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With the exception to Salmonella enterica serotype Typhimurium and S. Enteritidis (serogroups B [O:4] and D [O:9], respectively), there have been very few studies conducted on the respiratory tract as route of infection in chickens with salmonellas from serogroup C1 (O:6,7) . Therefore, the purpose of this present study was to determine the potential organ invasion by Salmonella enterica serotype Potsdam (SP), S. Mbandaka (SM), and S. Infantis (SI) from serogroup C1 (O:6,7) and compare their characteristics with those of S. Enteritidis (SE) on intratracheally (IT) challenged 3-week-old layer chicks. A total of 360 one-day-old White Leghorn layer chicks were acquired from a commercial hatchery and randomly assigned into four treatment groups (SP, SM, SI, and SE, respectively), consisting of three independent trials. Chicks were grown up to 21 days (3 weeks) and IT-challenged thereafter with 10 6 CFU of respective salmonella organisms per group ( n  = 30). Chicks ( n  = 5) were humanely sacrificed every 24 h for 6 days post-IT infection and organs such as lung, heart, liver, spleen, kidney and caecal content were cultured for salmonella. All treatment groups exhibited colonization of lungs and caecal contents at 1 d ( P  = 0.475) and 4 d ( P  = 0.696) post-IT infection, respectively. There was no isolation of SP, SM, and SI in heart, liver, spleen, and kidney. In contrast, SE was obtained from heart, liver, and spleen of IT-infected chicks. The findings of this study contribute to a better understanding of the importance of the respiratory route in salmonella infection in poultry.

There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.

Cases of poor egg production were investigated in 2 layer farms from Ibaraki Prefecture in eastern Japan. To identify any microbial agents that may have caused the problem, necropsy, bacterial isolation, histopathology, and virus detection were performed. Members of the avian adenoviruses was detected by PCR in oviduct samples from both farms; chicken anemia virus coinfection was also confirmed in one of the farms. Avian adenovirus was isolated from the oviducts of the affected chickens on each farm. Inoculation into chick embryos showed tropism for the chorio-allantoic membrane. Stunting and hemorrhaging was observed in all infected embryos, as well as death in a few. Inoculation of 1-day-old specific pathogen-free chicks, and 400-day-old commercial hens, did not result in any significant findings. The isolated viruses were analyzed by sequencing of the hexon gene and were confirmed as fowl adenovirus type-c serotype-4 (FAdV-4). The 2 virus strains were found to be 99.29% similar to each other. One of the strains, Japan/Ibaraki/Y-H6/2016, was 99.15% similar to the KR5 strain. The other, Japan/Ibaraki/M-HB2/2016, was 99.57% similar to the KR5 strain. Fiber-2 gene analysis confirmed the identity as FAdV-4 that is closely related to nonpathogenic strains. Although nonpathogenic to chicks and laying hens, this infection can possibly cause economic damage. Perhaps the bigger concern is the effect on infected breeder operations. Because the virus is fatal to 9.09% of infected embryos, this could translate to a considerable loss in chick production owing to embryonic death. This is the first report of detection and isolation of FAdV-4 from the chicken oviduct; however, further studies are needed to elucidate its impact on both layer and breeder flocks. Indeed, FAdV-4 has negative effects on the avian reproductive tract as well., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

From a total of 72 commercial layer and pullet farms that were monitored in the eastern Japan area, 4 farms had mild to severe respiratory disease accompanied by decreased feed intake and drop in egg production. Microbiological analysis showed that 3 of the 4 farms, particularly from Fukushima, Tochigi, and Ibaraki prefectures, were positive for Ornithobacterum rhinotracheale (ORT). Out of 65 birds examined, ORT was isolated in 21 birds (32.31%). All isolates were Gram-negative pleomorphic rods with a colony size of 0.05 mm, translucent with grayish coloration, and with butyric smell after 48 h of incubation in 10% chicken blood agar at 37°C under microaerophilic conditions. All isolates reacted positively in the p-nitrophenyl-β-d-galactopyranoside test within 3 h and were positive in cytochrome oxidase tests with an API 20NE identification system biocode of 0-0-2-0-0-0-4. An agar gel precipitation test showed that all isolates were serotype-A. All strains were positive in PCR by yielding a 784 bp amplicon of the 16S rRNA gene. All strains were resistant to amikacin, colistin, gentamicin, kanamycin, neomycin, polymyxin b, streptomycin, and sulfamethoxazole trimethoprim and susceptible to amoxicillin clavulanic acid, ampicillin, doxycycline, spectinomycin, and tetracycline. This study is the first characterization of ORT from commercial layer chickens in eastern Japan., (© 2017 Poultry Science Association Inc.)

