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Invasion of Salmonella enteritidis in the tissues of reproductive organs in laying Japanese quail: an immunocytochemical study
Invasion of Salmonella Enteritidis in the Tissues of Reproductive Organs in Laying Japanese Quail: An Immunocytochemical Study T. Takata, J. Liang, H. Nakano, and Y. Yoshimura1 Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima 739-8528, Japan in the fibroblast-like and macrophage-like cells in the stroma and surface layer of follicles. The SE immunoreaction products were identified on the mucosal surface, in the mucosal epithelium, and in the stromal tissues in all segments of the oviduct. Many of the bacteria were contained in the cytoplasm of mucosal epithelial cells and stromal cells in those tissues. The SE immunoreactions were also found in the tissues of kidney, spleen, and liver and in the large intestine. These results suggest that SE organisms introduced into the peritoneal cavity can invade and colonize the tissues of ovary and oviduct and may be responsible in the production of contaminated eggs.
(Key words: hen reproductive organs, immunocytochemical localization, Salmonella enteritidis, tissue infection) 2003 Poultry Science 82:1170–1173
INTRODUCTION Contamination of eggs by Salmonella organisms could occur either on the surface of the eggshell or in the contents of eggs. Previous studies have shown the presence of Salmonella organisms in yolk and albumin of eggs laid by birds that were experimentally inoculated with those organisms (Keller et al., 1997; Gast and Holt, 2000). Eggs internally contaminated by SE can cause salmonellosis in humans. Although the process of contamination of internal egg components has not been well explained, it is believed that internal contamination occurs in reproductive organs during egg formation. Hens orally injected with large dose of SE laid internally contaminated eggs with a peak during 12 to 15 d postinoculation followed by decreasing contamination until d 23 of postinoculation (Gast and Holt, 2001). The oviduct consists of the infundibulum, magnum, isthmus, uterus, and vagina. Because the cloaca is the common opening to the digestive and reproductive tracts, microorganisms in the digestive tract can reach the cloaca and then may migrate into the vagina. Sperm inseminated in the uterus are transported to the infundibulum by actions of the oviduct. Thus, it would
2003 Poultry Science Association, Inc. Received for publication December 18, 2002. Accepted for publication February 24, 2003. 1 To whom correspondence should be addressed: yyosimu@ hiroshima-u.ac.jp.
be possible that Salmonella that invaded the lower part of the oviduct are transported to the infundibulum, followed by movement through the peritoneal cavity to the ovary and other organs. It is also assumed that salmonella organisms invade circulating blood and are transported to the ovarian follicles (Keller et al., 1995; Thiagarajan et al., 1996). Thus Salmonella in reproductive organs may be incorporated into the yolk and albumen during the formation of eggs. The SE can be isolated from various organs including ovary and oviduct of the infected birds (Gast and Beard, 1990; Keller et al., 1997). It is possible that the SE invades the tissues of these organs; electron microscopic studies of chicks have shown passage of Salmonella through ileocecal mucosa (Popiel and Turnbull, 1985). The attachment and invasion of cultured chicken ovarian granulosa cells by SE (Thiagarajan et al., 1996) and colonization of SE in the ovarian and oviductal cells of birds inoculated with SE orally (Keller et al., 1995) also have been reported. However, further studies are needed to evaluate the possibility that SE in the peritoneal cavity can invade the tissues of reproductive organs. If SE colonizes tissues of reproductive organs, birds would lay contaminated eggs until the infection is eliminated. In mammalian animal models, cell-mediated immunity plays a major role in controlling Salmonella infection (George et al., 1987; Hsu, 1989). Although the immune mechanisms involved in the defense against Salmonella infection are less well understood in chickens, significance of phagocytosis by heterophils and
