Identification of Brachyspira spp. in the cecum of broiler chickens using histology and in situ diagnostic assays
DOI:
https://doi.org/10.5433/1679-0359.2021v42n5p2813Keywords:
Avian intestinal spirochetosis, Enteritis, Diagnosis, Histology, Immunohistochemistry, Fluorescence in situ hybridization, Brachyspira spp.Abstract
The genus Brachyspira corresponds to the group of bacteria formerly classified into the genus Serpulina and includes several commensal and pathogenic intestinal spirochetes that affect pigs, poultry, and other animal species, including humans. In birds, some pathogenic species of this genus causes a condition known as avian intestinal spirochetosis, which remains underdiagnosed, thereby causing serious economic losses. Brachyspira is a fastidious organism that necessitates the employment of fast and efficient identification techniques. The aim of this study was to identify Brachyspira spp. using histology, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in formalin-fixed paraffin embedded (FFPE) tissue samples from the cecum of commercial poultry. Samples were collected from 129 birds aged between 35 and 45 days from commercial broiler farms. For evaluation, routine histology processing (H&E) and the histochemical technique, periodic acid–Schiff (PAS) were done. Additionally, FFPE tissue samples were evaluated for FISH and IHC. The histological lesions were analyzed and graded after H&E staining, and the goblet cells were counted and compared using PAS staining with the positive and negative samples obtained through FISH and IHC. For FISH, probes labeled with Brachyspira spp., B. pilosicoli, B. hyodysenteriae, and B. intermedia were used, whereas rabbit polyclonal antibody specific for Brachyspira spp. was used for IHC. Of 129 samples, 82 were positive with IHC and 86 were positive with FISH. The samples positive for the genus Brachyspira in the FISH technique were tested for B. pilosicoli, B. hyodysenteriae, and B. intermedia in which 56 were positive for B. pilosicoli, 75 for B. hyodysenteriae and 80 for B. intermedia. There was an increase in goblet cells in the samples positive for FISH and IHC. The techniques used were effective and gave corresponding results, thus serving as a fast and efficient tool for diagnosis.References
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Phillips, N. D., La, T., & Hampson, D. J. (2006). Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces. Veterinary Microbiology, 116(1-3), 239-245. doi: 10.1016/j.vetmic.2006.03.020
Porter, R. E. (1998). Bacterial enteritides of poultry. Poultry Science, 77(8), 1159-1165. doi: 10.1093/ps/77. 8.1159
Pusztai, A., & Greer, F. (1984). Effect of dietary legume proteins on the morphology and secretory responses to the rat small intestine. Protides of the Biological Fluids,32, 347-350. doi: 10.1016/B978-0-08-03173 9-7.50088-4
Rojas, P., Petrich, A., Schulze, J., Wiessner, A., Loddenkemper, C., Epple, H. J.,... Moter, A. (2017). Distribution and phylogeny of Brachyspira spp. in human intestinal spirochetosis revealed by FISH and 16S rRNA-gene analysis. Anaerobe, 47, 25-32. doi: 10.1016/j.anaerobe.2017.03.012
Schmiedel, D., Epple, H. J., Loddenkemper, C., Ignatius, R., Wagner, J., Hammer, B.,... Moter, A. (2009). Rapid and accurate diagnosis of human intestinal spirochetosis by fluorescence in situ hybridization. Journal of Clinic Microbiology, 47(5), 1393-1401. doi: 10.1128/JCM.02469-08.
Shivaprasad, H. L., & Duhamel, G. E. (2005). Cecal spirochetosis caused by Brachyspira pilosicoli in commercial turkeys. Avian Diseases, 49(4), 609-613. doi: 10.1637/7383-052005.1.
Song, Y., & Hampson, D. J. (2009). Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens. Veterinary Microbiology, 137(1-2), 129-136. doi: 10.1016/j.vetmic.2008.12.020
Tolosa, E. M. C., Rodrigues, C. J., Behmer, O. A., & Freitas, A. G., Neto. (2003). Manual de técnicas para histologia normal e pathologia (2a ed., pp. 20-41). Barueri: Manole.
Venkatakrishnan, V., Quintana-Hayashi, M. P., Mahu, M., Haesebrouck, F., Pasmans, F., & Lindén, S. K. (2017). Brachyspira hyodysenteriae infection regulates mucin glycosylation synthesis inducing an increased expression of core-2 o-glycans in porcine colon. Journal of Proteome Research, 16(4), 1728-1742. doi: 10.1021/acs.jproteome.7b00002
Boye, M., Jensen, T. K., Møller, K., Leser, T. D., & Jorsal, S. E. (1998). Specific detection of the genus Serpulina, S. hyodysenteriae and S. pilosicoli in porcine intestines by fluorescent rRNA in situ hybridization. Molecular and Cellular Probes, 12(5), 323-330. doi: 10.1006/mcpr.1998
Burrough, E. R., Strait, E. L., Kinyon, J. M., Bower, L. P., Madson, D. M., Wilberts, B. L., Songer, J. G. (2012). Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs. Journal of Veterinary Diagnostic Investigation, 24(6), 1025-1034. doi: 10.1177/1040638712457927
Cooper, M., & Lummas, S. (2017). Antibodies for immunohistochemistry. In Immunohistochemistry and immunocytochemistry essential methods (2nd ed., p. 1-24). Chennai: Wiley Blackwell. Retrieved from https://books.google.com.br/books?id=LPAjDgAAQBAJ&dq=Antibodies+for+ Immunohistochemistry +in+Immunohistochemistry+and+Immunocytochemistry+Essential+Methods&hl=en-BR&lr
Culling, C. F. A., Allison, R. T., & Barr, W. T. (1985). Cellular pathology technique (4nd ed.). Vancouver, Canada: Butterworth & Co. Ltd.
