Proliferative changes in the adrenal medulla of aged Chinese native pigs.

Four aged retired Chinese native pigs, three females and one male, estimated as over 10-year-old, were subjected to autopsy because of infertility due to aging. Grossly, nodular lesions were found bilaterally in the adrenal medulla of all four pigs. Based on the gross and the histopathological findings, they were diagnosed as either medullary nodular hyperplasia or pheochromocytoma. Immunohistochemically, proliferating cells of all these lesions were immuno-positive for chromogranin-A, indicating adrenal medulla-derived. Ultrastructurally, cytoplasmic neurosecretory granules suggestive of secretion were observed in these proliferating cells. There have been only limited numbers of reports on adrenal medullar proliferative changes including pheochromocytoma in pigs. The present cases will provide a valuable information for the characterization of similar changes in animals and human.

Experimental induction of necrotic enteritis in chickens by a netB-positive Japanese isolate of Clostridium perfringens.

Necrotic enteritis (NE) is one of the most important bacterial diseases in terms of economic losses. Clostridium perfringens necrotic enteritis toxin B, NetB, was recently proposed as a new key virulent factor for the development of NE. The goal of this work was to develop a necrotic enteritis model in chickens by using a Japanese isolate of C. perfringens. The Japanese isolate has been found to contain netB gene, which had the same nucleotide and deduced amino acid sequences as those of prototype gene characterized in Australian strain EHE-NE18, and also expressed in vitro a 33-kDa protein identified as NetB toxin by nano-scale liquid chromatographic tandem mass spectrometry. In the challenge experiment, broiler chickens fed a commercial chicken starter diet for 14 days post-hatch were changed to a high protein feed mixed 50:50 with fishmeal for 6 days. At day 21 of age, feed was withheld for 24 hr, and each chicken was orally challenged twice daily with 2 ml each of C. perfringens culture (109 to 1010 CFU) on 5 consecutive days. The gross necrotic lesions were observed in 90 and 12.5% of challenged and control chickens, respectively. To our knowledge, this is the first study that demonstrated that a netB-positive Japanese isolate of C. perfringens is able to induce the clinical signs and lesions characteristic of NE in the experimental model, which may be useful for evaluating the pathogenicity of field isolates, the efficacy of a vaccine or a specific drug against NE.

Production of human apolipoprotein(a) transgenic NIBS miniature pigs by somatic cell nuclear transfer.

Most cases of ischemic heart disease and stroke occur as a result of atherosclerosis. The purpose of this study was to produce a new Nippon Institute for Biological Science (NIBS) miniature pig model by somatic cell nuclear transfer (SCNT) for studying atherosclerosis. The human apolipoprotein(a) (apo(a)) genes were transfected into kidney epithelial cells derived from a male and a female piglet. Male cells were used as donors initially, and 275 embryos were transferred to surrogates. Three offspring were delivered, and the production efficiency was 1.1% (3/275). Serial female cells were injected into 937 enucleated oocytes. Eight offspring were delivered (production efficiency: 0.9%) from surrogates. One male and 2 female transgenic miniature pigs matured well. Lipoprotein(a) was found in the male and one of the female transgenic animals. These results demonstrate successful production of human apo(a) transgenic NIBS miniature pigs by SCNT. Our goal is to establish a human apo(a) transgenic NIBS miniature pig colony for studying atherosclerosis.

Characterization of Eimeria brunetti Isolated from a Poultry Farm in Japan.

None of anticoccidial vaccines (Trivalent TAM™, monovalent Neca™ and imported pentavalent Paracox(®)-5) contain Eimeria brunetti in Japan, which has not been regarded as a cause of coccidiosis, because of its low prevalence. However, we have recently reported the evidence of a high nationwide prevalence of this species. In this report, we describe the characteristics of E. brunetti which have never been clearly defined in Japan. Mortality rates and other disease characteristics caused by our strain (Nb strain) were similar to those reported previously in other studies. Despite great reduction of body weight gains among groups infected with over 1 × 10(3) oocysts, the intestinal lesions in the infected chickens were rather mild compared to previous studies. Sulfa drugs and lasalocid were so effective that the E. brunetti infection was almost completely blocked. Consequently, it is suggested that the characteristics of E. brunetti are various among the strains, but the pathogenicity of the Japanese Nb strain is enough strong to cause clinical coccidiosis.

