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Rodents serve as amplifiers of Salmonella infections in poultry flocks and can serve as a source of Salmonella contamination in the environment even after thorough cleaning and disinfection. This study aims to determine the dynamics of Salmonella occurrence in rodents and its relation to Salmonella contamination in the layer farm environment, including air dusts and eggs. From 2008 to 2017, roof rats ( Rattus rattus ), environmental swabs, air dusts, and eggs were collected from an intensive commercial layer farm in East Japan and were tested for Salmonella spp. using standard procedures. In roof rat samples, the Salmonella isolation rate was reached at 10% (95% confidence interval [CI] 8.1-21.9) in which Salmonella Corvallis, Salmonella Infantis, Salmonella Potsdam, and Salmonella Mbandaka were the frequent isolates from the cecal portion of the intestines. On the other hand, the prevalence rate of Salmonella in environmental swabs was at 5.1% (95% CI 2.2-7.4) while air dusts were at 0.9% (95% CI 0.2-1.8). It was observed that the prevalence of predominant Salmonella serotypes shifted over time; in roof rats, it was noted that Salmonella Potsdam gradually replaced Salmonella Infantis. In environmental swabs and eggs, Salmonella Corvallis and Salmonella Potsdam increased significantly while Salmonella Infantis became less frequent. In air dusts, Salmonella Corvallis was observed to decrease and Salmonella Potsdam became more common. Based on our findings, the role of roof rats in the epidemiology of Salmonella in layer farms was expanded from being a reservoir and an amplifier host into a shifting vessel of the most predominant serotypes.

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.

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.

The efficacy of Salmonella enteritidis (SE) oil-emulsion bacterin (a commercially available vaccine) was evaluated in an intravaginal challenge model in hens producing a high rate of SE-contaminated eggs. Hens were vaccinated at 38 wk of age. A second (booster) bacterin injection was administered 4 wk later. Two weeks after the second vaccination, all hens were challenged intravaginally with 10(7) colony-forming units of SE. After challenge, 36 of 189 eggs (19.0%) in the vaccinated hens were positive for SE, and this contamination rate was significantly (P < 0.01) lower than that in the unvaccinated hens (61 of 165 eggs, 37.0%). SE was highly recovered from the cloacal and vaginal swabs of the unvaccinated and vaccinated hens, but the number of SE from the cloaca of the vaccinated hens was significantly (P < 0.05) lower than that in the unvaccinated hens at 7 days post-challenge (PC). The recoveries of SE from the spleen and ovary in the vaccinated hens were significantly (P < 0.05) lower than those in the unvaccinated hens at 7 days PC. At necropsy, SE was recovered from 2 of 15 forming eggs (13.3%) taken from the oviducts of the unvaccinated hens, whereas no SE was recovered from 17 forming eggs in the vaccinated hens. After vaccination, serum antibodies for SE in the vaccinated hens were significantly higher than those in the unvaccinated hens. Antibodies from the oviductal washing, especially immunoglobulin G isotype, in the vaccinated hens were higher than those in the unvaccinated hens after challenge. This intravaginal challenge model produced frequent contaminated eggs and clearly demonstrated the ability of the bacterin to protect against egg contamination. The present model may be a useful tool for further studies to evaluate the protective effect against SE contamination of eggs by potential vaccine candidates.

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.

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.

Eleven-day-old chickens received 10(8) colony-forming units Salmonella typhimurium orally for 2 consecutive days. The next day, the 13-day-old chickens were given a high dose of ochratoxin A (3 mg/kg) orally. The number of S. typhimurium in both the duodenal and cecal contents of chickens administered with high doses of ochratoxin A increased significantly when compared with control birds. Ochratoxin A was shown to be one of numerous factors that affect the susceptibility of chickens to salmonellae colonization.

