Growth, reproductive performance, carcass characteristics and meat quality in F1 and F2 progenies of somatic cell-cloned pigs.

The objective of this study was to examine the health and meat production of cloned sows and their progenies in order to demonstrate the application of somatic cell cloning to the pig industry. This study compared the growth, reproductive performance, carcass characteristics and meat quality of Landrace cloned sows, F1 progenies and F2 progenies. We measured their body weight, growth rate and feed conversion and performed a pathological analysis of their anatomy to detect abnormalities. Three of the five cloned pigs were used for a growth test. Cloned pigs grew normally and had characteristics similar to those of the control purebred Landrace pigs. Two cloned gilts were bred with a Landrace boar and used for a progeny test. F1 progenies had characteristics similar to those of the controls. Two of the F1 progeny gilts were bred with a Duroc or Large White boar and used for the progeny test. F2 progenies grew normally. There were no biological differences in growth, carcass characteristics and amino acid composition among cloned sows, F1 progenies, F2 progenies and conventional pigs. The cloned sows and F1 progenies showed normal reproductive performance. No specific abnormalities were observed by pathological analysis, with the exception of periarteritis in the F1 progenies. All pigs had a normal karyotype. These results demonstrate that cloned female pigs and their progenies have similar growth, reproductive performance and carcass quality characteristics and that somatic cell cloning could be a useful technique for conserving superior pig breeds in conventional meat production.

Il2rg gene-targeted severe combined immunodeficiency pigs.

A porcine model of severe combined immunodeficiency (SCID) promises to facilitate human cancer studies, the humanization of tissue for xenotransplantation, and the evaluation of stem cells for clinical therapy, but SCID pigs have not been described. We report here the generation and preliminary evaluation of a porcine SCID model. Fibroblasts containing a targeted disruption of the X-linked interleukin-2 receptor gamma chain gene, Il2rg, were used as donors to generate cloned pigs by serial nuclear transfer. Germline transmission of the Il2rg deletion produced healthy Il2rg(+/-) females, while Il2rg(-/Y) males were athymic and exhibited markedly impaired immunoglobulin and T and NK cell production, robustly recapitulating human SCID. Following allogeneic bone marrow transplantation, donor cells stably integrated in Il2rg(-/Y) heterozygotes and reconstituted the Il2rg(-/Y) lymphoid lineage. The SCID pigs described here represent a step toward the comprehensive evaluation of preclinical cellular regenerative strategies.

In situ hybridization and immunohistochemistry for the detection of porcine cytomegalovirus.

To establish in situ hybridization and immunohistochemistry based-assays for the detection of porcine cytomegalovirus, routinely processed renal tissue sections from 34 diseased piglets suspected of having the infection were obtained and examined. Using hematoxylin and eosin, porcine cytomegalovirus inclusion bodies were found in the nucleus of renal epithelial cells and capillary endothelial cells in the renal medulla in 30 cases. Inclusion bodies corresponding to porcine cytomegalovirus mRNA after in situ hybridization or porcine cytomegalovirus antigens after immunohistochemistry were easily determined. The cells were characterized by cytomegaly and basophilic intranuclear inclusion bodies. Using in situ hybridization, porcine cytomegalovirus mRNA were clearly detected in the nucleus and cytoplasm of the cells in 28 of the 30 (93.3%) cases. Using immunohistochemistry, porcine cytomegalovirus antigens were clearly detected in the cytoplasm of the cells in 21 of the 30 (70.0%) cases. Higher specificities and increased intensity of staining was observed with minimal background using in situ hybridization and immunohistochemistry compared with hematoxylin and eosin. Thus, the two established methods are useful and helpful tools for detecting the presence of a porcine cytomegalovirus infection.

The relationship between clinical pharmacokinetics of aripiprazole and CYP2D6 genetic polymorphism: effects of CYP enzyme inhibition by coadministration of paroxetine or fluvoxamine.