Feberwee, A., Hampson, D. J., Phillips, N. D., La, T., Van Der Heijden, H. M. J. F., Wellenberg, G. J., ... Landman, W. J. M. (2008). Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both. Journal of Clinical Microbiology, 46(2), 593-600. doi: 10.1128/JCM.01829-07
Hampson, D. J., Fellstron, M. C., & Thomson, J. R. (2006). Swine dysentery. In Diseases of swine (9nd ed., pp. 785-805). Ames, Iowa: Blackwell Publishing.
Hess, C., Zloch, A., Bilic, I., Hacksteiner, K., Kuchling, S., & Hess, M. (2017). High prevalence of Brachyspira spp. in layers kept in alternative husbandry systems associated with frequent species variations from end of rearing to slaughter. Avian Pathology, 46(5), 481-487. doi: 10.1080/03079457. 2017.1315049
Illanes, N. V., Tamiozzo, P. J., Cabral, A., Bertone, J., Romanini, S., Yaciuk, R, Pelliza, B. R. (2016). Detection of Brachyspira pilosicoli and other Brachyspira species in Argentine poultry farms. Revista Argentina de Microbiología, 48(1), 67-70. doi: 10.1016/j.ram.2015.12.002
Jensen, N. S., Stanton, T. B., & Swayne, D. E. (1996). Identification of the swine pathogen Serpulina hyodysenteriae in rheas (Rhea americana). Veterinary Microbiology, 52(3-4), 259-269. doi: 10.1016/S 0378-1135(96)00076-4
Mappley, L. J., La Ragione, R. M., & Woodward, M. J. (2014). Veterinary Microbiology, 168(2-4), 245-260. doi: 10.1016/j.vetmic.2013.11.019
Medhanie, G. A., McEwen, S. A., Weber, L., Sanei, B., Cooley, L., Houghton, S., Guerin, M. T. (2013). Risk factors associated with the colonization of Ontario layer chicken flocks with Brachyspira species. Preventive Veterinary Medicine, 109(3-4), 304- 311. doi: 10.1016/j.prevetmed.2012.09.017
Moter, A., & Göbel, U. B. (2000). Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms. Journal Microbiology Methods, 41(2), 85-112. doi: 10.1016/s0167-7012(00)00152-4
Paulovich, F. B., Borowski, S. M., Driemeier, D., Razia, L. E., Coutinho, T. A., Prates, A. B. H.,… Barcellos, D. E. S. N. (2004). Evaluation of the pathogenicity of Brachyspira pilosicoli strains by conventional histopathological techniques and by immunohistochemistry. Pesquisa Veterinária Brasileira, 24(3), 144-148. doi: 10.1590/s0100-736x2004000300005
Phillips, N. D., La, T., & Hampson, D. J. (2006). Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces. Veterinary Microbiology, 116(1-3), 239-245. doi: 10.1016/j.vetmic.2006.03.020
Porter, R. E. (1998). Bacterial enteritides of poultry. Poultry Science, 77(8), 1159-1165. doi: 10.1093/ps/77. 8.1159
Pusztai, A., & Greer, F. (1984). Effect of dietary legume proteins on the morphology and secretory responses to the rat small intestine. Protides of the Biological Fluids,32, 347-350. doi: 10.1016/B978-0-08-03173 9-7.50088-4
Rojas, P., Petrich, A., Schulze, J., Wiessner, A., Loddenkemper, C., Epple, H. J.,... Moter, A. (2017). Distribution and phylogeny of Brachyspira spp. in human intestinal spirochetosis revealed by FISH and 16S rRNA-gene analysis. Anaerobe, 47, 25-32. doi: 10.1016/j.anaerobe.2017.03.012
Schmiedel, D., Epple, H. J., Loddenkemper, C., Ignatius, R., Wagner, J., Hammer, B.,... Moter, A. (2009). Rapid and accurate diagnosis of human intestinal spirochetosis by fluorescence in situ hybridization. Journal of Clinic Microbiology, 47(5), 1393-1401. doi: 10.1128/JCM.02469-08.
Shivaprasad, H. L., & Duhamel, G. E. (2005). Cecal spirochetosis caused by Brachyspira pilosicoli in commercial turkeys. Avian Diseases, 49(4), 609-613. doi: 10.1637/7383-052005.1.
Song, Y., & Hampson, D. J. (2009). Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens. Veterinary Microbiology, 137(1-2), 129-136. doi: 10.1016/j.vetmic.2008.12.020
Tolosa, E. M. C., Rodrigues, C. J., Behmer, O. A., & Freitas, A. G., Neto. (2003). Manual de técnicas para histologia normal e pathologia (2a ed., pp. 20-41). Barueri: Manole.
Venkatakrishnan, V., Quintana-Hayashi, M. P., Mahu, M., Haesebrouck, F., Pasmans, F., & Lindén, S. K. (2017). Brachyspira hyodysenteriae infection regulates mucin glycosylation synthesis inducing an increased expression of core-2 o-glycans in porcine colon. Journal of Proteome Research, 16(4), 1728-1742. doi: 10.1021/acs.jproteome.7b00002
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2021-07-02
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Matos, M. R. de, Grzegozevski, A. P., Cruz, A. da, Cheng, A. C., Gerelli, A., Fusco, C., … Viott, A. de M. (2021). Identification of Brachyspira spp. in the cecum of broiler chickens using histology and in situ diagnostic assays. Semina: Ciências Agrárias, 42(5), 2813–2824. https://doi.org/10.5433/1679-0359.2021v42n5p2813
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