In the poultry industry, Eimeria spp. is one of the important pathogens which cause significant economic losses. We have previously generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina and with cross-reactive among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium spp. Furthermore, the protein of Cryptosporidium parvum recognized by the 6D-12-G10 has been identified as elongation factor-1α (EF-1α). In the present study, to identify the target molecule of E. acervulina by the mAb, we performed two-dimensional Western blotting analysis. Finally, we found two positive molecules which are identified as EF-1α and a related protein. Our previous finding using C. parvum and the results in this study suggest that EF-1α could be associated with the invasion facilitated by the cytoskeleton at the apical region of zoites.

Hemorrhagic diarrhea in poultry is caused by Eimeria tenella, the most pathogenic avian coccidian parasite, and new approaches to treat the disease are continually being sought. Although eimeripain, a cathepsin B-like cysteine protease from E. tenella, has recently been identified as a novel anticoccidial drug target, its localization during the intracellular development of parasites remains unclear. Here, we demonstrate the expression of eimeripain during asexual and sexual development of E. tenella in vivo. Promature eimeripain was detected only in the early immature second generation of schizonts. In contrast, the mature eimeripain was most strongly detected in the middle-sized immature second generation of schizonts. Both promature and mature eimeripain disappeared depending on the maturation level of second generation of schizonts, but were strongly expressed again in the third generation of schizonts. In the sexual stage, both promature and mature eimeripain were detected in the cytoplasm of micro- and macro-gametocytes and zygotes, but expression became weak in zoites forming oocysts. Collectively, our findings suggest that eimeripain might play a key role in the differentiation of intracellular zoites in the ceca and could be an interesting candidate to develop a novel, effective anti-coccidian drug.

Although there have been several reports on the efficacy assessment of a Salmonella enterica serovar Enteritidis vaccine against intestinal and parenchymatous organ diseases of laying hens, no public health risk characterization of its long-term effect on eggs has been reported. In this study, we attempted to assess the public health effect of an inactivated S. enterica serovar Enteritidis vaccine against serovar Enteritidis contamination of chicken eggs. We analyzed serovar Enteritidis isolation test results from four windowless farms in which inactivated-vaccine administration was initiated based on the sanitary monitoring program of a farm. When flocks with and without S. enterica serovar Enteritidis vaccine treatments were mixed, the application of an inactivated serovar Enteritidis vaccine decreased the most probable number (MPN) of bacteria by at least 100-fold in broken (liquid) egg samples positive for serovar Enteritidis, although a statistical difference between those MPNs could not be obtained. The isolation frequency after the vaccine application was less than 1/10 (P < 0.01). No S. enterica serovar Enteritidis bacteria were isolated approximately 1 year after all of the chickens had received the inactivated serovar Enteritidis vaccine. It was suggested that an adequate administration of an inactivated serovar Enteritidis vaccine reduced the contamination risk of eggs (the number of isolated serovar Enteritidis cells and detection frequency) compared to the contamination risk of eggs laid by nonvaccinated hens.

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.

The prevalence of Salmonella in four layer farms in eastern Japan was investigated between 2004 and 2006 to determine the role of roof rats (Rattus rattus) in the epizootology of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis). Persistent S. Enteritidis and S. Infantis contamination of the environment and pooled egg samples were detected in three out of four layer farms. A total of 113 (13.3%) and 158 (18.6%) out of 851 rats examined were positive for S. Enteritidis and S. Infantis, respectively. By pulsed-field gel electrophoresis, only one indistinguishable pulsed-field pattern was yielded by S. Enteritidis strains from rats, eggs and environmental samples from each of the two contaminated layer farms. Although, a variety of pulsed-field patterns were generated by S. Enteritidis isolates from rats, eggs, and the environment of the other contaminated farms, there are, however, some S. Enteritidis strains that are closely related clones. These results suggest that roof rats are carriers of S. Enteritidis and S. Infantis and that persistent S. Enteritidis and S. Infantis infections in a rat population may play an important role in the spread and maintenance of these pathogens inside the layer premises.