PURPOSE: To investigate the effects of coadministration of paroxetine or fluvoxamine on the pharmacokinetics of aripiprazole in healthy adult Japanese with different CYP2D6 genotypes. METHODS: Fourteen CYP2D6 extensive metabolizer (EM) and 14 CYP2D6 intermediate metabolizer (IM) subjects were coadministered a single oral dose of aripiprazole 3 mg after steady-state plasma concentrations of the SSRIs paroxetine (20 mg/day) or fluvoxamine (100 mg/day) were reached by repeated oral doses for 6-7 days. The pharmacokinetics of aripiprazole with and without coadministration of SSRIs were compared according to CYP2D6 genotypes. RESULTS: Coadministration of paroxetine, a potent CYP2D6 inhibitor, decreased systemic clearance (CL/F) of aripiprazole by 58 and 23% in CYP2D6 EMs and IMs, respectively, demonstrating that the percentage inhibition of CYP2D6 activity by coadministration of paroxetine was apparently greater in CYP2D6 EMs than in IMs. Coadministration of fluvoxamine, a less potent CYP3A4 inhibitor, decreased the CL/F of aripiprazole by 39% in CYP2D6 EMs and 40% in IMs, indicating the same inhibitory effect on CYP enzymes, regardless of the CYP2D6 genotype. Percent contribution of CYP2D6 to total CL/F (CYP2D6 plus CYP3A4) of aripiprazole estimated as a reduced percentage of CL/F by CYP enzyme inhibition was 62% for CYP2D6 EMs and 24% for IMs in paroxetine coadministration, and 40% for CYP2D6 EMs and 18% for IMs in fluvoxamine coadministration. CONCLUSIONS: There were marked differences in the degree of influence of paroxetine coadministration on the pharmacokinetics of aripiprazole between CYP2D6 EMs and IMs, but no apparent differences were found between two CYP2D6 genotypes in fluvoxamine coadministration. Aripiprazole can be used safely in combination with SSRIs that have a CYP enzyme-inhibitory action.

Emergence of avian influenza viruses with enhanced transcription activity by a single amino acid substitution in the nucleoprotein during replication in chicken brains.

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP (I)109(T)). By analyzing viruses constructed by the reverse-genetic method, we established that the NP (I)109(T) substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP (I)109(T) substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP (I)109(T) substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP (I)109(T) mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains.

Developmental ability of somatic cell nuclear transferred embryos aggregated at the 8-cell stage or 16- to 32-cell stage in cattle.

Aggregation of somatic cell nuclear transfer (SCNT) embryos in mice is reported to improve full-term development. In the present study, we attempted to improve the development of SCNT embryos by aggregation in cattle. In Experiment 1, to examine the effect of the timing of aggregation on in vitro development of cumulus-cell NT embryos, we aggregated two or three SCNT embryos (2X or 3X embryos) at the 1-cell, 8-cell and 16- to 32-cell stages. Irrespective of the timing of aggregation, 3X embryos developed to the blastocyst stage at a high rate. However, aggregation did not improve the total blastocyst formation rate of the embryos used. The cell numbers of 3X embryos aggregated at the 1-cell stage and 2X embryos tended to be higher than that of single NT embryos (1X embryos). Furthermore, a significant increase in cell number was observed in 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. In Experiment 2, we used fibroblast cells as nuclear donors and examined in vitro development of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. As a result, 3X embryos had high blastocyst formation rates and higher cell numbers than 1X embryos, which was consistent with the results of Experiment 1. In Experiment 3, we examined the full-term developmental ability of 3X embryos aggregated at the 8-cell stage and 16- to 32-cell stage. After transfer of fibroblast-derived NT embryos into recipient animals, a significantly higher pregnancy rate was obtained on Day 60 in 3X embryos than in 1X embryos. Two embryos aggregated at 8-cell stage and one embryo aggregated at the 16- to 32-cell stage developed to term, while no pregnancies derived from 1X embryos that lasted to Day 60. However, two of the cloned calves were stillborn. These results suggest that aggregation of the 8-cell stage or 16- to 32-cell stage SCNT embryos may improve the pregnancy rate, but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.

NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens.

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.

Granulomatous lymphadenitis associated with Actinobacillus pleuropneumoniae serotype 2 in slaughter barrows.