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ABSTRACT The aim of this study was to determine whether Salmonella enteritidis (SE) inoculated into the peritoneal cavity would colonize tissues of reproductive organs in Japanese quail hens. Quail hens regularly laying were intraperitoneally inoculated with 5 × 107 or 5 × 108 SE cells, and the ovary, oviduct, kidney, spleen, liver, and large intestine were excised 24 or 48 h after the treatment. Paraffin sections of these organs were immunostained for SE. Invasion of SE was found in the tissues of the ovarian stroma, the follicular wall including superficial and theca layers, and occasionally in the granulosa layer and yolk. The SE immunoreaction product frequently was found
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MATERIALS AND METHODS Female Japanese quail laying seven eggs or more in a sequence were used in this study. They were kept in individual cages under 14L:10D and provided with feed and water ad libitum. Approximately after 2 h of oviposition, the birds were intraperitoneally inoculated with PBS alone or with SE suspended in PBS. The SE strain used in this study was isolated from clinical sample by Hiroshima Prefectural Health and Environmental Center, Hiroshima, Japan. The specific gene sequence had been confirmed by PCR using a primer set SIN-1 and SIN-2.2 The amount of SE inoculated in one bird was 5 × 107 or 5 × 108 cells in 0.5 mL of PBS. The birds inoculated with 5 × 107 SE cells were examined 24 or 48 h after the treatment. Those inoculated with 5 × 108 SE cells were examined only 24 h after the treatment. Control birds were intraperitoneally injected with 0.5 mL PBS and examined 24 or 48 h after the treatment. Four birds were used in each experimental or control group. For the tissue collection, the birds were killed by carbon dioxide inhalation and then the ovary (stroma, the largest and third largest preovulatory follicle), oviduct (tubular region of the infundibulum and middle parts of magnum, isthmus, uterus, and vagina), kidney, liver, spleen, cecum, and rectum were collected and fixed with 4% paraformaldehyde in PBS. The tissues were embedded in paraffin and sections of them (6 µm in thickness, 10 sections at every 60 µm in a tissue) were prepared and air-dried on slides. Sections were deparaffinized and rehydrated in PBS, followed by incubation with 1% (wt/vol) blocking reagent3 in PBS for 30 min to block nonspecific immunoreactions. Then sections were incubated with anti-Salmonella O antibody4 (10 µg/mL in 1% BSA-PBS) for 2 h. They were washed three times for 5 min each in PBS, and the immunoreactions were detected with a Histofine SABPO (M) kit.5 Briefly, the sections were incubated with
2
Takara Shuzo Co., Ohtsu, Japan. Roche Diagnostics GmbH, Mannheim, Germany. 4 HyTest Co., Turku, Finland. 5 Nichirei Co., Tokyo, Japan. 3
FIGURE 1. Sections of ovaries from SE-inoculated Japanese quail immunostained for SE. The birds were intraperitoneally inoculated with 5 × 108 SE cells and examined 24 h after the inoculation. G = granulosa layer; O = oocyte; T = theca layer; S = stroma; SL = superficial layer; V = vein; Y = yolk. Arrows indicate the immunoreactive SE. Scale bar = 20 µm. a) Ovarian stroma. Note the SE immunoreaction products in the stromal cells. b) Follicular wall of the largest follicle. SE immunoreactions are observed in the macrophage-like cells in the superficial layer and theca cells. c) Follicular wall of the largest follicle. Note the SE immunoreactions in the granulosa cells. d) Atretic largest follicle. Immunoreactions are observed in the macrophage-like cells and in the yolk.
biotinylated anti-mouse IgG for 1 h followed by washing in PBS and incubating with avidin-peroxidase complex for 1 h. Then the immunoprecipitates were visualized by incubating the sections with a hydrogen-peroxide-diaminobenzidine mixture. Control sections were prepared in the same manner except the first antibodies were replaced with normal mouse IgG. Sections were examined under a light microscope.