Levels of Salmonella enterica subsp. enterica serovar Enteritidis infection and serum S. Enteritidis antibodies after experimental S. Enteritidis challenge and feed withdrawal were investigated in S. Enteritidis-vaccinated and unvaccinated hens. The results were used to determine whether formalin-inactivated S. Enteritidis vaccination can protect layer hens from S. Enteritidis challenge during feed withdrawal periods. S. Enteritidis infection rates were evaluated from cloacal swabs, eggs and organs. Serum antibody titers to deflagellated S. Enteritidis whole cells (DEWC) and S. Enteritidis FliC-specific 9-kDa polypeptide (SEp 9) were examined by commercial ELISA kits. Cloacal S. Enteritidis recovery rates were lower in the vaccinated than unvaccinated group. Recovery rates of S. Enteritidis from samples increased after feed withdrawal and decreased after re-introduction of feed. S. Enteritidis counts in cloacal swabs were lower in the vaccinated than in the unvaccinated group (P<0.05). More S. Enteritidis-positive eggs were detected from the unvaccinated group. Before S. Enteritidis challenge, the DEWC ELISA titer of the vaccinated group was higher (P<0.05) than the unvaccinated group; subsequently, the S. Enteritidis DEWC ELISA titers of both groups increased gradually. In contrast, only the vaccinated group elicited high SEp-9 antibody titer during post-challenge and feed withdrawal. Additionally, vaccinated hens yielded negative S. Enteritidis isolation rates from egg contents. There is a correlation between negative S. Enteritidis isolation rates and high SEp 9 titers in vaccinated layer hens challenged with S. Enteritidis and subjected to feed withdrawal regimens. These findings suggest the S. Enteritidis vaccination of pullets may protect against S. Enteritidis infection during forced molting and that SEp 9 titer could be a potential indicator of antibody protection against S. Enteritidis infection. The potential of the SEp 9 peptide as an antigen for S. Enteritidis vaccination in the future is worth noting.

Japanese layer farms were surveyed for Salmonella Enteritidis vaccination and infection with specific antigens for egg yolk antibodies with the use of vaccination-specific antigen Salmonella Enteritidis FliC-specific 9-kDa polypeptide (SEP9) and infection-specific antigen deflagellated Salmonella Enteritidis whole cell (DEWC). The specific antibodies in eggs from 201 commercial layer farms throughout Japan were surveyed. The percentages of farm flocks with a mean enzyme-linked immunosorbent assay (ELISA) titer of over 0.1 were 56.2% (113 of 201) in DEWC-ELISA and 22.3% (45 of 201) in SEP9-ELISA. Flocks indicating high titers in SEP9-ELISA always showed high titers in DEWC-ELISA. Because both specific antibody titers of the vaccinated flocks monitored long term remained high throughout life, flocks with high titers of both ELISAs in this survey must be vaccinated. On the other hand, 34.3% (69 of 201) of flocks had high titers of DEWC-specific antibody alone. Because Salmonella Enteritidis infection induces the DEWC-specific antibody but not the SEP9-specific antibody, detecting only high ELISA titers of DEWC-specific antibody can be an effective monitoring tool for Salmonella Enteritidis exposure rather than vaccination. These results suggest that vaccination programs in Japanese layer farms would be insufficient to control Salmonella Enteritidis infection, and egg screening to detect specific antibodies would be valuable in obtaining the necessary information to control Salmonella Enteritidis infection.

Salmonella Enteritidis is the predominant serovar associated with egg-borne salmonellosis in humans. The colonization of S. Enteritidis in the vagina may play a role in the production of S. Enteritidis-contaminated eggs. In the first experiment, the in vitro adhesion of S. Enteritidis in vaginal and follicular explants was compared with that of S. Typhimurium by bacteriological isolation methods. The mean number of S. Enteritidis associated with vaginal explants was significantly (P < 0.05) higher than S. Typhimurium associated with vaginal explants and both serovars associated with follicular explants. In the second experiment, the in vitro adhesion and invasion of S. Enteritidis strains in the vaginal epithelium was compared with that of several strains of S. Agona, S. Infantis, S. Hadar, S. Heidelberg, S. Montevideo and S. Typhimurium, by immunohistochemical methods. The mean number of Salmonella in the vaginal epithelium depended on their lipopolysaccharide (LPS) type, with the rank order as follows: LPS type O9 (S. Enteritidis) > LPS type O4 (S. Agona, S. Typhimurium and S. Heidelberg) > LPS type O7 (S. Montevideo and S. Infantis) and LPS type O8 (S. Hadar). This rank order of Salmonella invasiveness is in accordance with the frequency of Salmonella outbreaks involving contaminated eggs. These findings suggest that S. Enteritidis has a higher ability to colonize the vaginal epithelium than other serovars, and the Salmonella LPS type may play an essential role in tropism of the reproductive tract.

Specific antibody levels of laying hens and young chickens experimentally infected with Salmonella Enteritidis and vaccinated farm flocks were evaluated by enzyme-linked immunosorbent assays (ELISAs) with two different antigens, deflagellated S. Enteritidis whole cell (DEWC) and S. Enteritidis FliC-specific 9kDa polypeptide (SEP9). Infected laying hens excreted S. Enteritidis throughout the experimental period, and the specific antibody titers in DEWC-ELISA, were significantly higher than the uninfected group. It suggests that this DEWC-specific antibody will serve as an effective indicator of S. Enteritidis infection, especially for non-vaccinated laying flocks. SEP9-specific antibodies were detected in spray-inoculated young chickens but not in oral-inoculated young chickens. Compared with greatly high SEP9-specific antibody levels of vaccinated farm flocks, no response was observed in orally infected hens. These results indicate that S. Enteritidis discontinues expressing SEP9 once it has crossed the intestinal barrier, and that SEP9-ELISA will serve as a valuable monitoring tool for the status of S. Enteritidis vaccination on a flockwide basis, independent of stable S. Enteritidis infections.

