Eiichiroh Baba

Differences among six Salmonella serovars in abilities to colonize reproductive organs and to contaminate eggs in laying hens.

The abilities of Salmonella serovars to colonize the reproductive organs of chickens and to contaminate eggs were compared. Mature laying hens were inoculated intravenously with 10(5) colony-forming units of Salmonella enteritidis, Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to cause the systemic infection. Salmonella enteritidis was recovered from three yolks of the laid eggs (7.0%), suggesting egg contamination from the transovarian transmission of S. enteritidis. The liver, spleen, and cecum were colonized by each serovar similarly at 4 or 7 days postinoculation (PI), whereas the ovary and preovulatory follicles were colonized by S. enteritidis with significantly (P < 0.05) higher levels than by the other serovars at 4 and 7 days PI. Salmonella enteritidis was recovered from the cloaca and vagina at 2, 4, and 7 days PI and from the other portions of the oviduct at 4 and 7 days PI. In addition, S. enteritidis had been persistent in the peripheral blood for 7 days PI. These results suggest that S. enteritidis is the predominant serovar to colonize the reproductive organs of mature laying hens among six serovars used in this study, reflecting the field situatibn in which the predominant outbreaks of human salmonellosis were caused by S. enteritidis-contaminated eggs recently. The ability of S. enteritidis to colonize the reproductive organs may be one of the reasons that egg contamination with S. enteritidis has increased.

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.

Differences in Abilities to Colonize Reproductive Organs and to Contaminate Eggs in Intravaginally Inoculated Hens and In Vitro Adherences to Vaginal Explants Between Salmonella enteritidis and Other Salmonella Serovars

In Experiment 1, mature laying hens were inoculated intravaginally with 10(6) colony-forming units of Salmonella enterica serovar enteritidis (S. enteritidis), Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to compare their abilities to colonize the reproductive organs of chickens and to contaminate eggs. Salmonella enteritidis was more frequently recovered (from 11 of 40 eggs, 27.5%) than the other serovars, and especially the inner shell was contaminated with these organisms in 10 of 40 eggs (25.0%). The contamination rates and the viable counts in cloaca were significantly (P < 0.05) higher in hens inoculated with S. enteritidis than in those inoculated with the other serovars at 4 days postinoculation (PI). In the vagina, the positive rates were 90%-100% in hens inoculated with S. enteritidis, and the viable counts of the organisms in this portion were significantly (P < 0.05) higher than those of the other serovars at 2, 4, and 7 days PI. The ceca were colonized similarly by each serovar at 7 days PI. The spleen and ovary were infected with S. enteritidis in three and one hen, respectively. No Salmonella was recovered from liver and peripheral blood in any hen. Salmonella enteritidis was recovered from other oviductal portions than the vagina (10%-20%), whereas no forming egg was contaminated in the oviduct. In Experiment 2, the in vitro adherence of these six serovars to the vaginal epithelium was compared with vaginal explants. The mean number of S. enteritidis attaching to the secondary villi in the vaginal lumen was significantly (P < 0.05) higher than those of the other serovars. These results suggest that S. enteritidis has a specific advantage over the other Salmonella serovars by its capacity to colonize the vaginal tissues of hens, and this higher affinity of S. enteritidis to the vagina may play a significant role in the production of many S. enteritidis-contaminated eggs.

Effects of Eimeria brunetti Infection and Dietary Zinc on Experimental Induction of Necrotic Enteritis in Broiler Chickens

Broilers infected with Eimeria brunetti and given dietary zinc were examined for experimental induction of necrotic enteritis. Inoculation with sporulated E. brunetti oocysts at 7 days of age was followed by 5 consecutive days of oral inoculation with cultured Clostridium perfringens. Feed was supplemented with zinc at 1000 ppm. Upon necropsy of broilers 6 days after coccidial inoculation, necrotic enteritis was found in 20% (2/10) of birds given both organisms and dietary zinc. Coccidial lesion scores were also highest in that group. Birds infected with E. brunetti and C. perfringens with no dietary zinc had significantly higher coccidiosis lesion scores (P less than 0.05) than groups inoculated with E. brunetti only, regardless of zinc supplementation. Alpha toxin levels in intestinal contents were low in groups infected with both organisms, regardless of zinc supplementation. Zinc was tested for effects of alpha toxin production in vitro. In the mid-log phase (6 hours incubation), a high level of alpha toxin was produced in zinc-supplemented media, but this was lost quickly in the presence of trypsin. Addition of zinc partly protected the toxin from the action of trypsin.

Influence of Bacteria on Clostridium perfringens Infections in Young Chickens

When monoflora chickens with Lactobacillus acidophilus or Streptococcus faecalis were inoculated with Clostridium perfringens either in broth culture or resuspended in Gifu anaerobic medium broth or supernatant fluid, few or no chickens died. Approximately 50% of germ-free chickens died after inoculation of C. perfringens culture, whereas no conventional birds died after inoculation of broth culture. C. perfringens in the contents of duodenum from germ-free chickens numbered about 10(4) colony-forming units (CFU)/g after inoculation 10(8) CFU broth culture per bird, but in gnotobiotic and conventional chickens this organism decreased or was not detected. When C. perfringens was cultured in intestinal contents collected from germ-free chickens, C. perfringens proliferated but alpha toxin was not detected. These findings indicate that the pathogenicity of C. perfringens was suppressed by L. acidophilus or S. faecalis administered previously or inhibited by normal intestinal flora.

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.

Clostridial population and the intestinal lesions in chickens infected with Clostridium perfringens and Eimeria necatrix

Chickens infected with Clostridium perfringens and Eimeria necatrix were examined bacteriologically and pathologically. When chickens were inoculated with 1.0 x 10(8) C. perfringens and/or 2 x 10(4) E. necatrix sporulated oocysts, populations of C. perfringens in the intestinal contents were examined on 3, 5 and 7 days after E. necatrix inoculation. In both groups infected with E. necatrix, the mean clostridial counts were significantly higher than those of uninfected controls. The concurrent infection had no enhancing effects on increasing the clostridial population more than E. necatrix-alone. Mortality of 4-day-old chickens inoculated on 5 consecutive days with C.perfringens after receiving E. necatrix was higher than those of chickens inoculated with the both organisms. However, intestinal lesions of the concurrently infected group were not different from E. necatrix-alone-infected group on 5 and 7 days after the coccidial infection. When chickens received a large dose (1.5 x 10(9)) of C. perfringens after the inoculation with E. necatrix, edema in the duodenum through jejunum were observed early after the bacterial broth inoculation. These results suggest that the concurrent infection with E. necatrix and C. perfringens increases clostridial population in the intestine of the chickens and has synergic effects on mortality and edema in the upper intestine.

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.