The distribution of E-cadherin expression in listeric rhombencephalitis of ruminants indicates its involvement in Listeria monocytogenes neuroinvasion.

AIM: To investigate the expression of E-cadherin, a major host cell receptor for Listeria monocytogenes (LM) internalin A, in the ruminant nervous system and its putative role in brainstem invasion and intracerebral spread of LM in the natural disease. METHODS: Immunohistochemistry and double immunofluorescence was performed on brains, cranial nerves and ganglia of ruminants with and without natural LM rhombencephalitis using antibodies against E-cadherin, protein gene product 9.5, myelin-associated glycoprotein and LM. RESULTS: In the ruminant brain, E-cadherin is expressed in choroid plexus epithelium, meningothelium and restricted neuropil areas of the medulla, but not in the endothelium. In cranial nerves and ganglia, E-cadherin is expressed in satellite cells and myelinating Schwann cells. Expression does not differ between ruminants with or without listeriosis and does not overlap with the presence of microabscesses in the medulla. LM is observed in phagocytes, axons, Schwann cells, satellite cells and ganglionic neurones. CONCLUSION: Our results support the view that the specific ligand-receptor interaction between LM and host E-cadherin is involved in the neuropathogenesis of ruminant listeriosis. They suggest that oral epithelium and Schwann cells expressing E-cadherin provide a port of entry for free bacteria offering a site of primary intracellular replication, from where the bacterium may invade the axonal compartment by cell-to-cell spread. As E-cadherin expression in the ruminant central nervous system is weak, only very locally restricted and not related to the presence of microabscesses, it is likely that further intracerebral spread is independent of E-cadherin and relies primarily on axonal spread.

Identification of cancer stem cells derived from a canine lung adenocarcinoma cell line.

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.

An African pygmy hedgehog adenovirus 1 (AhAdV-1) outbreak in an African pygmy hedgehog (Atelerix albiventris) colony in Japan.

An African pygmy hedgehog adenovirus 1 (AhAdV-1) outbreak in a colony of 24 African pygmy hedgehogs (APHs) with a case of fatal pneumonia occurred in Japan. Thirteen out of a colony of 15 APHs with respiratory symptoms were diagnosed with AhAdV-1 infection based on the detection of AhAdV-1 genome in throat/nasal swabs and further one APH was diagnosed on isolation of the virus. Five infected APHs died during the outbreak and AhAdV-1 caused severe pneumonia and death in one case. After the outbreak, persistent AhAdV-1 infection was suggested in one surviving APH. AhAdV-1 is a novel adenovirus and is suspected to be an emerging pathogen.

Ectopic (subcutaneous) Paragonimus miyazakii infection in a dog.

Ectopic infection with Paragonimus miyazakii was determined to be the cause of a subcutaneous inguinal mass in a 15-month-old, male, boar-hunting dog. On histologic examination, the mass comprised granulomatous panniculitis, intralesional adult trematodes and eggs, and lymphadenitis. Extrapulmonary paragonimosis in animals is rare. This appears to be the first report in a dog of ectopic P. miyazakii infection with mature trematodes and eggs that involved the inguinofemoral lymphocenter and surrounding subcutis.

Disseminated Histiocytic Sarcoma in an African Hedgehog (Atelerix albiventris).

Disseminated histiocytic sarcoma (HS) was diagnosed on post-mortem examination of a 1.5-year-old African hedgehog (Atelerix albiventris) that was presented in poor physical condition and with diarrhoea. Leucocytosis and a hypoechoic abdominal mass were noted on haematological and ultrasonographical examinations. Gross pathological, histopathological, immunohistochemical and ultrastructural evaluation of the mass supported a diagnosis of disseminated HS. To our knowledge, this report represents the first documentation of disseminated HS in this species.

Radioresistance of cancer stem-like cell derived from canine tumours.

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.

Positive immunolabelling for feline infectious peritonitis in an African lion (Panthera leo) with bilateral panuveitis.

A 15-year-old male African lion (Panthera leo) was presented with blindness due to bilateral panuveitis with retinal detachment. Feline coronavirus (FCoV) antigen was identified immunohistochemically in ocular macrophages, consistent with a diagnosis of feline infectious peritonitis (FIP) infection. This is the first report of FIP in an African lion and the first report of ocular FIP in a non-domestic felid.

Cytosolic phospholipase A2 in rat decidual cells: evidence for its role in decidualization.

We investigated the existence and possible role of cytosolic phospholipase A2 (cPLA2) in rat decidualized uteri. PLA2 activity in the cytosol of a decidualized uterine horn, induced by intraluminal oil infusion, was significantly higher than that in contralateral intact horn. The activity was almost completely depressed by cPLA2 inhibitors including arachidonyl trifluoromethyl ketone (ATK). The immunoreactive signals for cPLA2 were intense in decidua and glandular epithelial cells. In vivo administration of ATK (0.1-100 microg) caused a dose-dependent inhibition of decidualization. These results show the presence of cPLA2 and its probable implication in decidualization in rat uterus.