RESULTS No specific staining was observed in any tissues of control birds that were injected with PBS. The ovarian stroma was a loose connective tissue where numerous cortical follicles were located. The follicular wall of preovulatory follicles consisted of the granulosa layer surrounding the yolk, theca layer, superficial layer extending from the ovarian stroma, and superficial epithelium. In the ovarian stroma the immunoreaction products for SE were identified in the connective tissue of 10 of the 12 inoculated hens (Figure 1a). The SE reaction products were also found in the superficial layer and occasionally in the theca layer of preovulatory follicles. Those were observed in the cytoplasm of fibroblast-like and macrophage-like cells in the superficial layer (Figure 1b). In some follicles, the products were observed in the theca cells and granulosa cells (Figure 1c). One of the largest follicles underwent atresia, and a markedly large number of SE-containing cells in the surface layer and SE-positive intrafollicular yolk granules were observed (Figure 1d). The oviductal mucosal tissue was surrounded by ciliated
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 12, 2015
response of T-cell subsets and B cells in defending against SE have been suggested (Kramer, 1998; Withanage et al., 1998; Andreasen et al., 2001). The presence of immunocompetent cells, including antigen-presenting cells and T and B cells, has been shown in the ovary (Barua et al., 1998a,b, 2001; Barua and Yoshimura, 1999) and oviduct (Yoshimura et al., 1997; Zheng et al., 1998, 2000, 2001; Zheng and Yoshimura, 1999). Determination of SE invasion into these tissues and the feature of their colonization are essential to analyze whether these immunocompetent cells respond to SE invasion. Thus, the goal of this study was to determine whether SE inoculated into the peritoneal cavity invades and colonizes the tissues of reproductive organs.
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FIGURE 2. Sections of oviducts from SE-inoculated Japanese quail immunostained for SE. The birds were intraperitoneally inoculated with 5 × 108 SE cells and examined 24 h after the inoculation. E = mucosal epithelium; M = muscle; S = stroma; Se = serosa; L = oviductal lumen. Arrows indicate the immunoreactive SE. Scale bar = 20 µm. a) and b) Mucosa and serosa layers of infundibulum, respectively. Note SE immunoreaction products in the subepithelial stroma in the mucosa and a large number of positive cells in the serosa. c) Magnum. Note SE immunoreactions mucosal epithelial and subepithelial cells. d) and e) Isthmus and uterus, respectively. Note SE immunoreactions on the mucosal surface and in the mucosal epithelial cells. f) Vagina. SE immunoreactions are observed on the mucosal surface and in the mucosal epithelium and stromal cells.
pseudostratified epithelium. The SE reaction products were identified on the mucosal surface, in the mucosal epithelium, the lamina propria, and serosa in all the segments of the oviduct (Figures 2, a to f). Those in the lamina propria were found in the cells of subepithelial stroma. The products of SE reaction were also identified in the epithelial cells of tubules in the kidney, spleen cells, and liver (Figures 3, a to c) as well as in the lamina propria of rectum and cecum (Figure 3d). The SE reaction products were located in the cells of each tissue. The tissues of the ovary, oviduct, and other organs did not show histological degradations even the SE reactions were positive.
