Makoto Haritani

Epizootic outbreaks of gizzard erosion associated with adenovirus infection in chickens.

Two outbreaks of gizzard erosion in slaughtered broiler chickens in Japan were examined pathologically and microbiologically. The prevalences of such lesions were 9%-11% and 4%-50% in the affected flocks. Affected chickens had no clinical signs. Group I fowl adenovirus (FAV) serotype 1 was isolated from gizzard lesions. Histologically, gizzard mucosa were necrotic. Intranuclear inclusion bodies were seen in the enlarged nuclei of degenerating epithelial cells of the gizzard. The keratinoid layer in the erosion was edematous and desquamated and contained degenerative cells. Moderate to marked inflammatory cell infiltration was observed in the lamina propria and perivascular connective tissue in the submucosa and muscle layer. Immunohistochemical staining showed evidence of FAV antigens in the intranuclear inclusion bodies within degenerating epithelial cells. Ultrastructurally, numerous viral particles were demonstrated in the inclusions.

An Improved Isolation Technique for Bovine Neospora Species

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Characterization of antibodies raised against bovine-PrP-peptides

To analyze the antigenicity of peptides derived from bovine prion protein (PrP) cDNA, we immunized rabbits with four synthetic peptides and compared the immunoreactivity of antibodies to PrPs from various species by immunoblotting and immunohistochemistry. Two of the antibodies reacted strongly with all PrPs. The other antibodies, raised against overlapping peptides close to two glycosylation sites, did not recognize PrPSc-mouse but did recognize PrPSc-sheep which contains two sugar residues and PrPCJD with or without a sugar residue. Our results suggest that these antibodies may have species-specificity for both glycosylation status and amino acid sequences of the protein. In conclusion, we identified two regions in bovine-PrP which appear suitable for raising antibodies that detect various kinds of PrPs, and one region (Ab103-121) which appears suitable for raising antibodies that detect several species of PrPs. These antibodies may be useful for diagnosing prion diseases and for researching their pathogenesis.

An epizootic of subcutaneous tumors associated with subgroup A avian leukosis/sarcoma virus in young layer chickens.

An outbreak of subcutaneous tumors in young layer chickens in a flock in Japan was investigated. Tumors appeared as extensive swelling or bulbous protrusions of the integument and were observed in the head or wing of chickens approximately 9 wk old, with a prevalence of 0.4% (157 of 42,000) in the affected flock. Histologically, two types of tumor were observed: myxoma containing abundant hyaluronic acid and neurofibroma with hyperplasia of the Herbst corpuscles. Ultrastructurally, type C retroviruses, such as viral particles, were found in the tumors. The tumors were specifically stained by immunohistochemistry using monoclonal antibodies against the subgroup A avian leukosis/sarcoma virus (ALSV) and yielded a positive reaction to primers specific for subgroup A ALSV by reverse transcriptase-polymerase chain reaction assay. The virus was isolated from the tumors. Seventeen of 20 clinically normal chickens in the affected flock showed antibodies against ALSV. These results suggest that subcutaneous tumors are associated with subgroup A ALSV infection.

Two Different Scrapie Prions Isolated in Japanese Sheep Flocks

Two different scrapie prion strains with different characteristics were obtained from two sheep naturally infected with scrapie in Japan. In mice transmission, one (Tsukuba‐1) showed shorter incubation periods (133±2 days) than the other (Tsukuba‐2) (288±5 days). Spongiform changes and accumulation of an abnormal isoform of prion protein (PrPSc) were observed throughout the brain in Tsukuba‐1 inoculated mice, while the lesions and the PrPSc accumulation were localized in the brain stem of mice with Tsukuba‐2. Western blot analysis showed that there was no strain‐specific glycoform of PrPSc within these two strains. A super‐infection experiment revealed that neither strain interfered with the others propagation.

