997). Recognition of two new species of intestinal spirochetes: Serpulina intermedia sp. nov. and Serpulinamurdochii sp. nov. International Journal of Systematic Bacteriology 47, 1007–1012.
Stanton, T.B., Postic, D. and Jensen, N.S. (1998) Serpulina alvinipulli sp. nov., a new Serpulina species enteropathogenic to chickens. International Journal of Systematic Bacteriology 47, 1007–1012.
Stephens, C.P., Hampson, D.J. (2001).Intestinal spirochaete infections in chickens: a review of disease associations, epidemiology and control.Animal Health Research Reviews
Stephens, C.P. and Hampson, D.J. (1999).Prevalence and disease association of intestinal spirochaetes in chickens in
eastern Australia. Avian Pathology 28,447–454.
Swayne, D.E. (1997). Avian intestinal spirochaetosis. In B.W. Calneck (Ed.), Diseases of Poultry 10th edn (pp. 325–
332) Ames, IA: Iowa State University Press.
Swayne, D.E. (2003). Avian intestinal spirochaetosis. In Y.M. Saif, H.J.Barnes, J.R. Glisson, A.M. Fadly, L.R.McDougaldand D.E Swayne (Eds.),Isolation and Identification of Brachyspira pilosicoli
Diseases of Poultry, 11th edn (pp. 826_836). Ames: Iowa State Press.
Swayne, D.E., Bermudez, A.J., Sagartz, J.E.,Eaton, K.A., Monfort, J.D.,Stoutenberg, J.W. and Hayes, J.R.(1992). Association of cecalspirochaetes with pasty vents and
dirty eggshells in layers. Avian Diseases 36, 776–781.
Swayne, D.E., Eaton, K.A., Stoutenburg, J.,Trott, D.J., Hampson, D.J. and Jensen,N.S. (1995). Identification of a new
intestinal spirochete with pathogenicity to chickens. Infection and Immunity 63,430–436.
Thomson J.R., Murray B.P., Henderson1 L.E.,Thacker J, Burch D.G.S. (2007).Brachyspira species isolated from UK poultry samples. Presented at the 4th International Conference on Colonic Spirochaetal Infections in Animals and Humans, Prague, Czech Republic Trampel, D.W., Jensen, N.S. and Hoffman,
L.J. (1994). Cecal spirochaetosis in commercial laying hens. Avian
Diseases 38, 895–898.
Trott D.J., Combs B.G., Oxberry S.L., Mikosza A.S.J., Robertson I.D., Passey M., Taime J., Sehuko R., Hampson D.J.(1997). The prevalence of Serpulina pilosicoli in humans and domestic
animals in the Eastern Highlands of Papua New Guinea. Epidemiology and Infection 119, 369-379.