Intracellular Localization of Mycoplasma bovis in the Bronchiolar Epithelium of Experimentally Infected Calves.

Lung tissues from calves infected experimentally with Mycoplasma bovis were examined by immunohistochemistry and electron microscopy. All inoculated calves had dark red areas of consolidation affecting both left and right lungs, which were characterized microscopically by subacute purulent bronchiolitis with hyperplasia of the surrounding lymphoid tissue. Immunohistochemically, M. bovis antigen was detected on the surface and inside the cytoplasm of bronchiolar epithelial cells in the pneumonic foci. The antigen was also found in the cytoplasm of phagocytes at the margin of bronchiolar exudates. Electron microscopically, numerous organisms were demonstrated in the immunohistochemically-positive sites. These findings suggest that M. bovis organisms adhere to the bronchiolar epithelium and at least some of them invade the epithelium.

Molecular cloning, characterization of porcine IZUMO1, an IgSF family member.

IZUMO1, belonging to the family of mammalian immunoglobulin proteins, has been well characterized in the mouse. Here, we describe the molecular cloning and expression analysis of porcine IZUMO1 (pIZUMO1). Partial sequence information published in the National Center for Biotechnology Information (NCBI) database was used to generate the full-length sequence for IZUMO1 using rapid amplification of cDNA ends (RACE). A search of the porcine genomic sequence in the NCBI database identified a bacterial artificial chromosome (BAC) encoding the pIZUMO1 gene. This BAC is derived from porcine chromosome 6 and is syntenic with the corresponding regions of mouse, bovine, and human genomes encoding the IZUMO gene family. This BAC was found to encode an IZUMO1 protein with a predicted amino acid sequence having high similarity with mouse and human IZUMO1. Western blot analysis of proteins from porcine tissues indicated that pIZUMO1 was specifically expressed in the sperm. Furthermore, to confirm whether pIZUMO1 forms complexes, we overexpressed pIZUMO1 in HEK293 cells. The recombinant pIZUMO1 from cell extracts was found to form complexes. Our finding suggests that pIZUMO1 forms homodimeric complex on the sperm membrane. Furthermore, an IVF inhibition assay with an antibody for the porcine IZUMO1 Ig-like domain showed that Ig-like domain effectively prevented pig sperm-egg interactions.

Unilateral ocular subalbinism in a laboratory Beagle dog.

Blue discoloration of the iris was found in the left eye of a male laboratory Beagle dog, which had a normal tricolor coat and clinically showed no visual impairment. Ophthalmoscopically, the affected eye revealed red-colored tigroid fundus, in which no tapetum was present. The retinal vasculature and the optic disc showed no noticeable changes. Histopathologically, in the left eye melanocytes had extremely few melanin granules in the anterior segment, including the anterior border layer, in the stroma and sphincter muscle of the iris and in the stroma of the ciliary body and choroid. However, the posterior pigment epithelium of the iris, the pigment epithelium of the ciliary body and the retinal pigment epithelium showed normal pigmentation. The tapetal elements were completely absent. Number and distribution of the S-100 protein-positive melanocytes with or without melanin granules in the iris, ciliary body and choroid of the left eye were similar to those of the normal right eye. Ultrastructurally, melanocytes in the anterior segment of the affected iris possessed no or few melanosomes which were incompletely melanized. In the right eye, no abnormal features were observed. Based on these results, the present case was diagnosed as unilateral ocular subalbinism with tapetal aplasia in a Beagle dog.

Lipoolygosaccharide indirectly enhances inflammatory lesions in lungs as a primary infection site by non-encapsulated and type B Haemophilus influenzae through production of cytokines.