DISCUSSION We have examined how the SE colonizes in the reproductive organs of Japanese quail hens that were intraperitoneally inoculated with SE by immunocytochemistry. The significant finding of this study is that the SE reaction products were localized in the tissues and cells of ovary
and oviduct. They were also localized in the other tissues including the kidney, liver, spleen, and large intestine. The SE reaction products were localized in the stromal connective tissue and follicular wall in the ovaries, as well as mucosal epithelium and stroma in the oviduct. The presence of SE in the ovarian stroma and follicular wall suggest that the SE in the peritoneal cavity invaded the tissues through superficial epithelium. Then the SE was likely to reach the intrafollicular yolk because in some cases they were found in the granulosa cells and yolk of follicles. The SE is also likely to pass through mucosal epithelium surrounding the lamina propria of oviductal mucosal tissue because the organisms were found in the epithelium and stroma. Popiel and Turnbull (1985) revealed the passage of Salmonella organisms from epithelium to lamina propria of chick ileocecal mucosa. Keller et al. (1995) described that the SE appeared in the medulla of ovary, cortical stromal tissue of atretic follicle, and mucosal epithelium of oviduct in orally inoculated hens and also suggested that egg invasion by bacterial translocation from the peritoneal cavity might not occur. The current results indicate that the SE in the peritoneal cavity can invade the ovarian and oviductal tissues from the surface of these organs and colonize cells of these tissues, as does SE in digestive organs. However, the current histological observations suggest that significant tissue damage may not occur during this process. Enhancement of immunity in the reproductive organs may be needed to protect them from infection. The SE reaction products were found in the liver, kidney, spleen, and large intestine of some birds, suggesting that the invasion of SE in the tissues can occur not only in reproductive organs but also in other organs. The SE might disperse in the peritoneal cavity, followed by migration in the oviductal lumen, cloaca, and then large
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 12, 2015
FIGURE 3. Sections of kidney (a), liver (b), spleen (c), and rectum (d) from SE-inoculated Japanese quail immunostained for SE. The birds were intraperitoneally inoculated with 5 × 108 SE cells and examined 24 h after the inoculation (a to c) or inoculated with 5 × 107 SE cells and examined 48 h after that (d). SE immunoreaction products are localized in all organs (arrows). Scale bar = 20 µm. E = mucosal epithelium; L = oviductal lumen; S = stroma.
SALMONELLA ENTERITIDIS IN HEN REPRODUCTIVE ORGANS
intestine. It remains unknown whether the reproductive organs are more susceptible than the other organs. In conclusion, the current results suggest that the SE in the peritoneal cavity can invade and colonize the ovarian and oviductal tissues. It is possible that they are incorporated into internal egg components during egg formation, leading to production of contaminated eggs.
ACKNOWLEDGMENT This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to Y. Y.
Andreasen, C. B., J. K. Akunda, and T. T. Kramer. 2001. Comparison of heterophil phagocytosis for heterophil-adapted Salmonella enteritidis (HASE) and wild-type Salmonella enteritidis (SE). Avian Dis. 45:432–436. Barua, A., H. Michiue, and Y. Yoshimura. 2001. Changes in the localization of MHC class II positive cells in hen ovarian follicles during the process of follicular growth, postovulatory regression and atresia. Reproduction 121:953–957. Barua, A., and Y. Yoshimura. 1999. Effects of aging and sex steroids on the localization of T cell subsets in the ovary of chicken, Gallus domesticus. Gen. Comp. Endocrinol. 114:28– 35. Barua, A., Y. Yoshimura, and T. Tamura. 1998a. Effects of aging and oestrogen on the localization of immunoglobulin containing cells in the ovary of chicken, Gallus domesticus. J. Reprod. Fertil. 114:11–16. Barua, A., Y. Yoshimura, and T. Tamura. 1998b. The effects of age and sex steroids on the macrophage population in the ovary of chicken, Gallus domesticus. J. Reprod. Fertil. 114:253–258. Gast, R. K., and C. W. Beard. 1990. Isolation of salmonella enteritidis from internal organs of experimentally infected hens. Avian Dis. 34:991–993. Gast, R. K., and P. S. Holt. 2000. Deposition of phage type 4 and 13a Salmonella enteritidis strains in the yolk and albumen of eggs laid by experimentally infected hens. Avian Dis. 44:706–710. Gast. R. G.., and P. S. Holt. 2001. The relationship between the magnitude of the specific antibody response to experimental