Estimation of bovine leukemia virus (BLV) proviral load harbored by lymphocyte subpopulations in BLV-infected cattle at the subclinical stage of enzootic bovine leucosis using BLV-CoCoMo-qPCR.

BACKGROUND: Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. BLV infection may remain clinically silent at the aleukemic (AL) stage, cause persistent lymphocytosis (PL), or, more rarely, B cell lymphoma. BLV has been identified in B cells, CD2+ T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, γ/δ T cells, monocytes, and granulocytes in infected cattle that do not have tumors, although the most consistently infected cell is the CD5+ B cell. The mechanism by which BLV causes uncontrolled CD5+ B cell proliferation is unknown. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method, BLV-CoCoMo-qPCR, which enabled us to demonstrate that the proviral load correlates not only with BLV infection, as assessed by syncytium formation, but also with BLV disease progression. The present study reports the distribution of BLV provirus in peripheral blood mononuclear cell subpopulations isolated from BLV-infected cows at the subclinical stage of EBL as examined by cell sorting and BLV-CoCoMo-qPCR. RESULTS: Phenotypic characterization of five BLV-infected but clinically normal cattle with a proviral load of > 100 copies per 1 × 105 cells identified a high percentage of CD5+ IgM+ cells (but not CD5- IgM+ B cells, CD4+ T cells, or CD8+T cells). These lymphocyte subpopulations were purified from three out of five cattle by cell sorting or using magnetic beads, and the BLV proviral load was estimated using BLV-CoCoMo-qPCR. The CD5+ IgM+ B cell population in all animals harbored a higher BLV proviral load than the other cell populations. The copy number of proviruses infecting CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells (per 1 ml of blood) was 1/34 to 1/4, 1/22 to 1/3, and 1/31 to 1/3, respectively, compared with that in CD5+ IgM+ B cells. Moreover, the BLV provirus remained integrated into the genomic DNA of CD5+ IgM+ B cells, CD5- IgM+ B cells, CD4+ T cells, and CD8+ T cells, even in BLV-infected cattle with a proviral load of <100 copies per 105 cells. CONCLUSIONS: The results of the recent study showed that, although CD5+ IgM+ B cells were the main cell type targeted in BLV-infected but clinically normal cattle, CD5- IgM+ B cells, CD4+ cells, and CD8+ T cells were infected to a greater extent than previously thought.

Production of cloned NIBS (Nippon Institute for Biological Science) and α-1, 3-galactosyltransferase knockout MGH miniature pigs by somatic cell nuclear transfer using the NIBS breed as surrogates.

BACKGROUND: Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. METHOD AND RESULTS: In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). CONCLUSIONS: These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.

BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status.

BACKGROUND: Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in BLV-infected animals. Indeed, the assay was highly effective in detecting BLV in cattle from a range of international locations. This assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. In this study, we compared the sensitivity of our BLV-CoCoMo-qPCR method for detecting BLV proviruses with the sensitivities of two real-time PCR systems, and also determined the differences of proviral load with serotests. RESULTS: BLV-CoCoMo-qPCR was found to be highly sensitive when compared with the real-time PCR-based TaqMan MGB assay developed by Lew et al. and the commercial TaKaRa cycleave PCR system. The BLV copy number determined by BLV-CoCoMo-qPCR was only partially correlated with the positive rate for anti-BLV antibody as determined by the enzyme-linked immunosorbent assay, passive hemagglutination reaction, or agar gel immunodiffusion. This result indicates that, although serotests are widely used for the diagnosis of BLV infection, it is difficult to detect BLV infection with confidence by using serological tests alone. Two cattle were experimentally infected with BLV. The kinetics of the provirus did not precisely correlate with the change in anti-BLV antibody production. Moreover, both reactions were different in cattle that carried different bovine leukocyte antigen (BoLA)-DRB3 genotypes. CONCLUSIONS: Our results suggest that the quantitative measurement of proviral load by BLV-CoCoMo-qPCR is useful tool for evaluating the progression of BLV-induced disease. BLV-CoCoMo-qPCR allows us to monitor the spread of BLV infection in different viewpoint compared with classical serotest.