Effect of a matrix metalloproteinase inhibitor on host resistance against Listeria monocytogenes infection.

Hydroxy acid-based matrix metalloproteinase (MMP) inhibitors have been shown to inhibit tumor infiltration and growth, endotoxin shock, and acute graft-versus-host disease. Blockade of the release of soluble tumor necrosis factor-alpha (TNF-alpha) and CD95 ligand (CD95L; FasL) from cell-associated forms is reportedly involved in the mechanism of the drug effect. We investigated the effect of a MMP inhibitor, KB-R7785, on host resistance against Listeria monocytogenes infection, in which TNF-alpha is essentially required for the defense, in mice. The administration of KB-R7785 exacerbated listeriosis, while the drug prevented lethal shock induced by lipopolysaccharide and D-galactosamine. KB-R7785 inhibited soluble TNF-alpha production in spleen cell cultures stimulated by heat-killed L. monocytogenes and the drug treatment reduced serum TNF-alpha levels in infected mice, whereas the compound was ineffective on the modulation of interferon-gamma and interleukin-10 production. The effect of KB-R7785 was considered to be dependent on TNF-alpha because the drug failed to affect L. monocytogenes infection in anti-TNF-alpha monoclonal antibody-treated mice and TNF-alpha knockout mice. Anti-CD95L monoclonal antibody was also ineffective on the infection. These results suggest that induction of infectious diseases, to which TNF-alpha is critical in host resistance, should be considered in MMP inhibitor-treated hosts.

Virulent and avirulent Rhodococcus equi infection in T-cell deficient athymic nude mice: pathologic, bacteriologic and immunologic responses.

We investigated the pathologic, bacteriologic and immunologic responses of BALB/c-nu/nu mice (nude mice) and BALB/c mice (euthymic mice) infected intravenously with virulent and avirulent Rhodococcus equi ATCC 33701, and its plasmid-cured derivative ATCC 33701P-, to evaluate the role of T lymphocytes. Adaptive transfer of immune and normal spleen cells into nude mice was also investigated. Nude and euthymic mice were inoculated with 10(6) ATCC 33701 or 10(6) ATCC 33701P- intravenously (i.v.) and killed at 0, 7, 14, 21, 28 and 35 days post-inoculation, except dead cases. In athymic nude mice infected with ATCC 33701, deteriorating systemic inflammatory responses developed during the experimental period and multiplication of the bacteria continued until the end of the experiment. Nude mice developed splenomegaly and multifocal gross hepatic necrosis with some mortality. Splenomegaly was caused by diffuse proliferation of bacteria-laden macrophages and epithelioid cells, and gross hepatic necrosis was caused by the formation of thromboses and granulomatous lesions. Infection of euthymic mice with a sublethal dose of ATCC 33701 resulted in transient granuloma formation in the liver and spleen, production of specific antibodies against the virulent bacteria and gradual elimination thereof. In contrast, infection with ATCC 33701P- produced few lesions after rapid elimination and no antibody production against bacteria in either normal or athymic nude mice. In nude mice given normal and immune spleen cells, histopathological lesions and granulomas formed only in the liver and spleen, in addition to specific antibodies against 15- to 17-kDa antigens. The pathological lesions observed in the nude mice given immune spleen cells were similar to those seen in the mice given normal spleen cells, but they were less severe than those in mice given normal spleen cells. Mice given immune spleen cells showed a significantly higher elevation of antibody production than mice given normal spleen cells. These results suggested that protection against virulent R. equi in mice depends mainly on cell-mediated immune responses, whereas avirulent R. equi in mice are cleared by innate immune responses.

Pathogenesis of Rhodococcus equi infection in mice: roles of virulence plasmids and granulomagenic activity of bacteria.

Virulence of Rhocococcus equi ATCC 33701 and its plasmid-cured derivative ATCC 33701P- was compared in BALB/c and C3H/HeJ mice in terms of bacterial growth kinetics and histological changes in the liver, spleen and lungs, and humoral immune responses. Injection with a sublethal dose of 10(6) ATCC 33701 in mice resulted in microabscess formation after rapid multiplication in the liver and spleen by day 4, and then the bacteria were gradually eliminated with the formation of granuloma and the production of specific antibodies against 15- to 17-kDa antigens of the virulent bacteria. By contrast, ATCC 33701P- was avirulent as shown by early elimination of viable bacteria and no evidence of net multiplication in the organs. Histopathological changes consisted of only slight, transient infiltration of neutrophils and macrophages in the liver. Although live ATCC 33701P- did not evoke any humoral or histological responses in the mice, a large inoculum (10(8)) of killed ATCC 33701 and ATCC 33701P- resulted in the formation of granuloma in the liver and accelerated extramedullary hemopoiesis in the spleen. These results suggest that the pathogenesis of R. equi infection involves at least two important virulence determinants, both of which play critical roles in the disease: one is the virulence plasmid, which is required for R. equi to resist and grow within host cells; and the other is the granulomagenic activity that is related to the lipids and nature of the cell wall of the species, which induces the characteristic pathological changes.