Experiments were conducted to determine the effect of Eimeria tenella infection on the production of Salmonella enteritidis-contaminated eggs and susceptibility of egg-laying hens to S. enteritidis infection. The dose of sporulated oocysts of E. tenella used in the present study was 2 x 10(5) per chicken. When hens were infected with 10(6) colony-forming units (CFU) of S. enteritidis, E. tenella infection resulted in a significant increase (P < 0.05) of S. enteritidis contamination in the shells of fresh eggs but not in the egg contents of fresh eggs and stored eggs. Significant effects of coccidial infection on the contamination of eggs (shells and contents) were not observed when hens were infected with 10(4) CFU of S. enteritidis. Surface-disinfected eggs pooled within each week from individual chickens infected with 10(4) CFU of S. enteritidis and E. tenella showed significantly (P < 0.05) more S. enteritidis contamination in eggshells, but not in egg contents, than did those from hens infected with S. enteritidis alone. Coccidial infection resulted in a significant increase of S. enteritidis in the cloacal swabs of hens infected with 10(4) CFU of S. enteritidis. In all the experiments, chickens infected with S. enteritidis and E. tenella showed significantly higher positive rates and counts of cecal S. enteritidis than did chickens infected with S. enteritidis alone. No significant differences of S. enteritidis-positive rates in ovary, oviduct, liver, and spleen were noted whether the hens were infected with a combination of E. tenella and S. enteritidis or with S. enteritidis alone.

Five experiments were conducted to determine the effects of dietary lactose and rye on cecal colonization of Clostridium perfringens in white leghorn chickens. Six days after oral inoculation of the organism, the numbers of C. perfringens organisms in the cecal contents were significantly lower in chickens on 2% and 10% lactose-supplemented feed than in chickens on unsupplemented feed. When C. perfringens was given in drinking water, 10% lactose supplementation was needed to significantly reduce the counts of C. perfringens 4, 6, and 8 days after feeding began. Effect of rye-ration on cecal colonization of C. perfringens was also examined. Counts of C. perfringens in cecal contents of chickens fed a diet containing 50% rye were significantly higher than control values 4, 6, and 8 days after feeding began. When chicks were fed a diet containing both 10% lactose and 50% rye, C. perfringens counts in cecal contents were lower than in chickens fed 50% rye only at 6 days after feeding began. Results led to the conclusion that dietary lactose is effective in reducing the cecal colonization of C. perfringens.

The effects of different levels of oocyst inocula of Eimeria acervulina, E. tenella, or E. maxima on plasma carotenoids, total plasma lipids, total plasma proteins, packed cell volume (PCV), bird performance, and coccidial lesion scores were determined in male Hubbard chickens. Each test consisted of an uninfected treatment and either four levels (E. tenella and E. maxima) or five levels (E. acervulina) of inocula. Carotenoids and lipids were significantly (P < or = 0.05) depressed by E. acervulina starting at 10(2) oocysts per bird, whereas 10(4) and higher inoculum levels significantly depressed plasma protein. Carotenoids and lipids were significantly depressed by E. tenella beginning at 10(4) oocysts per bird, whereas 10(2) oocysts per bird and greater levels significantly depressed plasma protein. E. maxima significantly depressed carotenoids and lipids beginning at 6.7 x 10(2) oocysts per bird and plasma protein at 6.7 x 10(4) oocysts per bird. PCV was depressed by E. acervulina and E. tenella starting at 10(4) oocysts per bird but was not affected by E. maxima. Weight gain was significantly depressed by E. acervulina beginning at 10(5) oocysts per bird, by E. tenella at 10(4) oocysts per bird, and by E. maxima at 6.7 x 10(4) oocysts per bird. Coefficients of determination (R2) were highest for carotenoids (0.96-0.99), followed by lipids (0.93-0.96), weight gain (0.89-0.91), feed:weight gain ratio (0.89-0.91), and protein and PCV (0.65-0.92). Results demonstrated that plasma carotenoids and lipids were excellent response variables for measuring the effects on broiler chickens of each of the Eimeria spp. tested.