A case of congenital hyperostosis in a newborn piglet.

One female newborn piglet showed prominent thickening of both forelimbs and died soon after birth. Histopathologically, thin and woven trabeculae of bone was extending out from the edge of cortical bone in the affected forelimbs, and diagnosed as congenital hyperostosis. The extent of radially proliferated trabeculae was most prominent in radioulna. Many round- to spindle-shaped cells were observed in periosteum, which were considered to be osteoblasts. Around the periosteum, the mesenchymal proliferation was extensive with abundant mucus, and cartilaginous metaplastic changes were observed mainly around the radioulna and humerus. Dilatation of vessels with fibrin deposition in vessel walls was often observed, which were considered to reflect the localized circulatory disturbance.

Malignant sertoli cell tumor in a goose (Anser cygnoides domesticus).

This paper describes the pathologic features of a malignant Sertoli cell tumor found in an adult goose (Anser cygnoides domesticus). At necropsy, in addition to one large tumor mass (15 cm in diameter), multiple small tumor masses were observed over the peritoneum and mesenterium in the coelomic cavity. The large tumor mass was composed of sheets, lobules, and small islands of tumor cells, and elongated tumor cells lying perpendicular to fibrous connective tissue were characteristic. Such histopathologic characteristics were common to all the tumors. The tumor cells were immunohistochemically positive for neuron-specific enolase and S-100, and some tumor cells contained fine intracytoplasmic pigments that stained red by oil red O staining. These findings, taken together with the fact that one testis was markedly atrophied and bore no tumor cells and the other testis was not discernible, the present case was diagnosed as unilateral malignant Sertoli cell tumor arising from the unilateral testis. To our knowledge, this is the first report of Sertoli cell tumor in the goose.

A case of renal oxalosis in a 3-month-old cat raised under controlled conditions.

The kidneys of a 3-month-old female cat were examined. The cat which had been raised under controlled conditions with no history of any poisoning showed progressive weight loss with increases in blood BUN and creatinine concentrations. At necropsy, both kidneys were firm in consistency with formation of focal scars. Histopathologically, widespread deposition of crystals was observed in the renal tubules (in both dilated lumina and degenerative epithelia) accompanying mild interstitial fibrosis with lymphocyte infiltration. The crystals were colorless or basophilic on the hematoxilin and eosin-stained section and could be visualized with polarized light as doubly fractile crystals. The crystals were identified as calcium oxalate crystals by histochemical examinations using von Kossa stain and alizarin red S stain under different conditions and by ultrastructural examination. Judging from the above-mentioned findings, the present renal lesion detected in an infant cat was diagnosed as renal oxalosis which was suspected to be hereditary in nature.

Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites.

At present, morphological characteristics of oocyst is the only achievable method for the identification of bovine coccidia to the species level. In this study, the internal transcribed spacer 1 (ITS-1) region of ribosomal RNA genes of six bovine Eimeria species; E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis and E. zuernii, were sequenced and analyzed the phylogenetic relationship among them. In pair-wise alignment, the sequences among the same species had high homology of over 90%. E. bovis and E. zuernii were closely related within the same cluster. This cluster and E. alabamensis were distant from major cluster of bovine coccidia that included E. auburnensis, E. cylindrica and E. ellipsoidalis. Species-specific PCR assays based on the amplification of the ITS-1 region were also developed to identify the 6 pathogens. The ITS-1 region of each Eimeria species had sufficient inter-specific sequence variation enough to design the primer sets that differentially amplified each target species. This PCR assay for the detection and differentiation of Eimeria parasite showed higher sensitivity when compared to the conventional oocyst-morphological examination. This is the first attempt for the identification of 6 bovine Eimeria parasites in the genomic level and may provide as useful methods for diagnosis and epidemiology of bovine coccidial infection.

