In vivo and in vitro evaluation of tissue colonization and survival capacity of Salmonella Oranienburg in laying hens

Authors: BAB, UMA - Us Food & Drug Administration (FDA); Proszkowiec-Weglarz, Monika; SHARMA, GIRDHARI - Us Food & Drug Administration (FDA); PEREIRA, MARION - Us Food & Drug Administration (FDA); BALAN, KANNAN - Us Food & Drug Administration (FDA)

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2018
Publication Date: 5/21/2018
Citation: Bab, U.S., Proszkowiec-Wegla, M.K., Sharma, G.M., Pereira, M., Balan, K.V. 2018. In vivo and in vitro evaluation of tissue colonization and survival capacity of Salmonella Oranienburg in laying hens. Poultry Science. 97:3230-3235. http://dx.doi.org/10.3382/ps/pey189.
DOI: https://doi.org/10.3382/ps/pey189

Interpretive Summary: Human salmonellosis is typically associated with the consumption of contaminated animal or plant-derived foods. Shell eggs and poultry meat are the predominant sources of salmonella and resulting infection. There are many Salmonella enterica serovars that have been implicated in human salmonellosis. Recently Salmonella enterica serovar Oranienburg was linked to a human illness outbreak in the Midwest (2015 and 2016) from consumption of eggs. In comparison to well-known and researched Salmonella enterica serovar Enteritidies, not much is known about S. Oranienburg's potential to colonize laying hens tissue and contaminate eggs. We used in vivo and in vitro models to evaluate tissue colonization and survival capacity of S. Oranienburg. Laying hens were challenged with an oral dose of 107 or 109 colony forming unit (cfu) of S. Oranienburg and evaluated one, two, and four weeks post-challenge. Standard microbiological methods were used for detection of S. Oranienburg in tissues, egg shell wash, internal egg contents, and excreta. S. Oranienburg presence was detected in spleen, ovaries, upper oviduct and lower oviduct at two weeks post-challenge, and only in spleen four weeks post-challenge. While the presence of S. Oranienburg on eggshells was only seen when there were traces of excreta, bacteria were not recovered from internal egg contents. Moreover, S. Oranienburg had the capacity to invade and proliferate in ovarian granulosa cells, similarly to S. Enteritidies. Infective potential of S. Oranienburg was also assessed by its survival in egg white over four weeks under refrigerated condition. S. Oranienburg was characterized by 65 % survivability in egg white after 4 weeks. Overall, our data suggested that S. Oranienburg infection in layers did not result in egg contamination and colonization of egg forming tissues was limited to two weeks post-challenge. Survival within ovarian granulosa cells and egg white shows the ability of this bacterium to withstand antibacterial factors and the potential to penetrate the yolk. Given that the S. Oranienburg-outbreak(s) resulted in 60 cases of illness, this is the first report of an oral challenge study in laying hens to assess in vivo colonization, egg contamination, and in vitro survival and persistence of the bacterium within the granulosa cells and egg albumen.

Technical Abstract: Salmonella Oranienburg (SO) was linked to a human salmonellosis outbreak in the Midwest in 2015 and 2016 from consumption of eggs. However, unlike Salmonella Enteritidis (SE), little is known regarding the potential of SO to colonize in laying hens and contaminate eggs. We used in vivo and in vitro models to evaluate tissue colonization and survival capacity of SO. Twenty-eight-week-old laying hens were challenged with an oral dose of 107 (n = 92) or 109 (n = 96) colony forming unit (cfu) and evaluated after one, two, and four weeks. Standard microbiological methods with pre-enrichment and enrichment in selective media were used for detection of SO in tissues, egg shell wash, internal egg contents, and excreta. Peak systemic presence of SO was detected between 1 -2 weeks post infection (pi), with maximum colonization of spleen (86.9%), ovaries (31.6%), upper oviduct (15.8%), and lower oviduct (34.3%), while at 4 weeks SO was only recovered from spleens (25%). While the presence of SO on eggshells was seen only when there were traces of excreta, bacteria was not recovered from internal egg contents; shedding in excreta was 92 and 100% in birds gavaged with 107and 109 cfu at two weeks pi, respectively. The invasion and proliferation of SO in ovarian granulosa cells (GC) was compared to that of SE. The results showed that the invasion of SO into GC was comparable to SE, but proliferation of SO was significantly lower. The infective potential of SO was also assessed by enumerating its survival in egg white over 4 weeks under refrigerated condition, resulting in 65% survival at four weeks. Overall, our data suggested that SO infection in layers did not result in egg contamination via vertical transmission, and colonization of egg forming tissues was limited to two weeks pi. Survival within GC and egg white shows the ability of SO to withstand antibacterial factors and the potential of SO to penetrate the yolk. (322 words)