Invasion of Chicken Anemia Virus in Specific-Pathogen-Free Chicken Flocks and Its Successful Elimination from the Colony.

A specific-pathogen-free (SPF) chicken colony was maintained with successive groups a month apart in age. The absence of specific pathogens, including chicken anemia virus (CAV), was confirmed through periodic serological tests for each group. However, some groups became CAV seropositive. The procedures of removing seropositive and the adjacent seronegative chickens followed with chemically disinfecting the housing did not halt CAV outbreaks. The full genome sequence of the CAV strain that appeared was closely related to low-virulence isolates in China. The outbreaks of CAV decreased with an increase in the seropositive chicken population, indicating that the progeny is protected from CAV infection by maternal anti-CAV antibodies. The persistence of CAV in erythroid and lymphoid tissues or reproductive tissues from CAV seropositive chickens was examined in chickens of various ages using polymerase chain reaction (PCR). Since a low persistence of CAV was observed in the colony, we isolated eggs from CAV seropositive hens through artificial insemination using semen collected from roosters and confirmed as CAV-free by PCR. Fertilized eggs were transferred to a new SPF facility and used for generating CAV-free progeny. To date, chickens reared in the new facility have been CAV-free for longer than two years. Redirection of eggs from seropositive hens was an effective means of eliminating CAV from chickens.

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.

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.

A gravimetric simplified method for nucleated marrow cell counting using an injection needle.

A simplified gravimetric marrow cell counting method for rats is proposed for a regular screening method. After fresh bone marrow was aspirated by an injection needle, the marrow cells were suspended in carbonate buffered saline. The nucleated marrow cell count (NMC) was measured by an automated multi-blood cell analyzer. When this gravimetric method was applied to rats, the NMC of the left and right femurs had essentially identical values due to careful handling. The NMC at 4 to 10 weeks of age in male and female Crj:CD(SD)IGS rats was 2.72 to 1.96 and 2.75 to 1.98 (x10(6) counts/mg), respectively. More useful information for evaluation could be obtained by using this gravimetric method in addition to myelogram examination. However, some difficulties with this method include low NMC due to blood contamination and variation of NMC due to handling. Therefore, the utility of this gravimetric method for screening will be clarified by the accumulation of the data on myelotoxicity studies with this method.

Expression in Escherichia coli and purification of the functional feline granulocyte colony-stimulating factor.

Feline granulocyte colony-stimulating factor (G-CSF) with an N-terminal histidine hexamer tag was expressed as inclusion bodies in E. coli. The G-CSF solubilized in 6 M guanidine solution was absorbed onto a Ni-NTA column and, after washing with decreasing concentrations of guanidine, eluted with imidazole in a soluble and apparently pure form. The activity of the recombinant feline G-CSF was 3 x 10(6)U/mg protein, as assayed by its stimulatory effect on NFS-60 cell proliferation. When a low level of purified feline G-CSF was administered once a day for two successive days to cats, the number of neutrophil increased 4-fold while the levels of other blood cell types remained virtually unchanged. Daily administration of G-CSF for a total of 11 days led to a more than 10-fold increase in neutrophils, an 8-fold increase in the number of monocytes and 2-fold increase in lymphocytes. No severe side effects or antibody production was observed in cats after administration of G-CSF.