Enhanced cell fusion activity in porcine epidemic diarrhea virus adapted to suckling mice.

Porcine epidemic diarrhea virus (PEDV) is the major causative agent of fatal diarrhea in piglets. To study the pathogenic features of PEDV using a mouse model, PEDV with virulence in mice is required. In pursuit of this, we adapted a tissue-culture-passed PEDV MK strain to suckling mouse brains. PEDV obtained after ten passages through the brains (MK-p10) had increased virulence for mice, and its fusion activity in cultured cells exceeded that of the original strain. However, the replication kinetics of MK and MK-p10 did not differ from each other in the brain and in cultured cells. The spike (S) protein of MK-p10 had four amino acid substitutions relative to the original strain. One of these (an H-to-R substitution at residue 1,381) was first detected in PEDV isolated after eight passages, and both this virus (MK-p8) and MK-p10 showed enhanced syncytium formation relative to the original MK strain and viruses isolated after two, four, and six passages, suggesting the possibility that the H-to-R mutation was responsible for this activity. This mutation could be also involved in the increased virulence of PEDV observed for MK-p10.

Hepatic Myelolipoma with systemic amyloidosis in a goose (Anser cygnoides domesticus).

We report here a case of hepatic myelolipoma with systemic amyloidosis in a goose (Anser cygnoides domesticus), which died suddenly following the short history of weakness and greenish diarrhea. At necropsy, multiple yellowish-white foci were observed on the surface of the prominently enlarged liver. Histologically, there were multiple foci of adipose tissue admixed with myeloid elements in various proportions in the liver as well as amyloid deposition in several organs including the liver, intestine, spleen, kidney, and ovary. Ultrastructurally, erythroblast-like cells and myelocytes, which showed various stages of differentiation, were observed in the foci of the liver. These findings shared characteristics of hepatic myelolipoma which is very rare in birds.

Development of quantitative real-time polymerase chain reaction for detection of and discrimination between Erysipelothrix rhusiopathiae and other Erysipelothrix species.

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

Prevalence of swine Torque teno virus genogroups 1 and 2 in Japanese swine with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease complex.

Torque teno virus (TTV) was first isolated from a human hepatitis patient in 1997. TTV was also identified in several animals, including pigs, cattle, sheep, cats and dogs. In this study, we analysed the prevalence of swine TTV genogroups 1 (TTV1) and 2 (TTV2) in Japanese swine populations with suspected post-weaning multisystemic wasting syndrome and porcine respiratory disease by using a nested polymerase chain reaction method. Of 153 serum samples from 16 different herds in Japan, TTV1 was detected in 46 samples (30%), TTV2 in 47 samples (31%) and both in 15 samples (10%). There was no significant difference in the detection rate among geographical regions. The overall prevalence rate of TTV genogroups was significantly lower in < or = 30-day-old pigs (11%) compared to that in older age groups (54-82%). These results suggest that swine TTV may be widespread in post-weaning pigs and could play aetiological roles in pig diseases in Japan. This is the first report on the prevalence of swine TTV in Japan.

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections.

Multiplex PCR and multiplex RT-PCR were developed to identify nine viruses in pigs with multiple infections. These viruses are: porcine circovirus type 2 (PCV2), suid herpesvirus 1, porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus, porcine rotavirus A (PoRV-A), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and Getah virus. These methods were shown to be high specificity and sensitivity. In the clinical application, a total of 75 field samples were examined by our methods and previously reported methods for PCV2, PRRSV, TGEV, and PEDV. As a result, the detection rates of our multiplex PCR and multiplex RT-PCR were higher than those of the previously reported methods. Furthermore, it was confirmed that 24 PCV2 positive samples were co-infected with other viruses, 11 with PRRSV, 10 with PPV, 2 with PoRV-A, and 1 with TGEV by a combination of multiplex PCR and multiplex RT-PCR. PPV and PoRV-A were newly detected by multiplex PCR and multiplex RT-PCR. These results suggest that the combination of our multiplex PCR and multiplex RT-PCR is useful for rapid and accurate identification of nine major pathogenic viruses in pigs with multiple infections.