Cutaneous malakoplakia in pigs inoculated with Rhodococcus equi.

Cutaneous malakoplakia was observed in pigs inoculated intramuscularly with Rhodococcus equi strains of intermediate virulence. Macroscopically, the inoculation sites showed the indurated swelling of the skin. Histopathologically, abscess formation with histiocytic granulomatous reaction was observed. Many macrophages contained target or owl-eye shaped hematoxyphil intracytoplasmic inclusions or calcosherites (Michaelis-Gutmann bodies) of various sizes. The Michaelis-Gutmann bodies were also seen outside of the macrophages. Histochemically, most Michaelis-Gutmann bodies stained positively with the von Kossa silver method and periodic acid Schiff. Immunohistochemically, some of Michaelis-Gutmann bodies were stained by two rabbit polyclonal antibodies (rabbit anti-A5 serum and rabbit anti-ATCC 33701 serum) and a mouse monoclonal antibody (anti-20-kDa antigen monoclonal antibody). This is the first report of cutaneous malakoplakia in domestic animals, which also revealed the relationship between R. equi infection and malakoplakia immunohistochemically. This experimental swine model is useful to investigate the morphogenesis of Michaelis-Gutmann bodies in malakoplakia through chronological skin biopsies.

The alkaline single cell electrophoresis assay with eight mouse organs: results with 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers.

The genotoxicity of 22 mono-functional alkylating agents (including 9 dialkyl N-nitrosoamines) and 10 DNA crosslinkers selected from IARC (International Agency for Research on Cancer) groups 1, 2A, and 2B was evaluated in eight mouse organs with the alkaline single cell gel electrophoresis (SCGE) (comet) assay. Groups of four mice were treated once intraperitoneally at the dose at which micronucleus tests had been conducted, and the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and/or 24 h later. All chemicals were positive in the SCGE assay in at least one organ. Of the 22 mono-functional alkylating agents, over 50% were positive in all organs except the brain and bone marrow. The two subsets of mono-functional alkylating agents differed in their bone marrow genotoxicity: only 1 of the 9 dialkyl N-nitrosoamines was positive in bone marrow as opposed to 8 of the 13 other alkylating agents, reflecting the fact that dialkyl N-nitrosoamines are poor micronucleus inducers in hematopoietic cells. The two groups of mono-functional alkylating agents also differ in hepatic carcinogenicity in spite of the fact that they are similar in hepatic genotoxicity. While dialkyl N-nitrosoamines produce tumors primarily in mouse liver, only one (styrene-7,8-oxide) out of 10 of the other type of mono-functional alkylating agents is a mouse hepatic carcinogen. Taking into consideration our previous results showing high concordance between hepatic genotoxicity and carcinogenicity for aromatic amines and azo compounds, a possible explanation for the discrepancy might be that chemicals that require metabolic activation show high concordance between genotoxicity and carcinogenicity in the liver. A high percent of the 10 DNA crosslinkers were positive in the SCGE assay in the gastrointestinal mucosa, but less than 50% were positive in the liver and lung. In this study, we allowed 10 min alkali-unwinding to obtain low and stable control values. Considering that DNA crosslinking lesions can be detected as lowering of not only positive but also negative control values, low control values by short alkali-treatment might make it difficult to detect DNA crosslinking lesions. In conclusion, although both mono-functional alkylating agents and DNA crosslinkers are genotoxic in mouse multiple organs, the genotoxicity of DNA crosslinkers can be detected in the gastrointestinal organs even though they were given intraperitoneally followed by the short alkali-treatment.

The comet assay in eight mouse organs: results with 24 azo compounds.

The genotoxicity of 24 azo compounds selected from IARC (International Agency for Research on Cancer) groups 2A, 2B, and 3 were determined by the comet (alkaline single cell gel electrophoresis, SCG) assay in eight mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8, and 24 h after treatment. For the 17 azo compounds, the assay was positive in at least one organ; (1) 14 and 12 azo compounds induced DNA damage in the colon and liver, respectively, (2) the genotoxic effect of most of them was greatest in the colon, and (3) there were high positive responses in the gastrointestinal organs, but those organs are not targets for carcinogenesis. One possible explanation for this discrepancy is that the assay detects DNA damage induced shortly after administration of a relatively high dose, while carcinogenicity is detected after long treatment with relatively low doses. The metabolic enzymes may become saturated following high doses and the rates and pathways of metabolic activation and detoxification may differ following high single doses vs. low long-term doses. Furthermore, considering that spontaneous colon tumors are very rare in rats and mice, the ability to detect tumorigenic effects in the colon of those animals might be lower than the ability to detect genotoxic events in the comet assay. The in vivo comet assay, which has advantage of reflecting test chemical absorption, distribution, and excretion as well as metabolism, should be effective for estimating the risk posed by azo dyes to humans in spite of the difference in dosage regimen.

