Increased lymphocyte subpopulations and macrophages in the ovaries and oviducts of laying hens infected with Salmonella enterica serovar Enteritidis.

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.

[Castleman lymphoma originated from the posterior mediastinum resected thoracoscopically: report of a case].

We report thoracoscopic resection of Castleman lymphoma originated from the posterior mediastinum. The patient was a 19-year-old woman, who was pointed out to show an abnormal shadow in the left upper lung in the chest X-ray photograph. It was diagnosed as a blood-rich posterior mediastinal tumor by dynamic MRI, and thoracoscopic surgery was performed. The tumor was adjacent to the vertebral body of the fourth thoracic vertebra. Although operation involved 2 hours and 40 minutes and 670 ml of bleeding due to a strong adhesion between the tumor and the intercostal muscle and considerable bleeding from the tumor itself, it was successfully removed under thoracoscopy. The tumor was elastic and hard, and 50 x 45 x 25 mm in size. The histopathological diagnosis was a Castleman lymphoma, hyaline vascular type. The postoperative course was satisfactory, and the patient was discharged from the hospital on the 7th postoperative day. Castleman lymphoma originated from the posterior mediastinum tends to bleed considerably during its resection. If a blood-rich posterior mediastinal tumor was found preoperatively, thoracoscopic operation must be advanced carefully keeping this disease in mind.

[Cases of death after surgery for pathologic stage I non-small cell lung cancer].

We clinically examined cases of death from pathologic stage I non-small cell lung cancer with the aim of improving the 5-year survival rate after surgery for this condition. The subjects were 70 patients with p-stage IA (20 cases of death) and 59 patients with p-stage IB (26 cases of death) from among those who underwent surgery for p-stage I non-small cell lung cancer between 1986 and 2000. 1) Of 30 patients who died from p-stage I lung cancer, 20 had distant metastases and 10 had recurrence in the thoracic cavity. Of 16 patients who died from other diseases, 5 had respiratory organ disease, 5 had cancers of other organs and 6 had circulatory organ disease. 2) Of 30 patients who died from p-stage I lung cancer, 20 (66.7%) had distant metastases, with lung metastasis occurring most frequently, in 10 of them (33.3%). The most common cause of death of patients with p-stage IB lung cancer was recurrence in the thoracic cavity. 3) The mean durations of survival (mean +/- standard deviation) after surgery for lung cancer of the patients who died from p-stage I lung cancer (30 patients) were 36.3 +/- 22.2 months for the 20 patients with distant metastases and 26.2 +/- 14.3 months for the 10 patients with recurrence in the thoracic cavity, the difference between groups was 10 months, but was not significant. 4) The 5-year survival rate in 45 patients who underwent p-stage IA mediastinal lymph node dissection was 83.1% whereas that in 25 patients without p-stage IB mediastinal lymph node dissection was 50.9% showing a significant difference of 32.2% (p < 0.01). 5) The patients in p-stage IA who died from other diseases were all men (10 patients). The mean durations of survival after surgery for lung cancer in the patients who died from other diseases were 35.2 +/- 19.0 months in the patients with respiratory organ disease, 37.0 +/- 23.9 months in those with cancers of other organs and 60 +/- 19.1 months in those with circulatory organ disease. 6) The 5-year survival rate after surgery in all cases of death was 76% in the patients in p-stage IA and 61.4% in those in p-stage IB. The 5-year survival rates in the patients excluding those who died from other diseases were 85% in the patients in p-stage IA (60 patients) and 60.3% in those in p-stage IB (53 patients) (p < 0.01). 7) To improve the 5-year survival rate in the patients with p-stage IA lung cancer, it is necessary to prevent death from other diseases in men. It is still possible to improve the 5-year survival rate in the patients with p-stage IB lung cancer by raising the accuracy of mediastinal lymph node dissection during surgery.

[A case with lung metastasis of osteosarcoma occurred 21 years after the operation of osteosarcoma on left tibia].

The patient was a 38-years-old woman. A chest X-ray film demonstrated the presence of an abnormal lesion. Her past history included osteosarcoma on the left tibia for which she received amputation of the left inferior limb at 17 years of age without any relapse thereafter. Considering that the patient might have lung metastasis of osteosarcoma on the basis of lung biopsy performed under CT guide, and then a tumor was removed under the thoracoscope. The tumor, 2.8 x 2.2 x 2.1 cm in size, was located right under the pleura at left S10 with its inside being filled up with fragile necrotic tissues. When compared pathohistologically with the primary lesion of osteosarcoma which had occurred 21 years before, the lung tumor was almost identical in terms of the tumor cell morphology but had a higher cell density without evidence of osteoid formation. The diagnosis of lung metastasis of osteosarcoma was established on the basis of the clinical course and the immunohistochemical staining. It is extremely rare case that osteosarcoma recurs in the form of lung metastasis 21 years after the operation of primary lesion. We report this case as a valuable one to identify the prognosis of osteosarcoma and the development mechanism of lung metastasis.

[Lobectomy of two or more lobes in patients with lung cancer].