salmonella enteritidis infection in laying hens and their production of contaminated eggs. Avian Dis. 45:425–431. George, A., R. Nair, S. Rath, S. N. Ghosh, and R. S. Kamat. 1987. Regulation of cell mediated immunity in mice immunized with Salmonella enteritidis. J. Med. Microbiol. 23:239–246. Hsu, H. S. 1989. Pathogenesis and immunity in murine salmonellosis. Microbiol. Rev. 53:390–409. Keller, L. H., C. E. Benson, K. Krotec, and R. J. Eckroade. 1995. Salmonella enteritidis colonization of the reproductive tract and forming and freshly laid eggs of chickens. Infect. Immun. 63:2443–2449. Keller, L. H., D. M. Schifferli, C. E. Benson, S. Aslam, and R. J. Eckroade. 1997. Invasion of chicken reproductive tissues and forming eggs is not unique to Salmonella enteritidis. Avian Dis. 41:535–539. Kramer, T. T. 1998. Effects of heterophil adaptation on Salmonella enteritidis fecal shedding and egg contamination. Avian Dis. 42:6–13. Popiel, I., and P. B. Turnbull. 1985. Passage of Salmonella enteritidis and Salmonella Thompson through chick ileocecal mucosa. Infect. Immun. 47:786–792. Thiagarajan D., M. Saeed, J. Turek, and E. Asem. 1996. In vitro attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis phage type 8. Infect. Immun. 64:5015–5021. Withanage, G. S. K., K. Sasai, T. Fukata, T. Miyamoto, E. Baba, and H. S. Lillehoj. 1998. T lymphocytes, B lymphocytes, and macrophages in the ovaries and oviducts of laying hens experimentally infected with Salmonella enteritidis. Vet. Immunol. Immunopathol. 66:173–184. Yoshimura, Y., T. Okamoto, and T. Tamura. 1997. Localisation of MHC class II, lymphocytes and immunoglobulins in the oviduct of laying and moulting hens. Br. Poult. Sci. 38:590–596. Zheng, W. M., J. Izaki, S. Furusawa, and Y. Yoshimura. 2000. Localization of immunoglobulin G γ-chain mRNA-expressing cells in the oviduct of laying and diethylstilbestrol-treated immature hens. Gen. Comp. Endocrinol. 120:345–352. Zheng, W. M., M. Nishibori, N. Isobe, Y. Yoshimura. 2001. An in situ hybridization study of the effects of artificial insemination on the localization of cells expressing MHC class II mRNA in the chicken oviduct. Reproduction 122:581–586. Zheng, W. M., and Y. Yoshimura. 1999. Localization of macrophages in the chicken oviduct: effects of age and gonadal steroids. Poult. Sci. 78:1014–1018. Zheng, W. M., Y. Yoshimura, T. Tamura. 1998. Effects of gonadal steroids on the localization of antigen-presenting cells, T and B cells in the chicken oviduct. J. Reprod. Fertil. 114:45–54.
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REFERENCES
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(Key words: hen reproductive organs, immunocytochemical localization, Salmonella enteritidis, tissue infection) 2003 Poultry Science 82:1170–1173
INTRODUCTION Contamination of eggs by Salmonella organisms could occur either on the surface of the eggshell or in the contents of eggs. Previous studies have shown the presence of Salmonella organisms in yolk and albumin of eggs laid by birds that were experimentally inoculated with those organisms (Keller et al., 1997; Gast and Holt, 2000). Eggs internally contaminated by SE can cause salmonellosis in humans. Although the process of contamination of internal egg components has not been well explained, it is believed that internal contamination occurs in reproductive organs during egg formation. Hens orally injected with large dose of SE laid internally contaminated eggs with a peak during 12 to 15 d postinoculation followed by decreasing contamination until d 23 of postinoculation (Gast and Holt, 2001). The oviduct consists of the infundibulum, magnum, isthmus, uterus, and vagina. Because the cloaca is the common opening to the digestive and reproductive tracts, microorganisms in the digestive tract can reach the cloaca and then may migrate into the vagina. Sperm inseminated in the uterus are transported to the infundibulum by actions of the oviduct. Thus, it would
2003 Poultry Science Association, Inc. Received for publication December 18, 2002. Accepted for publication February 24, 2003. 1 To whom correspondence should be addressed: yyosimu@ hiroshima-u.ac.jp.