We investigated the role of cytokines in differences in histopathologic changes in the lung between bronchopneumonia caused by non-encapsulated Haemophilus influenzae strain 770235f(0)b(0)and systemic disease caused by type b H. influenzae strain 770235f(0)b(+). Tumour necrosis factor-alpha (TNF-alpha), interleukin-(IL)-6 and IL-1 beta levels in bronchoalveolar lavage fluid (BALF) samples of mice infected with strain 770235f(0)b(0)were higher than in those infected with strain 770235f(0)b(+)until 24 h post-infection. Serum IL-6 rapidly increased in strain 770235f(0)b(0)infection after 72 h post-infection. Serum TNF-alpha level in strain 770235f(0)b(0)infection appeared earlier than in strain 770235f(0)b(+)infection. IL-1 beta production in strain 770235f(0)b(+)infection was later than in strain 770235f(0)b(0)infection. Moreover, a biphasic concentration pattern of TNF-alpha and IL-6 was noted in BALF of mice with strain 770235f(0)b(0)infection.

Anophthalmia and retinal degeneration associated with stenosis of the optic foramen in Fischer 344 rats.

Bilateral anophthalmia was discovered in a male rat (No. 1) and unilateral anophthalmia was found in the left eye of two female rats (Nos. 2 and 3) derived from a Fischer 344 inbred colony. One male rat (No. 4), a littermate of No. 3, had externally normal eyes, but his left eye had severe retinal atrophy, which was detected by ophthalmoscopy. The eyelids in both eyes of No. 1 and in the left eyes of Nos. 2 and 3 were present. Radiography of the skull revealed small optic foramina on both sides of No. 1 and on the affected side of Nos. 2, 3, and 4. Histologically, both globes and optic nerves (ONs) of No. 1 and the left globe and ONs of Nos. 2 and 3 were completely missing. Diffuse retinal degeneration in the left globe and axonal degeneration in the left ON and the right optic tract were observed in No. 4. Dysplasia of the sphenoid bone associated with stenosis of the optic foramen was detected on the affected side of all rats. Thus, ON aplasia in anophthalmic rats and atrophic ON in a rat with retinal degeneration seem to be closely related to stenosis of the optic foramen.

Spontaneous lymphoma in a Japanese White rabbit.

Lymphoma was observed in a 4-month-old female Japanese White rabbit. Grossly, the markedly enlarged mesenteric lymph nodes, prominent Peyer's patches of jejunum, splenomegaly, and enlargement of tracheobronchial lymph nodes, adrenal glands and ovaries were observed. Histologically, neoplastic lymphoid cells proliferated diffusely showing frequent mitotic figures and a characteristic 'starry sky' appearance. Their basophilic cytoplasm contained a few lipid droplets. The mesenteric lymph nodes, Peyer's patches of jejunum, and tracheobronchial lymph nodes were largely replaced by the tumor tissues. The stomach, small intestines, especially the jejunum, liver, spleen, ovaries, and adrenal glands were heavily infiltrated with neoplastic cells. These results suggest that the present lymphoma may have originated from the gastrointestinal lymphoid tissue.

Optic nerve dysplasia associated with meningeal defect in Sprague-Dawley rats.

Unilateral and bilateral dysplasias of the optic nerve (ON) were observed in 20/114 male and 14/110 female Sprague-Dawley rats at 12 weeks of age. Grossly, the intracranial segment of the affected ON had nodular thickening, bifurcation, and curvature. Nodular thickenings were seen in 20 males and 11 females. One female had a bifurcated ON. Curvature was observed in the left ONs of two females. Of 34 ON dysplasias, 12 ONs tapered off into a thin filament at the portion anterior to the dysplastic lesions. The intraorbital segments of the ONs in 33 rats were also reduced in size and were hardly recognizable in the meningeal sheath in 10 rats. Both eyeballs appeared normal in all the animals examined. Histologically, nerve fibers in intracranial and intraorbital segments of the ONs that appeared as slender filaments were markedly reduced in number. Nerve fibers in nodular thickenings were intertwined in haphazard fashion, forming scrollworklike structures. The meningeal sheaths in intracranial segments of the ONs in 15 rats and in intraorbital segments in eight rats were partially missing. The naked portion of the ON protruded into the meningeal spaces or gaps. The data indicate that developmental failures in the ON may have been induced due to insufficient blood supply through the meningeal covering or herniation of growing nerve fibers into the defective meninges. However, etiology and pathogenesis of this condition remain unclear.

Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry.

Enzymatic and non-enzymatic pretreatments for antigen retrieval (AR) on formalin-fixed, paraffin-embedded, deparaffinised sections were compared and optimised by immunostaining of tissues infected with 13 different pathogens. Enzymatic pretreatment was achieved by incubating slides in 0.25 per cent trypsin (TR) and non-enzymatic pretreatments by heating them in a microwave oven (MWO) or an autoclave (AC). Three different buffers of different pH and molarity including phosphate buffer (PB), citrate buffer (CB) and acetate buffer (AB) were used as AR solutions. All of them were effective regardless of molarity, for the MWO with a processing time of 20 minutes, and the AC with a processing time of 15 minutes. The detectability of many viral antigens was significantly improved by MWO or TR pretreatment, but that of most bacterial antigens was not improved and that of some viral antigens was decreased by TR pretreatment. These results suggest that AR pretreatment is a prerequisite procedure for immunohistochemical detection of many infectious agents but the optimal pretreatment has to be individualised for each agent.

Carbon tetrachloride-induced hepatotoxicity enhances the development of pulmonary foam cells in rats fed a cholesterol-cholic acid diet.

The effect of CCl4-hepatotoxicity on the development of pulmonary foam cells (PFCs) was studied using 4 groups of rats. Rats in the control group were fed a standard diet; rats in the second group were fed a hyper beta-lipoproteinemic (HB) diet consisting of the standard diet, 4% cholesterol, and 1% cholic acid; rats in the third group were fed the standard diet and administered with CCl4 (1.0 ml/kg, i.p., fortnightly) (CT); and rats in the fourth group were fed the HB diet and administered with CCl4 (HT). At feeding week 10, rats in the HB and HT groups developed hyper beta-lipoproteinemia. The ratios of foamy, lipid-ingested monocytes (FMs) to blood monocytes (BMs) and the histologic scores of PFC development were significantly greater in the HB and HT groups than in the control and CT groups, respectively. The ratio of PFCs to alveolar macrophages from bronchopulmonary lavage fluid was significantly higher in the HT group than in the CT group. At feeding week 20, rats in the CT and HT groups suffered hepatic injury and hypo beta-lipoproteinemia. Despite hypo beta-lipoproteinemia, the FM and PFC development, the ratio of BMs in differential leukocyte counts, and latex-phagocytotic activity of BMs were enhanced in the HT group. Furthermore, markedly enlarged FMs, described here as giant-FMs, that contained numerous lipid droplets in their abundant cytoplasm appeared in the peripheral blood from rats in the HT group. Histologically, embolism caused by giant-FMs in the pulmonary blood vessels and intraalveolar accumulation of PFCs were detected with a high incidence in the HT group. The present study suggests that CCl4-hepatotoxicity may affect lipoprotein synthesis in the liver of hyper beta-lipoproteinemic rats and defective or modified lipoproteins can be phagocytosed actively by BMs, and then BMs transform into FMs or giant-FMs, resulting in the PFC development and pulmonary embolism by giant-FMs.

Natural case of salpingitis apparently caused by Mycoplasma gallisepticum in chickens.

A natural case of salpingitis, apparently caused by Mycoplasma gallisepticum (MG), in layer chickens is described. In the flock from which the chickens originated, there was a 3 to 4% drop in egg production per month around 250 days old. The production was reduced 70% at 400 days of age, which was 77% of the predisease level. Salpingitis was characterized by marked thickening of the oviductal mucosa due to epithelial hyperplasia and marked lymphoplasmacytic infiltration. Colonization of MG on the epithelial surface was evidenced by electron microscopy and immunohistochemistry using rabbit hyperimmune serum against the S6 strain of MG. Antibodies to MG were detected in all the chickens examined..