We clinically examined patients who had undergone resection of two or more lobes for lung cancer. The subjects were 50 patients (25 who underwent pneumonectomy and 25 bilobectomy) who underwent lobectomy of two or more lobes from among those with primary non-small cell lung cancers in our hospital between 1975 and 1999; these individuals were assigned to Group A, and compared with 166 patients with lobectomy in Group B. The five-year survival rate was 27.7% in Group A, which differed significantly from the rate of 55.6% in Group B (p < 0.01, Kaplan-Meier method with log-rank test). The percentage of Stage I patients was 34% (17 patients) in Group A and 60.2% (100 patients) in Group B: this difference was significant (chi 2 test, p < 0.01). There were more patients with advanced cancer in Group A than in Group B. However, the five-year survival rates of Stage I patients were 52.4% in Group A and 77.6% in Group B, and significantly different (p < 0.05). In a comparison with respect to histological type, the five-year survival rates also differed significantly between Group A and B (p < 0.01 for adenocarcinoma, p < 0.05 for squamous cell carcinoma, with higher values in Group B for both). Resection of two or more lobes was indicated based on infiltration of the main tumor into adjacent lobes in 19 patients (38%), infiltration of lymph node metastasis into a bronchus or pulmonary artery in 14 (28%), direct infiltration of the main tumor into a bronchus in 10 (20%), and for other reasons in 7 (14%). The five-year survival rates for these groups were 15.8, 22.1, 54 and 42.9%, respectively. There was a significant difference between the patients with infiltration of cancer into adjacent lobes and those with direct infiltration into a bronchus (p < 0.05). The prognosis of patients with resection of two or more lobes was poorer than that of patients with lobectomy even in Stage I. In particular, infiltration of cancer into adjacent lobes accompanied lymph node metastasis in more than 50% in cases, and appeared to suggest a poor prognosis.

A case of primary squamous cell carcinoma of the breast with rapid progression.

Primary squamous cell carcinoma of the breast is a rare disease for which no definite treatment or prognosis has been established. This report describes our experience with one case of primary squamous cell carcinoma of the breast that ended in death after rapid progression. The patient was a 58 year old woman who visited our hospital complaining of a growing tumor and pain in the right breast. Ultrasonographically the tumor consisted of solid tumor and a large cyst. Squamous cell carcinoma was diagnosed by fine-needle aspiration cytology. Neoadjuvant CEF was given, but the tumor continued to grow, so CEF was discontinued after one course and modified radical mastectomy was performed subsequently. There was no evidence of metastasis to lymph nodes. About 5 months post operatively CT of the head and chest X-ray demonstrated metastatic lesions in the brain and lungs. Resection of the cerebral tumor was performed. About 1 month after the operation, however, a new metastatic brain tumor was found and the patient later died.

[A case of G-CSF-producing large cell carcinoma of the lung with gastric metastasis].

We report a rare surgically-treated case of G-CSF-producing large cell carcinoma of the lung with gastric metastasis. A 65-year-old male was admitted to our hospital because of fever, anemia and epigastralgia. Chest X-ray examination and CT scanning revealed a round mass shadow (8 cm) in contact with the chest wall in the right upper lung field and metastasis to the mediastinal lymph nodes. Laboratory examination showed a WBC of 16,800/mm3, CRP of 11.6 mg/dl, and a serum G-CSF of 90 pg/ml. Upper gastrointestinal series and gastroscopy showed an ulcerating submucosal tumorous lesion in the pyloric antrum. The lung carcinoma was treated by right upper lobectomy with chest wall resection. After 1 month, gastrectomy was performed. After the operation, the WBC normalized, and the CRP and serum G-CSF levels decreased. Histopathological examination demonstrated a poorly differentiated large cell carcinoma in the lung and a metastatic lesion in the stomach. Immunohistochemical staining with anti-G-CSF mono-clonal antibody showed negative results in the lung but positive results in the stomach. He was discharged 3 weeks after gastrectomy but died of aggravation of the general condition associated with local recurrence in the chest wall 2 months after discharge.

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.

'Babesia gibsoni' of dogs from North America and Asia belong to different species.

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0.5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related species.

Echocardiographic diagnosis of dirofilariasis in a ferret.

Heartworm disease is described in a 14-month-old neutered female ferret (Mustela putorius furo) which had a three-week history of sneezing, anorexia and dyspnoea. Echocardiography revealed the presence of heartworms as hyperechoic densities within the right atrial and ventricular cavities. At necropsy, four Dirofilaria immitis parasites (three females, one male) were found in the right heart, the cranial vena cava and the caudal vena cava. Histopathological findings were similar to those reported in canine heartworm disease. Echocardiography may be a useful method of diagnosis of heartworm disease in the ferret.

Lactobacillus flora in the cloaca and vagina of hens and its inhibitory activity against Salmonella enteritidis in vitro.

Lactobacilli in the cloaca and vagina of 40 normal laying hens were investigated quantitatively and qualitatively, and their ability to inhibit growth of Salmonella enteritidis (SE) was examined using a spot-the-lawn technique. All samples of cloacal contents and half the samples of vaginal mucus were positive for lactobacilli. The means +/- SD of total Lactobacillus counts in the cloaca and those in the vagina were log10 5.5 +/- 1.1 and 2.5 +/- 2.6 cfu/g, respectively. In the cloaca, Lactobacillus acidophilus was isolated from 92.5% of hens, and Lactobacillus salivarius was isolated from 85.0% of hens, whereas Lactobacillus fermentum was isolated from only one hen. In the vagina, L. acidophilus and L. salivarius were isolated from 42.5% of hens. In the inhibition assay in vitro, all strains of Lactobacillus from cloacal contents and vaginal mucus inhibited growth of SE. There was a wide range of the inhibitory activity even in the same species. No difference of the growth inhibition zone was observed between lactobacilli from cloaca and those from vagina. The present study suggested that lactobacilli in the cloaca and vagina of hens might have a protective effect against SE colonization.

Evaluation of the efficacy of Salmonella enteritidis oil-emulsion bacterin in an intravaginal challenge model in hens.

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.

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.

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.

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.

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.

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.

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.