Salmonella enterica serovar Enteritidis (SE) is a causative agent for human food poisoning cases throughout the world. The ovaries and the oviducts of the laying hens are the major sites of SE colonization from which vertical transmission to eggs occurs. In this study, Salmonella-induced changes in T lymphocytes, B lymphocytes and macrophages in the ovaries and oviducts were assessed after primary and secondary experimental inoculations of laying hen with SE. Statistically significant increases in the numbers of T cells (both CD4+ and CD8+) and macrophages were observed 7 to 14 days after primary inoculation, followed by a peak in B-cell numbers from the 14th day post-primary inoculation onwards in the secretory areas of the oviducts. The peak in lymphocyte numbers immediately preceded a decline in the rate of SE recovery from the reproductive tract beginning at day 14. The correlation of decreased Salmonella recovery with elevated lymphocyte and macrophage numbers strongly suggests that local cell-mediated immunity is involved in controlling SE injection in the ovaries and oviducts.

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.

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.

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.

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.

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.

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.

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.

Plasma chemistry and haematological studies were conducted on chickens with coccidiosis. Male White Leghorn chickens, of two weeks old, were inoculated with 5 x 10(4) Eimeria tenella sporulated oocysts or with 1 x 10(6) E acervulina sporulated oocysts. Blood samples were taken four, seven and 11 days after inoculation. A wet chemistry system was applied to measure the plasma activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase, creatine kinase, amylase and lactate dehydrogenase and the concentrations of creatine, total bilirubin, urate, total cholesterol, total protein, albumin, glucose and triglycerides. A dry chemistry system was applied to measure sodium, potassium, chloride and calcium. The number of red blood cells and packed cell volume were determined by a micro cell counter and blood pH was measured with a blood gas analyser. The erythrocyte count, packed cell volume, sodium and chloride levels in the chickens infected with E tenella were significantly (P < 0.05) lower than those of the uninfected controls. The significant decrease in blood pH of the chickens infected with E acervulina suggests malabsorption associated with duodenal lesions induced by the infection.

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.

Four experiments were conducted to examine the effect of Eimeria tenella infection on the recrudescence of Salmonella enteritidis in previously infected chickens. Significant recrudescence of cecal colonization in the birds challenged with E. tenella was observed at 3 wk after S. enteritidis infection in Experiment 1, at 3 and 4 wk in Experiment 2, and at 3, 4, and 5 wk in Experiments 3 and 4. The recrudescence was indicated by .81 to 6.31 logs increase in cecal S. enteritidis counts and by higher percentages of ceca that were culture-positive. The possible prolonged cecal S. enteritidis shedding from chickens infected with coccidia into the environment might be important for the perpetuation of S. enteritidis infectious cycle. Except for Experiment 1, in which a significant higher culture-positive rate of the liver was detected in the coccidia-infected group, no significant difference of culture-positive rate of liver and spleen between the treatments was observed. The recrudescence of previous S. enteritidis infection caused by E. tenella infection was obviously related to the initial S. enteritidis dose size and time of exposure to coccidia.

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.

To study increase of the Salmonella population in the cecum of chickens infected with Eimeria tenella, quantitative changes in mannose residues on the cecal mucosa were investigated. Inhibition of S typhimurium adherence to the cecum by a 2% carbohydrate (D-mannose, D-galactose, L-fucose, alpha-methyl-D-glucoside) in phosphate-buffered saline solution was examined. Only D-mannose had inhibitory effects. Whereas, D-galactose had somewhat enhancing effects on adherence of S typhimurium to the cecal mucosa of uninfected germ-free chickens. In infected and uninfected chickens, D-mannose inhibited adherence of S typhimurium. D-Mannose significantly (P < 0.05) increased adherence of Bacteroides sp. In infected and uninfected chickens, D-mannose did not have any effect on adherence of Clostridium perfringens and Bifidobacterium thermophilum. Under microscopic observation, only concanavalin A and Lens culinaris agglutinin, of 8 lectins examined, were recognized as lectin-positive staining lines or spots in the cecal mucosa, indicating presence of mannose residues on the cecal mucosa. In E tenella-infected chickens, lectin-positive staining was seen strongly on the coarse surface of damaged cells and at the bottom of the crypts. These results indicate that coccidial infection may induce increase of mannose residues on the intestinal surface and allow adhesion of more salmonellae to the intestine.

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.