Scierai ectasia associated with hereditary retinal dysplasia in a mutant strain of Japanese quail (Coturnix coturnix japonica).

Ocular defects and age-related lesions in mutant (GUB strain) Japanese quail (Coturnix coturnix japonica), phenotypically characterised by silver plumage, are described. Grossly, a circular area of hypopigmentation in the posterior retina with thinning of the subjacent sciera was observed in all GUB quails. As the birds matured, the thinned sciera progressed to scierai ectasia. Histologically, the sciera at the ectatic area consisted of an outer fibrous layer and was devoid of the inner cartilaginous shell. Atypical differentiation and duplication of the retina with absence of the choroid was common at the ectatic area. The retina, choroid, ciliary body and iris were all poorly pigmented. With increasing age, the ectatic area became cystic, and the duplicated retina degenerated and atrophied. In addition, there were mononuclear cell infiltration in the stroma of the iris and ciliary body, anterior and posterior synechiae, cataract and/or glaucoma in aged GUB quails. These findings suggest that posterior scierai ectasia in the GUB strain of Japanese quails may have developed secondarily to a congenital structural defect of the posterior portion of sciera associating with general ocular defects.

New murine model of bronchopneumonia due to cell-bound Haemophilus influenzae.

This murine model of nontypeable (unencapsulated) Haemophilus influenzae (NTHI) bronchopneumonia used organisms bound to mouse fetal lung (MFL) cells as an inoculum. Pretreatment of the mice with 40 microL of 1% formalin 3 days before intranasal instillation of the bacteria was necessary to allow infection. The number of NTHI recovered from the lungs plus trachea on day 7 after instillation was >100 times the number originally inoculated. Later, however, the number of recovered bacteria diminished gradually, and by day 14 it was almost identical to the original inoculum size. Serum IgM also peaked on day 7 after infection, after which IgG increased while IgM decreased. Histologically, bronchoalveolar infiltration of neutrophils was observed on day 3 after inoculation and continued at least for the following 4 days. The present experiment demonstrates that MFL cells can protect bacteria that have invaded the cells from the opsonizing and killing activities of host humoral defense mechanisms.

Establishment and characterization of cell lines, MeMS and MeFS, derived from spontaneous rat mesotheliomas.

New cell lines, designated MeMS and MeFS, were established from the epithelial type of mesotheliomas occurring spontaneously in aged male and female Fischer 344 rats, respectively. Morphologically, MeMS and MeFS expressed the epithelial phenotype. Coexpression of keratin and vimentin in both cell lines was confirmed by the immunocytochemistry, one-dimensional gel electrophoresis and immunoblotting for intermediate filament proteins. Hyaluronic acid synthesis of both MeMS and MeFS was demonstrated by the analysis using high-performance liquid chromatography. MeMS and MeFS seem to be useful models for studying the biological nature of rat mesotheliomas.

Histopathology and immunohistochemistry of renal lesions due to infectious bronchitis virus in chicks.

Two-day-old specific-pathogen-free chicks were inoculated intranasally with the MA-87 strain of infectious bronchitis virus, and trachea and kidney lesions studied histologically and immunohistochemically. Lesions and viral antigen were first detected in the trachea; severe damage was then observed in the kidney. Viral antigen appeared prior to the development of renal lesions and was detected in the cytoplasm of epithelial cells by 4 days post-inoculation (p.i.). The epithelial cells of the collecting ducts, collecting tubules and distal convoluted tubules were first affected, followed by involvement of Henle's loops, whereas the proximal convoluted tubules were only minimally affected. Antigen-positive cells of ducts and tubules were degenerated and desquamated. The severe epithelial cell damage resulted in infiltration of heterophils and macrophages in the interstitium, ducts and tubules. The detection of viral antigen was consistent with the distribution of histological lesions at 6 to 8 days p.i. At a later stage, antigen-positive cells disappeared and repair of epithelial cells was seen, accompanied by interstitial lymphoplasmacytic infiltration and lymphoid nodular formation.