Detection of five avian Eimeria species by species-specific real-time polymerase chain reaction assay.

To detect five different avian Eimeria species, we applied the SYBR Green-based real-time polymerase chain reaction (PCR) assay for the diagnosis of field-isolated parasites by using their individual species-specific primer sets. The primer sets were originally designed for Eimeria acervulina, E brunetti, E necatrix, and E tenella based on the sequence of the internal transcribed spacer 1 region of ribosomal DNA, whereas for E. maxima the primer sets were derived from sequences reported previously. The detection limit of these assays was defined at 10(2) or 10(1) oocysts depending on species. Melting curves from the real-time PCR assay showed that each species has a single peak and specific melting temperature value. Fecal samples from 32 poultry farms, which were endemic for coccidiosis, were examined using this assay. The data showed that E. brunetti was found in 21 farms, E maxima and E. necatrix in 16 farms, E. tenella in 12 farms, and E. acervulina in eight farms. This survey revealed that E brunetti was highly prevalent in Japan. This technique is not only easy and rapid but also available to detect Eimeria species specifically; therefore, it can be a valuable tool for diagnostic work for chicken coccidiosis.

An undifferentiated sarcoma in a rat resembling extraskeletal myxoid chondrosarcoma in man.

A soft tissue tumor occurring in the inguinal subcutaneous tissue was detected in a 109-week-old male F344 rat. Macroscopically, the tumor mass showed no skeletal relationship and a gelatinous multinodular appearance. Histologically, the tumor consisted of irregular lobules separated by scant fibrous septa. In each lobule, tumor cells were arranged in cords and strands in the plentiful myxoid stroma. The tumor cells showed marked pleomorphism, and had large, round to ovoid nuclei and abundant eosinophilic cytoplasm. Mitotic figures were often seen. Myxoid stroma of the tumor contained a large amount of acid mucopolysaccharides and collagen type II, which were demonstrated by histochemistry and immunohistochemistry respectively. The tumor cells showed positive immunoreactivities for vimentin and S100 protein. Ultrastructural examination revealed that the tumor cells had a large amount of mitochondria, Golgi complex and dilated rough endoplasmic reticulum containing amorphous material, and the myxoid stroma contained collagenous fibrils and proteoglycan particles. Based on these results, the present tumor in a rat resembled extraskeletal myxoid chondrosarcoma in man.

Actinomycosis of the brain and temporal bone in a goat.

A goat with neurologic signs had multifocal abscesses containing sulfur granules in the right brain and temporal bone. Histologically, the lesions consisted of pyogranulomas with several radiating bacterial colonies of various sizes. A tangled mass of filamentous and gram-positive bacteria was recognized in the central part of the colony. Actinomyces naeslundii antigen was detected in the colonies of bacteria in the brain and neighboring bone tissue by immunohistochemistry. Actinomycosis involving the central nervous system (CNS) and temporal bone is rare in animals. Cerebral infection with A. naeslundii may have resulted from direct extension from cervicofacial regions because the CNS lesions were distributed asymmetrically and were continuous with the right temporal bone.

Infectivity of porcine circovirus 1 and circovirus 2 in primary porcine hepatocyte and kidney cell cultures.

Infectivity of porcine circovirus (PCV) 1 and PCV2 was examined in primary porcine hepatocyte culture by comparing that of PCV in primary kidney cell culture. The virus titer of PCV2-infected hepatocyte cultures was higher than that of the PCV1-infected hepatocyte cultures and the PCV-infected kidney cell cultures. The number of virus-positive cells was most abundant in PCV2-infected hepatocyte cultures as determined by immunohistochemistry and/or in situ hybridization. The results of our data suggest that PCV2 preferably infects cultured hepatocytes as observed in the liver of pigs with postweaning multisystemic wasting syndrome.