The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP.

The genotoxicity of 30 aromatic amines selected from IARC (International Agency for Research on Cancer) groups 1, 2A, 2B and 3 and from the U.S. NTP (National Toxicology Program) carcinogenicity database were evaluated using the alkaline single cell gel electrophoresis (SCG) (Comet) assay in mouse organs. We treated groups of four mice once orally at the maximum tolerated dose (MTD) and sampled stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow 3, 8 and 24 h after treatment. For the 20 aromatic amines that are rodent carcinogens, the assay was positive in at least one organ, suggesting a high predictive ability for the assay. For most of the SCG-positive aromatic amines, the organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Organ-specific genotoxicity, therefore, is necessary but not sufficient for the prediction of organ-specific carcinogenicity. For the 10 non-carcinogenic aromatic amines (eight were Ames test-positive and two were Ames test-negative), the assay was negative in all organs studied. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative non-genotoxic (Ames test-negative) carcinogens. The alkaline SCG assay, which detects DNA lesions, is not suitable for identifying non-genotoxic carcinogens. The present SCG study revealed a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic non-carcinogens. These results suggest that the alkaline SCG assay can be usefully used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.

Endogenous tumor necrosis factor (TNF) production and modification of pathological lesions in experimental Escherichia coli endotoxemia of piglets.

We examined the kinetics of tumor necrosis factor (TNF) production induced by Escherichia coli lipopolysaccharide (LPS) in relation to LPS tolerance and endotoxemic lesions of piglets. The plasma of piglets demonstrated cytotoxicity to TNF-sensitive L929 cells between 0.5 and 4 h after inoculation with 200 micrograms kg-1 of LPS. This cytotoxicity was neutralized by anti-bovine TNF serum. These piglets had disseminated intravascular coagulation (DIC) and meningoencephalitis. However, if piglets were first treated with three doses of 40 micrograms kg-1 of LPS, both TNF production and the occurrence of DIC were inhibited when 200 micrograms kg-1 of LPS was inoculated into these piglets. Repetitive inoculation with increasing doses of LPS induced fibrinoid vasculitis, meningoencephalitis and pneumonitis, while hemorrhage was minimal. A very low amount of TNF activity was detected from most of the samples of a piglet after repeated LPS inoculation. These results suggested that severity of the hemorrhagic and thrombotic lesions might relate to the amount of endogenous TNF activity, and that LPS tolerance might relate to inhibition of TNF production.

Perocormus associated with segmental aplasia of the cervical spinal cord in a Japanese shorthorn calf.

A rare case of perocormus associated with segmental aplasia of the cervical spinal cord in a Japanese Shorthorn calf is reported. The severe reduction of body length with normal extremities combined to produce so the called "elk calf" appearance. Other associated malformations included hypoplasia of the lung, anorectal agenesis and a lobulated spleen.

Cerebellar hypoplasia associated with Arnold-Chiari malformation in a Japanese shorthorn calf.

A case of Arnold-Chiari malformation associated with severe cerebellar hypoplasia in a Japanese Shorthorn calf is reported. Histological examination revealed severe and extensive lesions in the cerebellar vermis. Hypoplasia of the cerebellum in Arnold-Chiari malformation appeared to indicate the abnormal embryonic development of the neural tissue, but the exact cause is not known.

Cuboidal epithelium lining of the parietal layer of Bowman's capsule in Afghan pikas (Ochotona rufescens rufescens).

Kidneys of 64 Afghan pikas (Ochotona rufescens rufescens) were examined histologically. Seven of 21 males and two of 21 females over 6 months of age had a cuboidal epithelium lining of the parietal layer of Bowman's capsule.

Pathology of excessive iron storage in the Afghan pika (Ochotona rufescens rufescens).

Iron deposition in the tissues of 30 Afghan pikas (Ochotona rufescens rufescens) was examined histopathologically. In all cases, iron deposits were present in the liver and in two-thirds of cases, there was portal fibrosis with tissue injury. In animals, in general, tissue injury induced by iron overload is usually mild and only in a few exceptional species does hepatic haemochromatosis occur. Thus, Afghan pikas are a rare example of reaction to iron overloading.