Olfactory undifferentiated carcinoma with endocranial extension in a Fischer-344 rat.

A spontaneous tumor in the nasal cavity, extending through the cribriform plate to the cranial cavity, was detected in a 103-wk-old female Fischer-344/DuCrj rat. The tumor appeared to arise from the olfactory epithelium and was composed of round to polygonal, undifferentiated cells arranged in solid sheet-like and lobular patterns with scant fibrovascular stroma. Ultrastructurally, the tumor cells often formed desmosomes between contiguous cells and had no neurosecretory granules. Immunohistochemically, the tumor cells did not react with any antibodies specific for intermediate filaments, neuronal, muscular, and endothelial elements. Based on these results, the tumor was diagnosed as undifferentiated olfactory epithelial carcinoma.

Establishment and characterization of a cell line (NB-YK) derived from a transplantable rat nephroblastoma.

A new cell line, designated NB-YK, was established from a transplantable rat nephroblastoma (NB-Y) which was derived from a spontaneous nephroblastoma in an aged Fischer 344 rat. NB-YK grew in a piling-up and noncohesive pattern on the plastic surface and formed colonies in a soft agar. The main cell type of NB-YK represented morphology of mesenchymal phenotype and most of the cells contained several secretory granules in their cytoplasm. Immunocytochemically the cells were positive for vimentin, cytokeratin, and laminin. Coexpression of vimentin and cytokeratin in the cells was confirmed by the one-dimensional gel electrophoresis and immunoblotting for intermediate filament proteins. NB-YK cells were tumorigenic and produced fibrosarcoma-like tumor when inoculated subcutaneously or intraperitoneally into syngeneic rats and nude mice. NB-YK seems to be a useful model for studying biological properties of nephroblastoma.

Induction of protective immunity in chickens orally immunized with inactivated infectious bursal disease virus.

The present studies were undertaken to examine the effects of oral administration of formalin-inactivated infectious bursal disease virus (IBDV) on the immune responses of chickens. Inactivated IBDV was suspended in phosphate-buffered saline containing sodium bicarbonate. This form of antigen, when administered by oral instillation, induced a serum antibody response against IBDV in chickens and these chickens were protected from subsequent viral challenge. The immunoglobulin class of IBDV-specific antibodies in serum was found to be IgG when determined by enzyme-linked immunosorbent assay. Cholera toxin, which has been reported to have potent mucosal adjuvant properties in mammals, did not enhance the serum antibody response. Oral followed by parenteral administrations of antigen induced an enhanced antibody response in chickens.

Occurrence of keratoconjunctivitis apparently caused by Mycoplasma gallisepticum in layer chickens.

Natural cases of keratoconjunctivitis, apparently caused by Mycoplasma gallisepticum (MG), in layer chickens are described. The disease occurred in a commercial flock consisting of 36,000 pullets (Babcock), first appearing around 30 days of age. Clinically, affected chickens showed unilateral or bilateral swelling of the facial skin and the eyelids, increased lacrimation, congestion of conjunctival vessels, and respiratory rales. Some of the severely affected chickens closed their eyes. The morbidity reached 27.8%, and it was estimated that approximately 10% died from reduced feed intake due to impaired vision. Ten 70-day-old chickens with clinical diseases were examined for lesions. There was acute to subacute keratoconjunctivitis in all of the chickens, and some exhibited laryngitis. Adherence of mycoplasma organisms to epithelial cells of the conjunctiva, cornea, and larynx was frequently observed. These organisms had an ultrastructure characteristic of MG and showed a positive reaction with rabbit polyclonal antibodies against the S6 strain of MG by immunohistochemical analysis. MG was isolated from tissue homogenates of the eyelids and tracheas of the affected chickens. Many of the chickens had atrophic bursae, and infectious bursal disease virus antigens were detected in necrotic bursal follicles by immunostaining. Therefore, immunosuppression due to infectious bursal disease was implicated in the pathogenesis of keratoconjunctivitis in the present cases.