be possible that Salmonella that invaded the lower part of the oviduct are transported to the infundibulum, followed by movement through the peritoneal cavity to the ovary and other organs. It is also assumed that salmonella organisms invade circulating blood and are transported to the ovarian follicles (Keller et al., 1995; Thiagarajan et al., 1996). Thus Salmonella in reproductive organs may be incorporated into the yolk and albumen during the formation of eggs. The SE can be isolated from various organs including ovary and oviduct of the infected birds (Gast and Beard, 1990; Keller et al., 1997). It is possible that the SE invades the tissues of these organs; electron microscopic studies of chicks have shown passage of Salmonella through ileocecal mucosa (Popiel and Turnbull, 1985). The attachment and invasion of cultured chicken ovarian granulosa cells by SE (Thiagarajan et al., 1996) and colonization of SE in the ovarian and oviductal cells of birds inoculated with SE orally (Keller et al., 1995) also have been reported. However, further studies are needed to evaluate the possibility that SE in the peritoneal cavity can invade the tissues of reproductive organs. If SE colonizes tissues of reproductive organs, birds would lay contaminated eggs until the infection is eliminated. In mammalian animal models, cell-mediated immunity plays a major role in controlling Salmonella infection (George et al., 1987; Hsu, 1989). Although the immune mechanisms involved in the defense against Salmonella infection are less well understood in chickens, significance of phagocytosis by heterophils and
1170
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 12, 2015
ABSTRACT The aim of this study was to determine whether Salmonella enteritidis (SE) inoculated into the peritoneal cavity would colonize tissues of reproductive organs in Japanese quail hens. Quail hens regularly laying were intraperitoneally inoculated with 5 × 107 or 5 × 108 SE cells, and the ovary, oviduct, kidney, spleen, liver, and large intestine were excised 24 or 48 h after the treatment. Paraffin sections of these organs were immunostained for SE. Invasion of SE was found in the tissues of the ovarian stroma, the follicular wall including superficial and theca layers, and occasionally in the granulosa layer and yolk. The SE immunoreaction product frequently was found
SALMONELLA ENTERITIDIS IN HEN REPRODUCTIVE ORGANS
1171
MATERIALS AND METHODS Female Japanese quail laying seven eggs or more in a sequence were used in this study. They were kept in individual cages under 14L:10D and provided with feed and water ad libitum. Approximately after 2 h of oviposition, the birds were intraperitoneally inoculated with PBS alone or with SE suspended in PBS. The SE strain used in this study was isolated from clinical sample by Hiroshima Prefectural Health and Environmental Center, Hiroshima, Japan. The specific gene sequence had been confirmed by PCR using a primer set SIN-1 and SIN-2.2 The amount of SE inoculated in one bird was 5 × 107 or 5 × 108 cells in 0.5 mL of PBS. The birds inoculated with 5 × 107 SE cells were examined 24 or 48 h after the treatment. Those inoculated with 5 × 108 SE cells were examined only 24 h after the treatment. Control birds were intraperitoneally injected with 0.5 mL PBS and examined 24 or 48 h after the treatment. Four birds were used in each experimental or control group. For the tissue collection, the birds were killed by carbon dioxide inhalation and then the ovary (stroma, the largest and third largest preovulatory follicle), oviduct (tubular region of the infundibulum and middle parts of magnum, isthmus, uterus, and vagina), kidney, liver, spleen, cecum, and rectum were collected and fixed with 4% paraformaldehyde in PBS. The tissues were embedded in paraffin and sections of them (6 µm in thickness, 10 sections at every 60 µm in a tissue) were prepared and air-dried on slides. Sections were deparaffinized and rehydrated in PBS, followed by incubation with 1% (wt/vol) blocking reagent3 in PBS for 30 min to block nonspecific immunoreactions. Then sections were incubated with anti-Salmonella O antibody4 (10 µg/mL in 1% BSA-PBS) for 2 h. They were washed three times for 5 min each in PBS, and the immunoreactions were detected with a Histofine SABPO (M) kit.5 Briefly, the sections were incubated with