This study evaluated the occurrence of granulomatous lymphadenitis and its association with Actinobacillus spp. in 151 653 slaughtered pigs. Markedly enlarged pulmonary hilar, mediastinal, mandibular or hepatic lymph nodes were detected in 6 castrated males. The cut surfaces showed multifocal yellow-white lesions. Histologically, gram-negative bacilli were visible in the centers of the lesions with asteroid bodies, epithelioid cells, and multinucleated giant cells. Dense fibrous connective tissue surrounded these granulomatous lesions. Immunohistochemically, the organisms reacted with polyclonal antibodies against Actinobacillus pleuropneumoniae serotype 2 in all 6 barrows. The organism was isolated from the lymph nodes of all 6 animals. The results indicate that the granulomatous lymphadenitis was associated with A. pleuropneumoniae serotype 2 and the disorder had a tendency to occur in slaughter barrows.

Needle-free jet injection of small doses of Japanese encephalitis DNA and inactivated vaccine mixture induces neutralizing antibodies in miniature pigs and protects against fetal death and mummification in pregnant sows.

Japanese encephalitis (JE) virus causes abortion and stillbirth in swine, and encephalitis in humans and horses. We have previously reported that immunogenicity of a DNA vaccine against JE was synergistically enhanced in mice by co-immunization with a commercial inactivated JE vaccine (JEVAX) under a needle-free injection system. Here, we found that this immunization strategy was also effective in miniature pigs. Because of the synergism, miniature pigs immunized twice with a mixture of 10 µg of DNA and a 1/100 dose of JEVAX developed a high neutralizing antibody titer (1:190 at 90% plaque reduction assay). Even using 1 µg of DNA, 3 of 4 pigs developed neutralizing antibodies. Following challenge, all miniature pigs with detectable neutralizing antibodies were protected against viremia. Pregnant sows inoculated with 10 or 1 µg of DNA mixed with JEVAX (1/100 dose) developed antibody titers of 1:40-1:320. Following challenge, fetal death and mummification were protected against in DNA/JEVAX-immunized sows.

Occurrence of a pig respiratory disease associated with swine influenza A (H1N2) virus in Tochigi Prefecture, Japan.

In February 2008, a feeder pig herd of the affected farm in Tochigi Prefecture, Japan, showed increasing respiratory symptoms; by April, the situation worsened with 12-16 pigs dying daily. Diagnostic tests revealed the presence of H1N2 subtype of swine influenza virus (SIV) and Pasteurella multocida from nasal swab and lung emulsion. Serological tests by hemagglutination inhibition method and enzyme-linked immunosorbent assay method (ELISA; imported from U.S.A.) indicated the spread of SIV into the pig herds of the affected farm around April 2008. The severe infection and subsequent damage were considered as a result of the combined infection of SIV (H1N2) and bacteria that may have been prevalent in the pig farm. Genetic homology search of sequences for the hemagglutinin (HA) and neuraminidase (NA) genes of A/swine/Tochigi/1/08 showed high homology to Japanese SIVs (H1N2) isolated in the 2000s. Therefore, we considered that Japanese SIV (H1N2) has established an independent stable lineage and participated in infecting pig populations as one of the factors of the pig respiratory disease complex. Consistent surveillance would contribute to clarifying the prevalence of dominant SIVs.

Prevalence of granulomatous pleuropneumonia associated with Actinobacillus pleuropneumoniae serotype 2 in slaughter pigs.

A total of 14,818 slaughtered pigs were examined macroscopically. Of these, 25 pigs with porcine pleuropneumonia were collected and the relations among Actinobacillus spp. and granulomatous lesions in organs (lungs and tonsils) were evaluated. In the lungs, only Actinobacillus pleuropneumoniae serotype 2 was isolated from 20 of the pigs. Histologically, granulomatous pneumonia with A. pleuropneumoniae antigen was detected in 8 of the pigs. The antigen was visible in the centers of the lesions along with asteroid bodies, epithelioid cells and multinucleated giant cells. In the tonsils, granulomatous lesions were not detected, although A. pleuropneumoniae serotype 2 (5 pigs), serotype 7 (1 pig), Actinobacillus porcitonsillarum (1 pig) and Actinobacillus minor (1 pig) were isolated. The present survey suggests that multifocal granulomatous pneumonia in slaughter pigs could be highly associated with A. pleuropneumoniae serotype 2 infection.

Antibody responses induced by experimental West Nile virus infection with or without previous immunization with inactivated Japanese encephalitis vaccine in horses.

