Kazumi Sasai

Inhibitory effects of competitive exclusion and fructooligosaccharide, singly and in combination, on Salmonella colonization of chicks.

The inhibitory effects of competitive exclusion (CE) and 0.1% concentration of fructooligosaccharide (FOS), singly and in combination, on Salmonella colonization of chicks were investigated. Moreover, quantitation of the major cecal flora (Bifidobacterium, Bacteroides, Lactobacillus, and Escherichia coli) was performed. One-day-old birds were divided into four groups: (i) control, (ii) CE, (iii) FOS, and (iv) CE plus FOS. Chicks received Salmonella Enteritidis at 7 days (experiment 1) or 21 days (experiment 2). Birds in each group were killed at 1 day, 7 days, and 14 days after inoculation of Salmonella Enteritidis for count of salmonella in cecal contents. In experiment 1, the mean number of Salmonella Enteritidis in the chicks inoculated with CE was significantly decreased compared with the other three groups at 1 day postinoculation. In experiment 2, the mean numbers of Salmonella Enteritidis in the chicks of the FOS group and the FOS plus CE group were significantly decreased compared with the control group at 1 day and 7 days postinoculation. On 7- and 21-day-old chicks, few changes on number of total bacteria, Bifidobacterium, Bacteroides, Lactobacillus, and E. coli were observed in the cecal contents of treated groups compared with the control group. Low-dose feeding of FOS in the diet of chicks with a CE treatment may result in reduced susceptibility to Salmonella colonization but may not lead to a shift in the intestinal gut microflora on 7- and 21-day-old chicks.

Salmonella enteritidis Contamination of Eggs from Hens Inoculated by Vaginal, Cloacal, and Intravenous Routes

Laying hens were inoculated intravaginally (IVg) once (IVg-single) or three times (IVg-triple), intracloacally (IC), or intravenously (IV) with Salmonella enteritidis (SE) phage type 4. Eggs tested were significantly (P < 0.05) fewer positive in group IC than in other groups. SE was recovered from egg contents in the groups IVg-single (9.6%), IVg-triple (4.2%), and IV (11.5%). IVg and IC inoculation resulted in colonization of the cloaca and lower portions of the oviduct but not the portion above the isthmus, whereas IV inoculation resulted in colonization of the entire oviduct. Only IV inoculation resulted in colonization of the ovary. In group IV, SE was recovered from three of six eggs found in the oviduct at necropsy, but in other groups, SE was not recovered from 53 eggs in the oviduct. The results suggested that the SE infection of vagina resulted in a frequent incidence of contaminated eggs and that SE adhered to the eggs from the contaminated vagina might pass through shells and shell membranes.

Salmonella penetration through eggshell associated with freshness of laid eggs and refrigeration

Effects of egg age after laying and refrigeration on penetration of the eggshell by Salmonella enteritidis (SE) and Salmonella typhimurium (ST) were examined. Eggs 0.25 to 3 h, 3.25 to 6 h, 1 day, and 7 days old held at two temperatures were immersed in SE or ST suspensions containing 10(3) or 10(6) CFU/ml at 25 degrees C for 10 min. After holding at 25 degrees C for 2 h, the inner eggshell and egg contents were examined for Salmonella cells. The recovery rates of Salmonella cells from both the inner eggshell and egg contents of the 0.25- to 3-h-old eggs were significantly higher than those of other groups, especially at the high-exposure dose. There was no significant difference noted between SE and ST in ability to penetrate through eggshell. Salmonella penetration was significantly decreased by cooling the eggs at 4 degrees C for 15 min prior to immersing them in SE or ST suspension. The data suggested that Salmonella cells readily penetrated through the shell of freshly laid eggs, but that this penetration was suppressed by cooling the eggs before they were exposed to Salmonella suspensions.

Dynamics of lymphocyte subpopulation changes in the cecal tonsils of chickens infected with Salmonella enteritidis.

Salmonella enteritidis (SE)-induced changes in various T and B lymphocyte subpopulations in the cecal tonsils of chickens were analyzed using flow cytometry. At 1 day post-SE inoculation, the percentages of CD3(+) and CD8(+) T lymphocytes were significantly decreased in the group inoculated with 1x10(9) SE colony-forming units (CFU) (SE high) and in the group inoculated with 1x10(6) SE CFU (SE low) compared with the uninfected control group. The percentage of CD4(+) T lymphocytes was significantly increased in the SE high group compared to the uninfected and the SE low groups at 4 days after SE inoculation. The percentage of IgG(+) B lymphocytes was also significantly increased in both SE high and low groups compared to the uninfected control at 6 days post-SE inoculation. In contrast, the SE low group showed significantly fewer IgM(+) B lymphocytes compared to the uninfected and SE high groups. These results show that SE infection induces significant changes in the cecal tonsil lymphocytes subpopulations shortly following SE inoculation.

T lymphocytes, B lymphocytes, and macrophages in the ovaries and oviducts of laying hens experimentally infected with Salmonella enteritidis.

Subsets of T lymphocytes, B lymphocytes, and macrophages in the ovaries and oviducts of laying hens were enumerated by immunohistochemistry after intravenous inoculation with Salmonella enteritidis. Almost all T cell subsets in the ovaries and different regions of the oviduct increased in number at 7 days post-inoculation and reached a peak by day 10. This T cell surge was followed by a peak in B cell numbers at day 14. The number of macrophages declined initially but recovered to preinoculation levels by day 21. At day 21, the numbers of T and B cells also returned to normal levels, except for IgG+ B cells in the infundibulum, isthmus, and vagina where they remained consistently elevated. The T and B cell proliferation at 10-14 days post-inoculation immediately preceded a decline in the number of S. enteritidis positive tissues from infected hens beginning at day 14 suggesting that these lymphocytes play a major role in the local immune response to S. enteritidis. The Salmonella-oviduct model will be useful for future studies on local immunity to various infectious agents.