2
Takara Shuzo Co., Ohtsu, Japan. Roche Diagnostics GmbH, Mannheim, Germany. 4 HyTest Co., Turku, Finland. 5 Nichirei Co., Tokyo, Japan. 3
FIGURE 1. Sections of ovaries from SE-inoculated Japanese quail immunostained for SE. The birds were intraperitoneally inoculated with 5 × 108 SE cells and examined 24 h after the inoculation. G = granulosa layer; O = oocyte; T = theca layer; S = stroma; SL = superficial layer; V = vein; Y = yolk. Arrows indicate the immunoreactive SE. Scale bar = 20 µm. a) Ovarian stroma. Note the SE immunoreaction products in the stromal cells. b) Follicular wall of the largest follicle. SE immunoreactions are observed in the macrophage-like cells in the superficial layer and theca cells. c) Follicular wall of the largest follicle. Note the SE immunoreactions in the granulosa cells. d) Atretic largest follicle. Immunoreactions are observed in the macrophage-like cells and in the yolk.
biotinylated anti-mouse IgG for 1 h followed by washing in PBS and incubating with avidin-peroxidase complex for 1 h. Then the immunoprecipitates were visualized by incubating the sections with a hydrogen-peroxide-diaminobenzidine mixture. Control sections were prepared in the same manner except the first antibodies were replaced with normal mouse IgG. Sections were examined under a light microscope.
RESULTS No specific staining was observed in any tissues of control birds that were injected with PBS. The ovarian stroma was a loose connective tissue where numerous cortical follicles were located. The follicular wall of preovulatory follicles consisted of the granulosa layer surrounding the yolk, theca layer, superficial layer extending from the ovarian stroma, and superficial epithelium. In the ovarian stroma the immunoreaction products for SE were identified in the connective tissue of 10 of the 12 inoculated hens (Figure 1a). The SE reaction products were also found in the superficial layer and occasionally in the theca layer of preovulatory follicles. Those were observed in the cytoplasm of fibroblast-like and macrophage-like cells in the superficial layer (Figure 1b). In some follicles, the products were observed in the theca cells and granulosa cells (Figure 1c). One of the largest follicles underwent atresia, and a markedly large number of SE-containing cells in the surface layer and SE-positive intrafollicular yolk granules were observed (Figure 1d). The oviductal mucosal tissue was surrounded by ciliated
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 12, 2015
response of T-cell subsets and B cells in defending against SE have been suggested (Kramer, 1998; Withanage et al., 1998; Andreasen et al., 2001). The presence of immunocompetent cells, including antigen-presenting cells and T and B cells, has been shown in the ovary (Barua et al., 1998a,b, 2001; Barua and Yoshimura, 1999) and oviduct (Yoshimura et al., 1997; Zheng et al., 1998, 2000, 2001; Zheng and Yoshimura, 1999). Determination of SE invasion into these tissues and the feature of their colonization are essential to analyze whether these immunocompetent cells respond to SE invasion. Thus, the goal of this study was to determine whether SE inoculated into the peritoneal cavity invades and colonizes the tissues of reproductive organs.