A group of horses immunized with inactivated Japanese encephalitis (JE) vaccine (JE-Immune Group) and a group of non-immunized horses (Non-Immune Group) were infected with West Nile virus (WNV). After WNV infection, neutralizing (Nt) antibody (Ab) titers to WNV were higher than those to JE virus (JEV) in the Non-Immune Group, but the NtAb titers to JEV were higher than those to WNV during most of the post-challenge observation period in the JE-Immune Group. Immunoglobulin M (IgM) Abs to WNV tested positive in the Non-Immune Group but negative in the JE-Immune Group, except for in one horse. These results suggest that diagnosis of WNV infection in JE-immunized horses requires serological tests for NtAb and IgM titers to both WNV and JEV.

Experimental West Nile virus infection in aigamo ducks, a cross between wild ducks (Anas platyrhynchos) and domestic ducks (Anas platyrhynchos var. domesticus).

Four 2-wk-old and four 4-wk-old aigamo ducks, a cross between wild and domestic ducks (Anas platyrhynchos and Anas platyrhynchos var. domesticus, respectively), were infected with the NY99 strain of West Nile virus (WNV) to investigate WNV's pathogenicity in aigamo ducks and the possibility that they could transmit WNV. In the group of infected 2-wk-old aigamo ducks (2w-infection group), all of the ducks ate and drank less and showed decreased activity, some showed ataxia, and one died. Meanwhile, the group of infected 4 wk olds (4w-infection group) showed no clinical signs during the experimental period. Viremia was observed in all of the ducks in both age groups. Peak viral titers in the three surviving members of the 2w-infection group were 10(3.7)-10(5.3) plaque-forming units (PFU)/ml serum; the peak was 10(7.1) PFU/ml serum in the 2w duck that died from the infection. Peak viral titers in the 4w-infection group were 10(4.1)-10(4.9) PFU/ml serum. Viral shedding in the oral and/or cloacal cavity was observed in all four members of the 2w-infection group and in three of the four members of the 4w-infection group. These results suggest that WNV-infected aigamo ducks can transmit WNV. Although aigamo ducks are reared in East Asia, where WNV is an exotic pathogen, the virus could be introduced and spread there in the future; thus it is important to take precautions against an introduction, and measures to prevent infection to aigamo duck operations should be prepared.

Characteristics of novel insect defensin-based membrane-disrupting trypanocidal peptides.

Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH(2)), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite's plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 microM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs.

Association of increased pathogenicity of Asian H5N1 highly pathogenic avian influenza viruses in chickens with highly efficient viral replication accompanied by early destruction of innate immune responses.

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.

Multiple functions of short synthetic enantiomeric peptides based on beetle defensins.

Four enantiomeric 9-mer peptides, D-peptides A (RLYLRIGRR-NH(2)), B (RLRLRIGRR-NH(2)), C (ALYLAIRRR-NH(2)), and D (RLLLRIGRR-NH(2)), were designed and synthesized on the basis of a beetle defensin antimicrobial peptide. These D-9-mer peptides have been reported to exhibit multiple functions, including antimicrobial and antiprotozoan activity, without cytotoxicity on normal fibroblasts and leukocyte cells. In this study, we found that the D-9-mer peptides inhibited telomerase activity (IC(100) = 40 microM). A new peptide, D-peptide C2 (ALYLAIRRRRRRRR-NH(2)), designed from D-peptide C to translocate into the cytoplasm by a penetrating sequence (octa-arginine), showed extremely strong telomerase inhibitory activity (IC(100) = 0.1 microM). D-Peptide C2 exhibited a great increase in cytotoxicity against various cancer cell lines (IC(50) = 3.4-26.4 microM). However, the immediate death of the cells suggested that the high cytotoxicity was not an effect of telomerase inhibitory activity. Mitochondrial swelling assay and microscopical observations of mitochondria indicated that the major target of the D-peptide C2 was the mitochondrial membrane.

Synthetic nonamer peptides derived from insect defensin mediate the killing of African trypanosomes in axenic culture.