Ultrastructural characteristics and molecular identification of Entamoeba suis isolated from pigs with hemorrhagic colitis: implications for pathogenicity

Protozoan parasites of the genus Entamoeba infect many classes of vertebrates and are primarily classified based on morphological criteria. To date, only a few species have been proven to cause disease. Here, we examined the pathology of infected pigs with hemorrhage and detected Entamoeba parasites. Isolates were characterized genetically and ultrastructurally to identify the species. Histopathologically, bleeding and thrombus formation were seen only in the large intestine mucosa, where a large number of trophozoites or some Entamoeba cysts were observed around breakdowns in the lamina propria. No screw-shaped bacteria were detected in the lesions, and no pathogenic bacteria such as Brachyspira spp. were detected in fecal cultures. Interestingly, electron microscopy revealed that the parasites possessed mitochondrial organelles, unlike other Entamoeba spp. The isolates were identified as Entamoeba suis by PCR analysis and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. In phylogenetic analyses based on the actin gene, the E. suis isolate formed a cluster with Entamoeba histolytica and Entamoeba invadens, as well as with other parasites of the Amoebidae. Whether the pathogenicity of the E. suis isolate is affected by the severity of infection or host health status remains unclear; however, our results suggest that E. suis could cause or exacerbate clinical symptoms such as hemorrhagic colitis or diarrhea.

Genetic identification of Entamoeba polecki subtype 3 from pigs in Japan and characterisation of its pathogenic role in ulcerative colitis

To date, three Entamoeba spp. (E. suis, zoonotic E. polecki and E. histolytica) have been identified in pigs, but their pathogenicity and molecular classification have not been fully determined. Examination and pathological analysis of pigs (n=3) with diarrhoea was conducted and revealed the presence of Entamoeba organisms. We performed a genetic analysis of the isolate using the small-subunit ribosomal RNA (SSU rRNA) gene region to identify the species. A severe ulcerative colitis was observed histopathologically with inflammatory cells, including macrophages and neutrophils, infiltrating the mucous membranes of the cecum and colon. Many Entamoeba trophozoites were found at the erosion site or at ulcerative lesions. Pathogenic viruses or bacteria were not detected. The SSU rRNA sequence of the Entamoeba isolate was found to be completely homologous to that of E. polecki subtype 3.

In Situ Detection of Cellular and Abnormal Isoforms of Prion Protein in Brains of Cattle with Bovine Spongiform Encephalopathy and Sheep with Scrapie by Use of a Histoblot Technique

To detect prion protein, brains from 5 cattle naturally affected with bovine spongiform encephalopathy (BSE) and 3 sheep naturally affected with scrapie were examined and compared with brains of normal cattle and sheep using a histoblot technique. The technique enabled the in situ distinctive detection of the cellular (PrPC) and abnormal (PrPSc) isoforms of the prion protein. In BSE- or scrapie-affected brains, the PrPC signal decreased, especially in those areas where the PrPSc signal was detected.

Effects of Antigen-Retrieval Pretreatments for Immunohistochemical Detection of Akabane Viral Antigen

Effects of various antigen-retrieval pretreatment techniques for immunohistochemical detection of Akabane virus antigen were examined with formalin-fixed, paraffin-embedded tissues taken from mice intracerebrally inoculated with the virus. The results indicated that no pretreatment is necessary for the detection of the antigen, and excessive enzymatic pretreatment of sections may lead to false-negative results.

First report of mixed Entamoeba polecki (ST 1) and E. suis infection in piglets shedding abnormalfeces by histopathological and molecular surveys

Of the three species of Entamoeba found in swine, namely E. histolytica, E. polecki and E. suis, E. histolytica can also infect humans, producing colitis or abscesses of liver and leading to death. However, the pathogenicity of other species of Entamoeba has not been fully characterized. Here, we conducted histopathology and molecular surveys on a pig farm where piglets had blackish feces or muddy diarrhea. Histopathological examination of two piglets showed necrosis of the mucous surface at the ileum, cecum, or colon, infiltration of neutrophils, and formation of ulcers. Based on morphological characteristics, E. polecki and E. suis trophozoites were mainly detected at lamina propria and surface of the lesion, respectively, and Lawsonia intracellularis, a bacterial pathogen, was also detected. Molecular analysis using the small subunit ribosomal RNA gene on other piglets and a sow revealed infection with both E. polecki and E. suis. These findings corroborate our previous reports that the two Entamoeba spp. are pathogenic in pigs as aggravations of symptoms with L. intracellularis. This is the first report about mixed infection with E. polecki and E. suis.