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FIGURE 2. Sections of oviducts from SE-inoculated Japanese quail immunostained for SE. The birds were intraperitoneally inoculated with 5 × 108 SE cells and examined 24 h after the inoculation. E = mucosal epithelium; M = muscle; S = stroma; Se = serosa; L = oviductal lumen. Arrows indicate the immunoreactive SE. Scale bar = 20 µm. a) and b) Mucosa and serosa layers of infundibulum, respectively. Note SE immunoreaction products in the subepithelial stroma in the mucosa and a large number of positive cells in the serosa. c) Magnum. Note SE immunoreactions mucosal epithelial and subepithelial cells. d) and e) Isthmus and uterus, respectively. Note SE immunoreactions on the mucosal surface and in the mucosal epithelial cells. f) Vagina. SE immunoreactions are observed on the mucosal surface and in the mucosal epithelium and stromal cells.
pseudostratified epithelium. The SE reaction products were identified on the mucosal surface, in the mucosal epithelium, the lamina propria, and serosa in all the segments of the oviduct (Figures 2, a to f). Those in the lamina propria were found in the cells of subepithelial stroma. The products of SE reaction were also identified in the epithelial cells of tubules in the kidney, spleen cells, and liver (Figures 3, a to c) as well as in the lamina propria of rectum and cecum (Figure 3d). The SE reaction products were located in the cells of each tissue. The tissues of the ovary, oviduct, and other organs did not show histological degradations even the SE reactions were positive.
DISCUSSION We have examined how the SE colonizes in the reproductive organs of Japanese quail hens that were intraperitoneally inoculated with SE by immunocytochemistry. The significant finding of this study is that the SE reaction products were localized in the tissues and cells of ovary
and oviduct. They were also localized in the other tissues including the kidney, liver, spleen, and large intestine. The SE reaction products were localized in the stromal connective tissue and follicular wall in the ovaries, as well as mucosal epithelium and stroma in the oviduct. The presence of SE in the ovarian stroma and follicular wall suggest that the SE in the peritoneal cavity invaded the tissues through superficial epithelium. Then the SE was likely to reach the intrafollicular yolk because in some cases they were found in the granulosa cells and yolk of follicles. The SE is also likely to pass through mucosal epithelium surrounding the lamina propria of oviductal mucosal tissue because the organisms were found in the epithelium and stroma. Popiel and Turnbull (1985) revealed the passage of Salmonella organisms from epithelium to lamina propria of chick ileocecal mucosa. Keller et al. (1995) described that the SE appeared in the medulla of ovary, cortical stromal tissue of atretic follicle, and mucosal epithelium of oviduct in orally inoculated hens and also suggested that egg invasion by bacterial translocation from the peritoneal cavity might not occur. The current results indicate that the SE in the peritoneal cavity can invade the ovarian and oviductal tissues from the surface of these organs and colonize cells of these tissues, as does SE in digestive organs. However, the current histological observations suggest that significant tissue damage may not occur during this process. Enhancement of immunity in the reproductive organs may be needed to protect them from infection. The SE reaction products were found in the liver, kidney, spleen, and large intestine of some birds, suggesting that the invasion of SE in the tissues can occur not only in reproductive organs but also in other organs. The SE might disperse in the peritoneal cavity, followed by migration in the oviductal lumen, cloaca, and then large
Downloaded from http://ps.oxfordjournals.org/ at National Institute of Education Library, Serials Unit on June 12, 2015
FIGURE 3. Sections of kidney (a), liver (b), spleen (c), and rectum (d) from SE-inoculated Japanese quail immunostained for SE. The birds were intraperitoneally inoculated with 5 × 108 SE cells and examined 24 h after the inoculation (a to c) or inoculated with 5 × 107 SE cells and examined 48 h after that (d). SE immunoreaction products are localized in all organs (arrows). Scale bar = 20 µm. E = mucosal epithelium; L = oviductal lumen; S = stroma.
SALMONELLA ENTERITIDIS IN HEN REPRODUCTIVE ORGANS
intestine. It remains unknown whether the reproductive organs are more susceptible than the other organs. In conclusion, the current results suggest that the SE in the peritoneal cavity can invade and colonize the ovarian and oviductal tissues. It is possible that they are incorporated into internal egg components during egg formation, leading to production of contaminated eggs.
ACKNOWLEDGMENT This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to Y. Y.
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