Synthetic antimicrobial 9-mer peptides (designated as peptides A and B) designed on the basis of insect defensins and their effects on the growth of African trypanosomes were examined using two isolates of Trypanosoma congolense, IL1180 and IL3338, and two isolates of Trypanosoma brucei brucei, ILTat1.1and GUTat 3.1, under axenic culture conditions. Both peptides inhibited the growth of all bloodstream form (BSF) trypanosomes at 200-400 microg/mL in the complete growth medium, with peptide A being more potent than peptide B. In addition, these peptides exhibited efficient killing at 5-20 microg/mL on BSF trypanosomes suspended in phosphate-buffered saline, whereas procyclic insect forms in the same medium were more refractory to the killing. Electron microscopy revealed that the peptides induced severe defects in the cell membrane integrity of the parasites. The insect defensin-based peptides up to either 200 or 400 microg/mL showed no cell killing or growth inhibition on NIH3T3 murine fibroblasts. The results suggest that the design of suitable synthetic insect defensin-based 9-mer peptides might provide potential novel trypanocidal drugs.

Prevalence and characteristics of eae- and stx-positive strains of Escherichia coli from wild birds in the immediate environment of Tokyo Bay.

The prevalence and characteristics of eae- and stx-positive Escherichia coli strains in wild birds in the immediate environment of Tokyo Bay, Japan, was examined using cloacal swab samples taken from 447 birds belonging to 62 species. PCR screening showed that the prevalences of stx- and eae-positive strains of Escherichia coli were 5% (23/447) and 25% (113/447), respectively. Four strains of stx(2f)-positive E. coli were isolated from two feral pigeons, an oriental turtle dove and a barn swallow. In contrast, 39 eae-positive E. coli strains were isolated, and most of the strains possessed a subtype of intimin that is classified as a minor group of human intimins, such as intimin upsilon, kappa, and mu. Moreover, these strains did not possess any of the other pathogenic genes tested, such as stxs, ehxA, bfp, or irp. Thus, wild birds were considered to be a reservoir of atypical enteropathogenic E. coli.

Experimental West Nile virus infection in jungle crows (Corvus macrorhynchos).

We experimentally infected jungle crows (Corvus macrorhynchos), which are representative corvids in East Asia, with West Nile virus (WNV) to study their susceptibility toward WNV infection. Six jungle crows were subcutaneously inoculated with 1,000 plaque-forming units (PFU) of the WNV NY99 strain. Within 7 days after inoculation, five of the six infected crows died, and peak viremias ranged from 10(6.5) to 10(10.9) PFU/mL serum. In addition, infected crows shed WNV in the oral cavity and cloaca, and the virus was widely disseminated in the organs of the crows. Based on these findings, we conclude that jungle crows are highly susceptible to WNV infection, and they could serve as amplifying hosts in the transmission of WNV. Although WNV has not been detected in East Asia, the virus could spread rapidly on introduction into this region because of the large number of potential amplifying hosts and vector mosquitoes that inhabit this region.

Bovine nuclear transfer using fresh cumulus cell nuclei and in vivo- or in vitro-matured cytoplasts.

We examined the effects of the source of recipient oocytes and timing of fusion and activation on the development competence of bovine nuclear transferred (NT) embryos derived from fresh cumulus cells isolated immediately after collection by ovum pickup (OPU). As recipient cytoplasts, we used in vivo-matured oocytes collected from hormone-treated heifers by OPU, or in vitro-matured oocytes from slaughterhouse-derived ovaries. NT embryos were chemically activated immediately (simultaneous fusion and activation, FA) or 2 h (delayed activation, DA) after fusion. When in vitro-matured oocytes were used as recipient cytoplasts, the development rate to the blastocyst stage of NT embryos produced by the DA method (23%) tended to be higher than those by the FA method (15%), but the difference was not significant. NT embryos derived from in vivo-matured cytoplasts have a high blastocyst yield (46%). Pregnancy rate at day 35 did not differ with the timing of fusion and activation (FA vs. DA; 50% vs. 44%) or oocyte source (in vivo- vs. in vitro-matured; 50% vs. 44%). Subsequently, the high fetal losses (88% of pregnancies) were observed with in vitro-matured cytoplasts, whereas no abortions were observed in NT fetuses from in vivo-matured cytoplasts. A total of three embryos derived from fresh cumulus cells developed to term. However, all three cloned calves were stillborn. These results indicate that improvement of development competence after NT is possible by using in vivo-matured oocytes as recipient cytoplasts in bovine NT.