Black leaf spot of Japanese persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis>), a new disease caused by <Emphasis Type="Italic">Adisciso kaki</Emphasis> sp. nov.

A new defoliating disease with severe leaf spot of Japanese persimmon (Diospyros kaki) was found in Shimane Prefecture. A fungus, isolated from the ascospores that had formed on the leaf spot, was demonstrated to reproduce the symptoms on leaves and described as a new species, Adisciso kaki, based on morphological and molecular phylogenetic analyses. This fungus is characterized by small ascomata without clypeus, obclavate to broadly cylindrical asci with an amyloid apical apparatus, and hyaline ascospores with a submedian septum. We coined the name black leaf spot (Kurohoshi-rakuyo-byo, in Japanese) for the new disease.     

Successful use of an acceleration rate response pacemaker with a transvenous steroid-eluting screw-in lead for third-degree atrioventricular block in a labrador retriever

Permanent pacemakers are commonly used in veterinary practice and can have a dramatic effect on the treatment of heart block. A Labrador Retriever dog suffering from exercise intolerance secondary to third degree atrioventricular block was treated with a new pacemaker system. A steroid-eluting screw-in type lead that has the advantage of being more fixed to the myocardial wall without increasing the pacing threshold was used. The heart rate was regulated with an acceleration sensing pacemaker generator that included several automatic modulation systems. Nineteen months after implantation, the dog has a normal level of activity. The present case suggests that this pacemaker design may offer important advantages for canine patients.     

High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system

Bioactive recombinant bovine interleukin-18 (rboIL-18) was expressed using a baculovirus system. Normally, IL-18 is translated as a precursor form of a 24kDa polypeptide and processed by IL-1beta converting enzyme (ICE) to a mature bioactive form of 18kDa protein. Hence, to express active form IL-18, we constructed two recombinant baculoviruses containing boIL-18 and human ICE (hICE) genes, respectively, and superinfected these viruses into insect cells. Superinfection of both recombinant viruses into the cells resulted in the expression of a 24kDa precursor form and an 18kDa mature form detectable in the supernatant by immunoblotting using anti-porcine IL-18 antibody. Culture supernatant from the superinfected cells showed a synergistic effect with recombinant boIL-12 for production of interferon-gamma (IFN-gamma) in bovine peripheral mononuclear cells. By addition of histidine hexamer at the C-terminal of boIL-18, the mature IL-18 was purified. Bioactivity remained after purification.     

Production and in vivo testing of a recombinant bovine IL-12 as an adjuvant for Salmonella typhimurium vaccination in calves

A recombinant bovine interleukin-12 (boIL-12) that contains a histidine hexamer, rboIL-12His, was produced, purified and administered to calves. We first tried the purification of heterodimer IL-12 from a mixture of p40 homodimer, p40 monomer, and p40-p35 heterodimer with a p35 subunit tagged with a histidine hexamar at its C-terminal (p35His). A recombinant baculovirus expressing p35His was generated and used for superinfection with a recombinant baculovirus expressing p40 subunit. The expressed subunits, p40 and p35His, were assembled into a 70kDa heterodimer in insect cells, released into culture medium, and then purified using a nickel chelate column. The purified rboIL-12His was bioactive for induction of IFN-gamma in bovine peripheral blood mononuclear cells (PBMCs) in vitro.The purified rboIL-12His was then administered to calves with inactivated Salmonella Typhimurium (ST). When sera were assayed by ELISA, specific anti-ST IgG1 antibodies were detected in all ST immunized calves, but, specific anti-ST IgG2 antibodies were detected only in calves administered ST along with rboIL-12His, indicating a possible switch to a Th1 response. Administration of commercially available Salmonella vaccine did not elicit IgG2 antibodies in calves. These results suggest that co-administration of IL-12 with inactivated ST cells could induce a Th1-type response in calves.     

Surveillance of avian influenza viruses in Northern pintails (Anas acuta) in Tohoku District, Japan

Among winter migratory waterfowl, Northern pintails (Anas acuta), in one of the largest flocks in Tohoku district, northeast Japan, were surveyed for influenza A viruses at five wintering sites in three prefectures, viz., Aomori, Akita, and Miyagi. A total of 38 influenza A viruses were isolated from 2066 fecal samples collected during November 2006 through March 2007. The overall isolation rate was 1.84%. Eleven different subtypes were isolated, including nine H5N2, seven H6N8, seven H10N1, four H4N6, three H6N1, three H11N9, and one each of H1N1, H6N2, H6N5, H10N9, H11N1. Only the H4N6 subtype was detected during two successive months, November and December, from Lake Ogawara of Aomori prefecture. One wintering site, Lake Izunuma of Miyagi prefecture, was negative for virus isolation throughout the study period. During the sampling period, the highest virus isolation rate was in December (4.90%) followed by November (2.18%), January (0.91%), and February (0.30%). Virus isolation was negative for samples collected in March 2007. These results suggest that influenza viruses are introduced by Northern pintail when they migrate into Japan, but the viruses are not maintained in the flocks, most likely because the birds are not breeding during the winter. We believe that this relatively large data set creates a strong foundation for future studies of avian influenza virus (AIV) prevalence, evolution, and ecology in wintering sites, along with the role of Northern pintails in the spread of AIV during their migration from northern Russia and Asia to Japan.     

Use of herpesvirus papio 2 as an alternative antigen in immunoblotting assay for B virus diagnosis

Herpesvirus papio 2 (HVP2), which infects baboons, is much more closely related genetically and antigenically to monkey B virus (BV) than to human herpes simplex virus 1(HSV1) and other related herpes viruses. The usefulness of HVP2 as an alternative test antigen in immunoblotting assays to detect BV-antibody in macaque monkey sera was assessed. Six HVP2 proteins reacted with BV-positive sera in immunoblotting. No specific bands could be detected with BV-negative sera. These results show the usefulness of HVP2 antigen as an alternative and safer antigen than authentic BV antigen in detecting BV antibody in immunoblotting.     

Transgenic Pigs with Pancreas-specific Expression of Green Fluorescent Protein

The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47–65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in β-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.     

Molecular phylogeny and diversity of Myanmar and Bhutan mithun based on mtDNA sequences

The mithun (Bos frontalis), synonymous with mithan and gayal, is considered to be a domesticated form of gaur (B. gaurus). However, there has been a controversy concerning its origin. In an effort to address this issue, the mitochondrial cytochrome b (cytb) genes of 20 mithun from Myanmar and 13 from Bhutan were sequenced to trace its maternal origin. Seven cytb haplotypes were found in the 33 mithun, and the phylogenetic tree for these haplotypes clearly showed three embranchments involving five gaur types, a B. indicus type, and a B. taurus type. Sixteen Myanmar and 12 Bhutan mithun had gaur haplotypes, while a B. indicus haplotype was found in three Myanmar and one Bhutan mithun. The B. taurus haplotype was detected in a single Myanmar animal. These results demonstrated that the principal maternal origin of mithun was gaur and suggested that it was directly domesticated from gaur. However, some introgression of domestic cattle existed in current mithun populations. The presence of cattle mtDNA raised the question of how many cattle nuclear genes might have been integrated into the gene pool of mithun.