Analysis of splenic and thymic lymphocyte subpopulations in chickens infected with Salmonella enteritidis.

Lymphocytes expressing CD3, CD4, CD8, pan lymphocyte, IgA, IgG and IgM cell surface antigens were assessed by in the spleen and thymus of chickens following infection with Salmonella enteritidis using flow cytometric analysis. At 6 days post primary infection and 2 days post secondary infection with S. enteritidis, the percentages of IgA+ and IgM+ lymphocytes in the spleen were significantly increased (P < 0.05). At 2 days post secondary infection with S. enteritidis, the percentage of CD4+ T lymphocyte in the spleen and CD8+ T lymphocyte percentage in the thymus were significantly increased (P < 0.05). These results indicate that S. enteritidis infection induces changes in the spleen and thymus that reflect the dynamics of the host protective immune response.

The detection of a novel type of Cryptosporidium andersoni oocyst in cattle in Japan

Cryptosporidium muris, found in rodents and cattle, has been recognized as a valid species. However, this organism from cattle was recently separated from C. muris infecting rodents based on molecular data and a transmission study. As a consequence, it has been proposed as a new species, C. andersoni. More recently, C. andersoni, which has infectivity to rodents, was detected in cattle in Japan, where it has been designated as a novel type. However, isolates from cattle in Japan have not been analyzed genetically, and therefore it remains unclear whether a novel type of C. andersoni is distributed widely in Japan. In the present study, we detected Cryptosporidium oocysts from cattle reared in a different area than those examined previously in Japan. These were identified by molecular analysis and experimental transmission. The 18S ribosomal RNA gene sequence of the isolate examined was identical with that of C. andersoni reported previously, and the isolate was successfully transmitted to severe combined immunodeficiency mice. Therefore, the isolate from cattle examined in the present study was identified as a novel type of C. andersoni. Our data suggests that it is widespread in cattle in Japan.

Secretion of Salmonella-specific antibodies in the oviducts of hens experimentally infected with Salmonella enteritidis

The production and secretion of Salmonella enteritidis whole cell antigen-specific antibodies in the oviducts and in the serum of laying hens experimentally infected with Salmonella enteritidis, was analyzed by ELISA. The dynamics of the antibody levels in the oviducts were identical to that in the serum. Subclasses of antibodies (IgA, IgG, and IgM) in the infected hens were found to increase significantly (p < 0.01) compared to those in the control uninfected hens throughout the experiment. IgG and IgM levels in both oviducts and in sera reached to a peak by 14 days post-inoculation, and remained elevated throughout. The secretion of IgA seemed to be transient since the IgA levels increased to a peak 7 days after both primary and secondary inoculations, and declined rapidly. The elevated levels of antibodies were followed by partial clearance of Salmonella organisms from the oviducts. The present results indicate a significant local immune reaction against the Salmonella infection and suggest an association of the local antibodies with the clearance of Salmonella from the oviducts at least partially.

A chicken anti-conoid monoclonal antibody identifies a common epitope which is present on motile stages of Eimeria, Neospora, and Toxoplasma.

The chicken monoclonal antibody (mAb) 6D12-G10, raised against Eimeria acervulina sporozoites, has previously been shown to recognize the conoid of E. acervulina sporozoites and inhibit sporozoite invasion of lymphocytes in vitro. In indirect immunofluorescent assay, the mAb 6D12-G10 also reacted with merozoites from E. acervulina and identified a 21-kDa merozoite protein on western blots. By confocal laser scanning microscopy, the conoid of sporozoites from 6 different avian Eimeria species (E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. tenella) were reactive with 6D12-G10 mAb. Furthermore, the 6D12-G10 mAb also showed cross-reactivity with motile stages of 2 closely related apicomplexans, Neospora, and Toxoplasma. These results indicate that the mAb 6D12-G10 identifies a conserved epitope on the conoid that is important in host cell invasion by the apicomplexan parasites.

Changes in microflora of the cloaca and oviduct of hens after intracloacal or intravaginal inoculation with Salmonella enteritidis.

Quantitative and qualitative microbiological examination was carried out on cloacal and oviductal contents pre- and postinfection with Salmonella enteritidis (SE) intracloacally or intravaginally. Before inoculation with SE, the means +/- standard deviation (SD) of total bacterial counts, anaerobic bacterial counts, and aerobic bacterial counts in the cloaca were log10 7.7 +/- 0.7, 7.4 +/- 0.2, and 6.9 +/- 0.8 colony-forming units (CFU)/g, respectively. The predominant bacteria were Bacteroidaceae, Lactobacillus, and Escherichia coli. Before inoculation with SE, the means +/- SD of total bacterial counts, anaerobic bacterial counts, and aerobic bacterial counts in the vagina were log10 5.7 +/- 1.4, 5.5 +/- 1.3, and 3.6 +/- 2.7 CFU/g, respectively. Bacteroidaceae and Lactobacillus were predominant. Following inoculation with SE, only the cloacal population of Lactobacillus in hens inoculated intracloacally was significantly increased compared to that before the inoculation. Other indigenous microflora were stable even after the inoculation. In the uterus, very few bacteria, Lactobacillus and Staphylococcus, were isolated. Five of 20 eggs (25%) from hens inoculated with SE intravaginally were positive for SE, whereas no SE was recovered from 22 eggs in hens inoculated with SE intracloacally. SE was recovered from the uterus after intravaginal inoculation with SE and from the vagina after intracloacal inoculation with SE. Contamination may ascend from the cloaca into the lower parts of the oviduct and subsequently contaminated eggs may occur.